Comparison of modification sites in glycated crystallin in vitro and in vivo.

Glycation of α-crystallin is responsible for age- and diabetic-related cataracts, which are the main cause of blindness worldwide. We optimized the method of identification of lysine residues prone to glycation using the combination of LC-MS, isotopic labeling, and modified synthetic peptide standards with the glycated lysine derivative (Fmoc-Lys(i,i-Fru,Boc)-OH). The in vitro glycation of bovine lens α-crystallin was conducted by optimized method with the equimolar mixture of [(12)C6]- and [(13)C6]D-glucose. The in vivo glycation was studied on human lens crystallin. The glycated protein was subjected to proteolysis and analyzed using LC-MS. The results of in vitro and in vivo glycation of α-crystallin reveal a different distribution of the modified lysine residues. More Amadori products were detected as a result of the in vitro reaction due to forced glycation conditions. The developed method allowed us to identify the glycation sites in crystallin from eye lenses obtained from patients suffering from the cataract. We identified K166 in the A chain and K166 in the B chain of α-crystallin as major glycation sites during the in vitro reaction. We found also two in vivo glycated lysine residues: K92 in the B chain and K166 in the A chain, which are known as locations for Amadori products. These modification sites were confirmed by the LC-MS experiment using two synthetic standards. This study demonstrates the applicability of the LC-MS methods combined with the isotopic labeling and synthetic peptide standards for analysis of post-translational modifications in the biological material.

PPARg2 Ala¹² variant protects against Graves' orbitopathy and modulates the course of the disease.

Orbital fibroblast differentiation to adipocytes is a peroxisome proliferator-activated receptor g (PPARg)-dependent process essential for pathogenic tissue remodeling in Graves' orbitopathy (GO). PPARg2 Pro¹²Ala polymorphism modulates expression and/or function of the molecule encoded by this gene and is a promising locus of GO. Here, we analyzed associations of PPARg2 Pro¹²Ala with clinical manifestation of GO in 742 Polish Caucasians including 276 Graves' disease (GD) patients. In our study, the Ala¹² allele and Ala¹² variant (Ala¹²Ala and/or Pro¹²Ala genotype) decreased the risk of GO (p = 0.000012 and p = 0.00013). Moreover, Ala¹²Ala genotype was observed only in patients without GO (p = 0.002). GD patients with Ala¹² variant had less active and less severe eye symptoms. Female carriers of the Ala¹² allele rarely developed GO, but the marker was not related to symptoms of GO. The opposite finding was recorded in males, in whom the studied polymorphism was related to activity, but not to the development, of GO. In Ala¹² variant carriers without familial history of thyroid disease, risk of GO was lower than in persons with a familial background. The Ala¹² allele seemed to protect smokers from GO, but in nonsmokers, such a relation was not obvious. A multivariate analysis indicated the Pro¹²Ala marker as an independent risk factor of eye symptoms (p = 0.0001) and lack of Ala increases the risk of GO 3.24-fold. In conclusion, the gain-of-function Ala¹² variant protects against GO and modulates the course of the disease.

[Intravitreal Bevacizumab injections in a child with choroidal neovascularization--case report]. / Iniekcje doszklistkowe bevacizumabu u dziecka z podsiatkówkowa neowaskularyzacja--opis przypadku.

A healthy 11 year old boy with active choroidal neovascularization (CNV) was referred to the Department of Ophthalmology in Wroclaw for unilateral visual deterioration from 2 weeks. The best-corrected visual acuity was counting fingers from 4 meters. Boy was successfully treated with three intravitreal injections of bevacizumab. Intravitreal bevacizumab was well tolerated, choroidal neovascularization involuted, and best-corrected visual acuity improved to 20/32. The boy remained stable for 6 months after injections.