A variety of monomeric PRG4 proteins and a disulfide-bonded dimer/multimer are secreted by chondrocytes in bovine cartilage explants. The observed decrease in M(W)'s of monomeric PRG4 species upon RA may be due to the release of post-translationally cleaved fragments. Further study of these species will provide insight into the PRG4 molecular structure and function relationship.

We developed a novel testing system that allows quantification of joint loading and permits analysis of changes in total protein and PRG4 contents in joint fluid of intact knees in live mice. A sequence of 15 repeat, isometric muscular contractions of "low" intensity (less than 50% of the maximal isometric muscular force), and "high" intensity (greater than 55% of maximal) were applied repeatedly (up to five times with a 15 min rest between contractions) to the mouse knee. Increases in knee joint loading were accompanied with significant increases in total protein (p<0.0001) and PRG4 concentrations in the synovial fluid. Total protein and PRG4 concentrations decreased with repeated "high" intensity loading. However, the addition of cell secretion inhibitors to the knee prior to muscular loading resulted in PRG4 levels that remained below the detection limit for all loading conditions. These results suggest that changes in synovial fluid proteins and PRG4 concentrations upon joint loading are mediated by cells within the joint, and that these changes may be used as quantitative indicators for the intensity and duration of acute joint loading, and might serve as a powerful clinical tool to assess the effectiveness of rehabilitation and prevention exercise programs.

Total internal reflection-fluorescence correlation spectroscopy (TIR-FCS) is an emerging technique that is used to measure events at or near an interface, including local fluorophore concentrations, local translational mobilities and the kinetic rate constants that describe the association and dissociation of fluorophores at the interface. TIR-FCS is also an extremely promising method for studying dynamics at or near the basal membranes of living cells. This protocol gives a general overview of the steps necessary to construct and test a TIR-FCS system using either through-prism or through-objective internal reflection geometry adapted for FCS. The expected forms of the autocorrelation function are discussed for the cases in which fluorescent molecules in solution diffuse through the depth of the evanescent field, but do not bind to the surface of interest, and in which reversible binding to the surface also occurs.

Fluorescence recovery after photobleaching and fluorescence correlation spectroscopy are the primary means for studying translational diffusion in biological systems. Both techniques, however, present numerous obstacles for measuring translational mobility in structures only slightly larger than optical resolution. We report a new method using through-prism total internal reflection fluorescence microscopy with continuous photobleaching (TIR-CP) to overcome these obstacles. Small structures, such as prokaryotic cells or isolated eukaryotic organelles, containing fluorescent molecules are adhered to a surface. This surface is continuously illuminated by an evanescent wave created by total internal reflection. The characteristic length describing the decay of the evanescent intensity with distance from the surface is smaller than the structures. The fluorescence decay rate resulting from continuous evanescent illumination is monitored as a function of the excitation intensity. The data at higher excitation intensities provide apparent translational diffusion coefficients for the fluorescent molecules within the structures because the decay results from two competing processes (the intrinsic photobleaching propensity and diffusion in the small structures). We present the theoretical basis for the technique and demonstrate its applicability by measuring the diffusion coefficient, 6.3 ± 1.1 µm2/sec, of green fluorescent protein (GFP) in Escherichia coli cells.

The pregnane X receptor (PXR), a member of the nuclear receptor superfamily, regulates the expression of drug-metabolizing enzymes in a ligand-dependent manner. The conventional view of nuclear receptor action is that ligand binding enhances the receptor’s affinity for coactivator proteins, while decreasing its affinity for corepressors. To date, however, no known rigorous biophysical studies have been conducted to investigate the interaction among PXR, its coregulators, and ligands. In this work, steady-state total internal reflection fluorescence microscopy (TIRFM) and total internal reflection with fluorescence recovery after photobleaching were used to measure the thermodynamics and kinetics of the interaction between the PXR ligand binding domain and a peptide fragment of the steroid receptor coactivator-1 (SRC-1) in the presence and absence of the established PXR agonist, rifampicin. Equilibrium dissociation and dissociation rate constants of ~5 μM and ~2 s−1, respectively, were obtained in the presence and absence of rifampicin, indicating that the ligand does not enhance the affinity of the PXR and SRC-1 fragments. Additionally, TIRFM was used to examine the interaction between PXR and a peptide fragment of the corepressor protein, the silencing mediator for retinoid and thyroid receptors (SMRT). An equilibrium dissociation constant of ~70 μM was obtained for SMRT in the presence and absence of rifampicin. These results strongly suggest that the mechanism of ligand-dependent activation in PXR differs significantly from that seen in many other nuclear receptors.