Comparing intra-articular CTXII levels assessed via magnetic capture or lavage in a rat knee osteoarthritis model.

OBJECTIVE: Parallel measures of osteoarthritis (OA) across species can help evaluate OA models relative to humans. Toward this need, our group recently developed a magnetic nanoparticle-based technology, termed magnetic capture, to analyze biomarkers within a rat knee. The objectives of this study were to directly compare magnetic capture to lavage, and assess c-telopeptide of collagen type II (CTXII) in the rat medial meniscus transection (MMT) model of knee OA. DESIGN: MMT surgery was performed in 30 male Lewis rats (3 months, 250 g). Using lavage or magnetic capture, CTXII was assessed in the OA-affected and contralateral knee at 1 week (n = 6 per group) or 4 weeks (n = 8 per group) after surgery. RESULTS: While lavage detected elevated CTXII concentrations in the OA-affected knee at 1 week (P = 0.002), magnetic capture detected elevated CTXII levels in the OA-affected knee at 4 weeks (P = 0.016). While magnetic capture did not detect significant elevation of CTXII at week 1, five of six rats evaluated with magnetic capture had higher CTXII levels in the OA-affected joint relative to the contralateral limb. Moreover, with magnetic capture, CTXII levels increased from 1 week to 4 weeks, corresponding to histological damage. CTXII concentrations evaluated via lavage were relatively constant across time. CONCLUSIONS: Magnetic capture and lavage evaluate CTXII in different ways: Magnetic capture measures total CTXII in the joint, while lavage measures concentration. Our data indicate magnetic capture may be advantageous at later time points, where CTXII can be diluted by effusions.

Exogenous thymosin beta4 prevents apoptosis in human intervertebral annulus cells in vitro.

Loss of cells in the human disc due to programmed cell death (apoptosis) is a major factor in the aging and degenerating human intervertebral disc. Our objective here was to determine if thymosin beta(4) (TB4), a small, multifunctional 5 kDa protein with diverse activities, might block apoptosis in human annulus cells cultured in monolayer or three-dimensional (3D) culture. Apoptosis was induced in vitro using hydrogen peroxide or serum starvation. Annulus cells were processed for identification of apoptotic cells using the TUNEL method. The percentage of apoptotic cells was determined by cell counts. Annulus cells also were treated with TB4 for determination of proliferation, and proteoglycan production was assessed using cell titer and 1,2 dimethylmethylamine (DMB) assays and histological staining. A significant reduction in disc cell apoptosis occurred after TB4 treatment. The percentage of cells undergoing apoptosis decreased significantly in TB4 treated cells in both apoptosis induction designs. TB4 exposure did not alter proteoglycan production as assessed by either DMB measurement or histological staining. Our results indicate the need for further studies of the anti-apoptotic effect of TB4 and suggest that TB4 may have therapeutic application in future biological therapies for disc degeneration.

Formation and implications of a ternary complex of profilin, thymosin beta 4, and actin.

Data from affinity chromatography, analytical ultracentrifugation, covalent cross-linking, and fluorescence anisotropy show that profilin, thymosin beta(4), and actin form a ternary complex. In contrast, steady-state assays measuring F-actin concentration are insensitive to the formation of such a complex. Experiments using a peptide that corresponds to the N terminus of thymosin beta(4) (residues 6-22) confirm the presence of an extensive binding surface between actin and thymosin beta(4), and explain why thymosin beta(4) and profilin can bind simultaneously to actin. Surprisingly, despite much lower affinity, the N-terminal thymosin beta(4) peptide has a very slow dissociation rate constant relative to the intact protein, consistent with a catalytic effect of the C terminus on conformational change occurring at the N terminus of thymosin beta(4). Intracellular concentrations of thymosin beta(4) and profilin may greatly exceed the equilibrium dissociation constant of the ternary complex, inconsistent with models showing sequential formation of complexes of profilin-actin or thymosin beta(4)-actin during dynamic remodeling of the actin cytoskeleton. The formation of a ternary complex results in a very large amplification mechanism by which profilin and thymosin beta(4) can sequester much more actin than is possible for either protein acting alone, providing an explanation for significant sequestration even if molecular crowding results in a very low critical concentration of actin in vivo.

Actin filament cross-linking by MARCKS: characterization of two actin-binding sites within the phosphorylation site domain.

We recently identified conformational changes that occur upon phosphorylation of myristoylated alanine-rich protein kinase C substrate (MARCKS) that preclude efficient cross-linking of actin filaments (Bubb, M. R., Lenox, R. H., and Edison, A. S. (1999) J. Biol. Chem. 274, 36472-36478). These results implied that the phosphorylation site domain of MARCKS has two actin-binding sites. We now present evidence for the existence of two actin-binding sites that not only mutually compete but also specifically compete with the actin-binding proteins thymosin beta(4) and actobindin to bind to actin. The effects of substitution of alanine for phenylalanine within a repeated hexapeptide segment suggest that the noncharged region of the domain contributes to binding affinity, but the binding affinity of peptides corresponding to each binding site has a steep dependence on salt concentration, consistent with presumed electrostatic interactions between these polycationic peptides and the polyanionic N terminus of actin. Phosphorylation decreases the site-specific affinity by no more than 0.7 kcal/mol, which is less than the effect of alanine substitution. However, phosphorylation has a much greater effect than alanine substitution on the loss of actin filament cross-linking activity. These results are consistent with the hypothesis that the compact structure resulting from conformational changes due to phosphorylation, in addition to modest decreases in site-specific affinity, explains the loss of cross-linking activity in phosphorylated MARCKS.

