Jasplakinolide paradoxically stabilizes actin filaments in vitro, but in vivo it can disrupt actin filaments and induce polymerization of monomeric actin into amorphous masses. A detailed analysis of the effects of jasplakinolide on the kinetics of actin polymerization suggests a resolution to this paradox. Jasplakinolide markedly enhances the rate of actin filament nucleation. This increase corresponds to a change in the size of actin oligomer capable of nucleating filament growth from four to approximately three subunits, which is mechanistically consistent with the localization of the jasplakinolide-binding site at an interface of three actin subunits. Because jasplakinolide both decreases the amount of sequestered actin (by lowering the critical concentration of actin) and augments nucleation, the enhancement of polymerization by jasplakinolide is amplified in the presence of actin-monomer sequestering proteins such as thymosin  4 . Overall, the kinetic parameters in vitro define the mechanism by which jasplakinolide induces polymerization of monomeric actin in vivo. Expected consequences of jasplakinolide function are consistent with the experimental observations and include de novo nucleation resulting in disordered polymeric actin and in insufficient monomeric actin to allow for remodeling of stress fibers.Jasplakinolide is a cyclic peptide isolated from the marine sponge, Jaspis johnstoni, that we have previously shown to bind to and stabilize filamentous actin in vitro (1). In vivo data suggests that jasplakinolide-treated prostate cancer cells have both decreased labeling of F-actin and decreased amounts of rhodamine-phalloidin bound to cell extracts (2), results that could be explained by the observation that jasplakinolide and phalloidin bind competitively to actin (1). In addition, however, in vivo data also convincingly show that jasplakinolide disrupts actin filaments with alterations in cellular architecture (2, 3), an effect that cannot be explained simply by competitive binding. We now present kinetic data characterizing the steady state and time-dependent in vitro interactions between jasplakinolide and actin that provide a plausible explanation for the effects of jasplakinolide on actin distribution in cultured cells. EXPERIMENTAL PROCEDURESMaterials-Rabbit skeletal muscle actin was prepared from frozen muscle (Pel-Freez, Rogers, AR) in buffer G (5.0 mM Tris, 0.2 mM ATP, 0.2 mM dithiothreitol, 0.1 mM CaCl 2 , and 0.01% sodium azide, pH 7.8) (4). Non-muscle actin from bovine brain was prepared by the method of Ruscha and Himes (5). Muscle and non-muscle pyrenyl-labeled actins 1 were prepared with 0.67-0.95 mol of label/mol of protein using the method of Kouyama and Mihashi (6). Labeled and unlabeled actins were further purified by gel filtration on Superose 12 (Amersham Pharmacia Biotech). Thymosin  4 cDNA was a gift from Dr. Vivian Nachmias and was inserted in a pET-11a vector, expressed in BL21(DE3) Escherichia coli, and purified as described previously (7). Jasplakinolide was a gift from ...
Significance: Oxidative stress is recognized as playing a role in stem cell mobilization from peripheral sites and also cell function. Recent Advances: This review focuses on the impact of hyperoxia on vasculogenic stem cells and elements of wound healing. Critical Issues: Components of the wound-healing process in which oxidative stress has a positive impact on the various cells involved in wound healing are highlighted. A slightly different view of woundhealing physiology is adopted by departing from the often used notion of sequential stages: hemostatic, inflammatory, proliferative, and remodeling and instead organizes the cascade of wound healing as overlapping events or waves pertaining to reactive oxygen species, lactate, and nitric oxide. This was done because hyperoxia has effects of a number of cell signaling events that converge to influence cell recruitment/chemotaxis and gene regulation/ protein synthesis responses which mediate wound healing. Future Directions: Our alternative perspective of the stages of wound healing eases recognition of the multiple sites where oxidative stress has an impact on wound healing. This aids the focus on mechanistic events and the interplay among various cell types and biochemical processes. It also highlights the areas where additional research is needed.
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