Increased neuronal nitric oxide synthase activity in retinal neurons in early diabetic retinopathy

PURPOSE. There are increased levels of nitric oxide (NO) in diabetic retinas. The purpose of this study was to determine the extent that neuronal nitric oxide synthase (nNOS) contributes to the increased levels of retinal NO in early diabetic retinopathy by examining the expression and activity of nNOS in retinal neurons after 5 weeks of diabetes. METHODS. Changes in NO levels were measured using NO imaging of retinal neurons in mice with streptozotocin-induced diabetes for five weeks. NO imaging was compared to nNOS localization using immunocytochemistry, and nNOS message and protein levels were measured using quantitative real-time PCR and western blots. RESULTS. There was a close anatomic correlation between the localization of the increased NO production and the nNOS immunoreactivity in the retinal plexiform layers of diabetic retinas. There was no change in nNOS message, but nNOS protein was decreased and its subcellular localization was altered. Treatment with insulin or aminoguanidine partially ameliorated the increase in NO in diabetic retinas. CONCLUSIONS. These results suggest that increased nNOS activity is responsible for the majority of increased NO in retinal neurons in early diabetic retinopathy. This supports a role for increased nNOS activity in the early neuronal dysfunction in the diabetic retina.National Institutes of Health (NEI EY04785 to WDE

Density Contrast Sedimentation Velocity for the Determination of Protein Partial-Specific Volumes

The partial-specific volume of proteins is an important thermodynamic parameter required for the interpretation of data in several biophysical disciplines. Building on recent advances in the use of density variation sedimentation velocity analytical ultracentrifugation for the determination of macromolecular partial-specific volumes, we have explored a direct global modeling approach describing the sedimentation boundaries in different solvents with a joint differential sedimentation coefficient distribution. This takes full advantage of the influence of different macromolecular buoyancy on both the spread and the velocity of the sedimentation boundary. It should lend itself well to the study of interacting macromolecules and/or heterogeneous samples in microgram quantities. Model applications to three protein samples studied in either H2O, or isotopically enriched H218O mixtures, indicate that partial-specific volumes can be determined with a statistical precision of better than 0.5%, provided signal/noise ratios of 50–100 can be achieved in the measurement of the macromolecular sedimentation velocity profiles. The approach is implemented in the global modeling software SEDPHAT