Cleavage of the small amyloidogenic protein b 2 -microglobulin after lysine-58 renders it more prone to unfolding and aggregation. This is important for dialysis-related b 2 -microglobulin amyloidosis, since elevated levels of cleaved b 2 -microglobulin may be found in the circulation of dialysis patients. However, the solution structures of these cleaved b 2 -microglobulin variants have not yet been assessed using single-residue techniques. We here use such methods to examine b 2 -microglobulin cleaved after lysine-58 and the further processed variant (found in vivo) from which lysine-58 is removed. We find that the solution stability of both variants, especially of b 2 -microglobulin from which lysine-58 is removed, is much reduced compared to wild-type b 2 -microglobulin and is strongly dependent on temperature and protein concentration.1 H-NMR spectroscopy and amide hydrogen ( 1 H ⁄ 2 H) exchange monitored by MS show that the overall threedimensional structure of the variants is similar to that of wild-type b 2 -microglobulin at subphysiological temperatures. However, deviations do occur, especially in the arrangement of the B, D and E b-strands close to the D-E loop cleavage site at lysine-58, and the experiments suggest conformational heterogeneity of the two variants. Two-dimensional NMR spectroscopy indicates that this heterogeneity involves an equilibrium between the native-like fold and at least one conformational intermediate resembling intermediates found in other structurally altered b 2 -microglobulin molecules. This is the first single-residue resolution study of a specific b 2 -microglobulin variant that has been found circulating in dialysis patients. The instability and conformational heterogeneity of this variant suggest its involvement in b 2 -microglobulin amyloidogenicity in vivo.Abbreviations b2m, b 2 -microglobulin; CE, capillary eletrophoresis; cK58-b2m, b 2 -microglobulin cleaved after lysine-58; dK58-b2m, b 2 -microglobulin with lysine-58 deleted; DRA, dialysis-related amyloidosis; DN3-b2m, b 2 -microglobulin devoid of N-terminal tripeptide; FID, free induction decay.

Aqueous humor has been shown to influence the proliferation of various ocular cell types, but the effect on immature retinal cells is not known. Here, the effect of pig aqueous humor on the proliferation of rat retinal precursor cells (RPCs) was investigated. RPCs were prepared from embryonic day 19 SpragueDawley rats and cultured in the presence or absence of aqueous humor from healthy pigs along with a medium consisting of Dulbecco's modified Eagle's medium:Ham's F-12 medium, N2 supplement, and epidermal growth factor. Proliferation was quantified by [ 3 H]thymidine incorporation under different treatment conditions, and any associated morphological changes were noted. Potential active components of porcine aqueous humor were partially characterized by gel filtration chromatography, and the effect on RPC proliferation was determined. Results showed that adding 20% aqueous humor increased [3 H]thymidine incorporation by as much as 317%, as compared with controls. Aqueous supplementation also increased both the number and size of RPC spherical aggregates ("spheres") over the first 4 days, consistent with increased proliferative activity. Using gel filtration and the in vitro proliferation assay, the growth-promoting activity of aqueous humor was localized to two different molecular mass ranges, namely, around 30 kDa and less than 1 kDa. Ascorbic acid was present in the lower molecular mass fraction, and use of this molecule reproduced some, but not all, of the proliferative activity present in aqueous humor. These results highlight the potential role of soluble factors present in the cellular microenvironment with respect to modulation of endogenous progenitor cell activity. STEM CELLS 2006;24:2766 -2775

Intravitreal injection is an important delivery route for studies involving the transplantation of various types of precursor cells to the retina; however, the effect on these cells of exposure to the vitreous microenvironment has not been specifically investigated. Here vitreous humor was evaluated for the potential to influence the proliferation of rat retinal precursor cells in vitro. Cells were isolated at embryonic day 19 and plated in standard proliferation medium in the presence or absence of fluid expressed from porcine vitreous humor. Cellular proliferation at different concentrations of vitreous fluid supplementation was quantified by using a (3)H-thymidine incorporation assay. Active components of vitreous fluid were partially characterized by gel filtration chromatography (GFC) and UV spectral analysis. The effect of each vitreous fraction on proliferation was determined as well. Results showed that addition of 20% vitreous fluid to primary rat retinal cultures significantly increased (3)H-thymidine incorporation compared with growth medium without vitreous supplementation. A vitreous fraction showing growth-promoting activity was localized to a molecular mass range <1000 Da, consistent with ascorbic acid. Ascorbic acid was confirmed in vitreous fluid by UV spectral analysis. Growth-augmenting activity was present in higher molecular mass vitreous fractions, consistent with protein components. Albumin, the major protein in vitreous fluid, was found to augment proliferation. Because vitreous-associated augmentation of retinal precursor proliferation remains an epidermal growth factor-dependent phenomenon, the proliferative status of transplanted cells in the vitreous cavity is likely determined by a combination of factors.

Five of 21 H-2 Kb/Db-binding retinal protein-derived peptides were found to be immunogenic, suggesting that these peptides could function as autoantigenic epitopes in the development of inflammatory eye diseases, such as uveitis.