Reduction of photobleaching and photodamage in single molecule detection: observing single actin monomer in skeletal myofibrils.

Recent advances in detector technology make it possible to achieve single molecule detection (SMD) in a cell. SMD avoids complications associated with averaging signals from large assemblies and with diluting and disorganizing proteins. However, it requires that cells be illuminated with an intense laser beam, which causes photobleaching and cell damage.

Decreasing photobleaching by silver nanoparticles on metal surfaces: application to muscle myofibrils.

Recently it has become possible to study single protein molecules in a cell. However, such experiments are plagued by rapid photobleaching. We recently showed that the interaction of fluorophores with localized surface plasmon polaritons (LSPs) induced in the metallic nanoparticles led to a substantial reduction of photobleaching.

Rotation of actin monomers during isometric contraction of skeletal muscle.

Cyclic interactions of myosin and actin are responsible for contraction of muscle. It is not self-evident, however, that the mechanical cycle occurs during steady-state isometric contraction where no work is produced. Studying cross-bridge dynamics during isometric steady-state contraction requires an equilibrium time-resolved method (not involving application of a transient).

Oldest coelacanth, from the Early Devonian of Australia.

Coelacanths are well-known sarcopterygian (lobe-finned) fishes, which together with lungfishes are the closest extant relatives of land vertebrates (tetrapods). Coelacanths have both living representatives and a rich fossil record, but lack fossils older than the late Middle Devonian (385-390 Myr ago), conflicting with current phylogenies implying coelacanths diverged from other sarcopterygians in the earliest Devonian (410-415 Myr ago). Here, we report the discovery of a new coelacanth from the Early Devonian of Australia (407-409 Myr ago), which fills in the approximately 20 Myr 'ghost range' between previous coelacanth records and the predicted origin of the group.

Measuring rotations of a few cross-bridges in skeletal muscle.

The ability to measure properties of a single cross-bridge in working muscle is important because it avoids averaging the signal from a large number of molecules and because it probes cross-bridges in their native crowded environment. Because the concentration of myosin in muscle is large, observing the kinetics of a single myosin molecule requires that the signal be collected from small volumes. The introduction of small observational volumes defined by diffraction-limited laser beams and confocal detection has made it possible to limit the observational volume to a femtoliter (10(-15) liter).

Post-Electrophoretic Identification of Oxidized Proteins.

The oxidative modification of proteins has been shown to play a major role in a number of human diseases. However, the ability to identify specific proteins that are most susceptible to oxidative modifications is difficult. Separation of proteins using polyacrylamide gel electrophoresis (PAGE) offers the analytical potential for the recovery, amino acid sequencing, and identification of thousands of individual proteins from cells and tissues.

Anti-apoptotic proteins are oxidized by Abeta25-35 in Alzheimer's fibroblasts.

We have examined the effects of the beta-amyloid peptide (Abeta(25-35)) on fibroblasts derived from subjects with Alzheimer's disease (AD) and from age-matched controls. The peptide was significantly more cytotoxic to the AD-derived fibroblasts. The level of protein oxidation was also greater in the cells from AD subjects.

Vitamin E prevents oxidation of antiapoptotic proteins in neuronal cells.

Oxidative damage to neuronal proteins appears to be central to the toxicity associated with a number of neuropathologies, including Alzheimer's disease. We have examined this by using oxidative stress to induce apoptosis in a mouse hippocampal neuronal cell line (HT-22). Oxidatively modified proteins were measured by high-resolution two-dimensional gel electrophoresis coupled with oxidation-specific immunostains.

Flavones from Scutellaria baicalensis Georgi attenuate apoptosis and protein oxidation in neuronal cell lines.

The oxidative modification of proteins plays a major role in a number of human diseases including Alzheimer's disease (AD). Flavones in extracts of Scutellaria baicalensis (SbE) have been reported to have exceptional antioxidant properties. We examined the effects of SbE on neuronal cells exposed to oxidative stress.

Identification of oxidized plasma proteins in Alzheimer's disease.

The modification of proteins by reactive oxygen species is central to the pathology of Alzheimer's disease (AD). Previously, we have observed specific oxidized proteins in blood plasma of AD subjects [Biochem. Biophys.