GdnHCl-induced kinetic unfolding curves of Sto-RNase HI.

<p>Lines represent the fit of Eq. (4). (A) C58/145A. Curve represents the unfolding trace to a final concentration of 5.8 M GdnHCl. (B) ΔC6. Curve represents the unfolding trace to a final concentration of 5.0 M GdnHCl.</p

Crystal structure of So-RNase HI, Ec-RNase HI, and Sto-esterase.

<p>(A) So-RNase HI, (B) Ec-RNase HI and (C) Sto-esterase.</p

Crystal structure of wild-type Sto-RNase HI.

<p>C-terminal seven residues (cyan); hydrophobic side-chains (blue); hydrogen bonds (thin red lines); and disulfide bond (thick red line).</p

Thermodynamic parameters for denaturation of wild-type, C58/145A and ΔC6 Sto-RNase HI.

a<p>Data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016226#pone.0016226-You2" target="_blank">[14]</a>.</p>b<p>Errors are standard error values from the data fitting using Eq. (3).</p

GdnHCl-induced equilibrium unfolding curves and thermodynamic stability profiles (temperature dependence of ΔG(H2O)) of Sto-RNase HI.

<p>Wild-type (solid line and circles), C58/145A (dashed line and triangles), and ΔC6 (dot-dashed line and squares). (A) GdnHCl-induced equilibrium unfolding at 20°C. The apparent fraction of unfolded protein is shown as a function of GdnHCl concentration. Lines are best fits to a two-state equation. (B) Thermodynamic stability profiles. Closed symbols are the T_m value from the heat-induced unfolding experiment <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016226#pone.0016226-You2" target="_blank">[14]</a>. Lines represent the fit of Eq. (3) using both equilibrium and heat-induced unfolding data.</p

CD spectra and crystal structure of Sto-RNase HI variants.

<p>(A) CD spectra of wild-type (solid line), C58/145A (dashed line), and ΔC6 (dot-dashed line) Sto-RNase HI. (B) Crystal structure of ΔC6 Sto-RNase HI. (C) Crystal structure of wild-type Sto-RNase HI. The C-terminal seven residues are in cyan.</p

Denaturation temperatures of chimeric proteins.

a<p>Errors are ±0.3°C.</p>b<p>Data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016226#pone.0016226-Tadokoro2" target="_blank">[16]</a>.</p>c<p>Data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016226#pone.0016226-Haruki1" target="_blank">[28]</a>.</p

Statistics on data processing and structure determination of ΔC6 Sto-RNase HI.

<p>Values in parentheses are the highest-resolution bin of respective data.</p>a<p>R_merge = Σ |I_hkl - hkl>|/Σ I_hkl, where I_hkl is the intensity measurement for reflection with indices hkl and hkl> is the mean intensity for multiply recorded reflections.</p>b<p>R_{work, free} = Σ ||F_obs| - |F_calc||/Σ |F_obs|, where the R-factors are calculated using the working and free reflection sets, respectively. The free reflections comprise a random 10% of the data held aside for unbiased cross-validation throughout refinement.</p

SDS-PAGE and heat-induced unfolding curves of chimeric proteins.

<p>(A) SDS-PAGE. Lanes 1, 3, 5, are a low-molecular weight marker kit (GE Healthcare). Lanes 2, 4, 6 are purified chimeric So-RNase HI, Ec-RNase HI and Sto-esterase. (B) Heat-induced unfolding. The apparent fraction of unfolded protein is shown as a function of temperature. Curves 1, 2 and 3 represent the unfolding traces of chimeric So-RNase HI (closed circles), Ec-RNase HI (open circles) and Sto-esterase (cross). Lines are best fits to a two-state equation.</p

Neuroprotective effect of bilberry extract in a murine model of photo-stressed retina

<div><p>Excessive exposure to light promotes degenerative and blinding retinal diseases such as age-related macular degeneration and retinitis pigmentosa. However, the underlying mechanisms of photo-induced retinal degeneration are not fully understood, and a generalizable preventive intervention has not been proposed. Bilberry extract is an antioxidant-rich supplement that ameliorates ocular symptoms. However, its effects on photo-stressed retinas have not been clarified. In this study, we examined the neuroprotective effects of bilberry extract against photo-stress in murine retinas. Light-induced visual function impairment recorded by scotopic and phototopic electroretinograms showing respective rod and cone photoreceptor function was attenuated by oral administration of bilberry extract through a stomach tube in Balb/c mice (750 mg/kg body weight). Bilberry extract also suppressed photo-induced apoptosis in the photoreceptor cell layer and shortening of the outer segments of rod and cone photoreceptors. Levels of photo-induced reactive oxygen species (ROS), oxidative and endoplasmic reticulum (ER) stress markers, as measured by real-time reverse transcriptase polymerase chain reaction, were reduced by bilberry extract treatment. Reduction of ROS by N-acetyl-L-cysteine, a well-known antioxidant also suppressed ER stress. Immunohistochemical analysis of activating transcription factor 4 expression showed the presence of ER stress in the retina, and at least in part, in Müller glial cells. The photo-induced disruption of tight junctions in the retinal pigment epithelium was also attenuated by bilberry extract, repressing an oxidative stress marker, although ER stress markers were not repressed. Our results suggest that bilberry extract attenuates photo-induced apoptosis and visual dysfunction most likely, and at least in part, through ROS reduction, and subsequent ER stress attenuation in the retina. This study can help understand the mechanisms of photo-stress and contribute to developing a new, potentially useful therapeutic approach using bilberry extract for preventing retinal photo-damage.</p></div