Actin-latrunculin A structure and function. Differential modulation of actin-binding protein function by latrunculin A.

Latrunculin A is used extensively as an agent to sequester monomeric actin in living cells. We hypothesize that additional activities of latrunculin A may be important for its biological activity. Our data are consistent with the formation of a 1:1 stoichiometric complex with an equilibrium dissociation constant of 0.2 to 0.4 micrometer and provide no evidence that the actin-latrunculin A complex participates in the elongation of actin filaments. Profilin and latrunculin A bind independently to actin, whereas binding of thymosin beta(4) to actin is inhibited by latrunculin A. Potential implications of this differential effect on actin-binding proteins are discussed. From a structural perspective, if latrunculin A binds to actin at a site that sterically influences binding by thymosin beta(4), then the observation that latrunculin A inhibits nucleotide exchange on actin implies an allosteric effect on the nucleotide binding cleft. Alternatively, if, as previously postulated, latrunculin A binds in the nucleotide cleft of actin, then its ability to inhibit binding by thymosin beta(4) is a surprising result that suggests that significant allosteric changes affect the thymosin beta(4) binding site. We show that latrunculin A and actin form a crystalline structure with orthorhombic space group P2(1)2(1)2(1) and diffraction to 3.10 A. A high resolution structure with optimized crystallization conditions should provide insight regarding these remarkable allosteric properties.

Low-temperature magnetic circular dichroism investigation of the active site of chloroperoxidase.

Magnetic circular dichroism (MCD) spectra for near UV and visible spectral regions of chemically reduced chloroperoxidase from Caldariomyces fumago have been recorded at temperatures from near 293 to 2.15 K. The spectra of reduced chloroperoxidase at 4.2 K were compared with those of photolysis products of its carbon monoxide complexes. Obtained results give evidence for high rigidity of the active site in chloroperoxidase and strongly suggest that thiolate is a protein-derived ligand of the heme iron in the reduced enzyme. The unusual high-spin ferrohemoproteins temperature dependence of the Soret MCD closely resembles that of the substrate-bound cytochrome P-450cam.

Contribution of protein conformation to heme stereochemistry and reactivity. Low-temperature magnetic circular dichroism data.

Visible and near infrared magnetic circular dichroism (MCD) spectra of heme proteins and enzymes as well as those of a protein-free heme bound to 2-methylimidazole were recorded and compared at 4.2 K in unrelaxed metastable and relaxed equilibrium heme stereochemistry. The relaxed and unrelaxed stereochemistries of a 5-coordinate ferrous heme were generated by chemical reduction of iron at room temperature before freezing the sample and by photolysis of CO or O2 complexes at 4.2 K, respectively. The results are discussed in terms of a protein contribution into energies of the Fe-N epsilon(His) and Fe-N(pyrrols) bonds and their change on a ligand binding. We observed and analyzed cases of weak (myoglobin, hemoglobin) and strong (leghemoglobin, peroxidases) constraints imposed by the protein conformation on the proximal heme stereochemistry by comparing the bond energies in proteins with those in the protoheme-(2-methylimidazole) model compound. The role of a protein moiety in modulating the ligand binding properties of leghemoglobin and the heme reactivity of horseradish peroxidase is discussed.

Heme-linked ionization and ligand binding produce identical changes of proximal heme stereochemistry in reduced horseradish peroxidase. Evidence for existence of two protein conformations.

The visible and near infrared magnetic circular dichroism spectra of chemically reduced horseradish peroxidase at neutral and alkaline pH values and 5-coordinate protoheme-(2-methylimidazole) at pH 9.1 were compared at 4.2 K with those of photolysis products of their carbon monoxide complexes. From the results obtained we concluded that: (i) there are two protein conformations of HRP which determine the geometry of the Fe-N(His) bond; (ii) the transition from one conformation (heme stereochemistry) to another can be induced by either heme-linked ionization or ligand binding; (iii) a trigger mechanism for switching between two conformations has to exist.

Osmotic pressure of DNA solutions and effective diameter of the double helix.

A simple osmometer with nuclear filters (polymer films with pores of a preset diameter) were used to measure the osmotic pressure of Col E1 plasmid DNA solutions in the concentration range of 1-4 mg/ml DNA. Linear and open circular DNA forms proved to have the same osmotic pressure within the experimental accuracy. The results of the measurements were used for calculating the second virial coefficient A2 of the solution of DNA segments and the effective chain diameter d eff in the ionic strength range of 10(-2)-0.1 M. As the ionic strength is lowered from 0.1 to 10(-2) M the effective diameter of DNA increases from 80 to 220 A. The results are in rather good agreement with theory and with other experimental data.