Magnetic resonance molecular imaging of post-stroke neuroinflammation with a P-selectin targeted iron oxide nanoparticle.

We have developed a magnetic resonance molecular imaging method using a novel iron-oxide contrast agent targeted towards P-selectin - MNP-PBP (magnetic nanoparticle-P-selectin binding peptide) - to image endothelial activation following cerebral ischemia/reperfusion. MNP-PBP consists of approximately 1000 PBP ligands (primary sequence: GSIQPRPQIHNDGDFEEIPEEYLQ GGSSLVSVLDLEPLDAAWL) conjugated to a 50 nm diameter aminated dextran iron oxide particle. In vitro P- and E-selectin binding was assessed by competition ELISA. Transient focal cerebral ischemia was induced in male C57/BL 6 mice followed by contrast injection (MNP-PBP; MNP-NH2; Feridex; MNP-PBP-FITC) at 24 h after reperfusion and T(2) magnetic resonance imaging at 9.4 T was performed. Infarction and microvasculature accumulation of contrast agent was assessed in coronal brain sections. MNP-PBP attenuated antibody binding to P-selectin by 34.8 +/- 1.7%. P-selectin was preferentially increased in the infarct hemisphere and MNP-PBP-FITC accumulation in the infarct hemisphere microvasculature was observed. Compared with the nontargeted iron oxide agents MNP-NH2 and Feridex, MNP-PBP showed a significantly greater T(2) effect within the infarction. MR imaging of P-selectin expression with a targeted iron oxide nanoparticle contrast agent may reveal early endothelial activation in stroke and other neuroinflammatory states.

Volumetric and spectroscopic characterizations of the native and acid-induced denatured states of staphylococcal nuclease.

We have characterized the acid-induced denaturation of staphylococcal nuclease (SNase) at different urea concentrations by a combination of ultrasonic velocimetry, high precision densimetry, and CD spectroscopy. Our CD spectroscopic results suggest that, at low salt and acidic pH, the protein is unfolded with disrupted secondary and tertiary structures. Furthermore, as judged by far UV CD spectra, the protein is further unfolded at acidic pH upon the addition of urea up to the concentration of 1.5 M. The midpoint of the transition shifts to more neutral pH values and the cooperativity of the transition decreases as the acid-induced denaturation of SNase occurs at higher urea concentrations. We find that the change in volume, Deltav, accompanying the acid-induced denaturation of SNase increases from -0.013 cm(3) g(-1) (-218 cm(3) mol(-1)) in the absence of urea to 0.011 cm(3) g(-1) (185 cm(3) mol(-1)) at 1.5 M urea. At all urea concentrations, the partial specific adiabatic compressibility, k(o)(s), of the protein decreases upon its unfolding with the values of Deltak(o)(s) equal to -6.3x10(-6) (-0.106 cm(3) mol(-1) bar(-1)), -4.5x10(-6) (-0.076 cm(3) mol(-1) bar(-1)), -4.6x10(-6) (-0.077 cm(3) mol(-1) bar(-1)), and -3.8x10(-6) (-0.064 cm(3) mol(-1) bar(-1)) cm(3) g(-1) bar(-1) at urea concentrations of 0, 0.5, 1.0, and 1.5 M, respectively. In general, our volumetric results suggest that the acid-induced denatured state of SNase is only partially unfolded with the solvent-exposed surface area equal to 70-80 % of that expected for the fully extended conformation.