Lens proteome map and -crystallin profile of the catfish Rita rita.

Crystallins are a diverse group of proteins that constitute nearly 90% of the total soluble proteins of the vertebrate eye lens and these tightly packed crystallins are responsible for transparency of the lens. These proteins have been studied in different model and non-model species for understanding the modifications they undergo with ageing that lead to cataract, a disease of protein aggregation. In the present investigation, we studied the lens crystallin profile of the tropical freshwater catfish Rita rita. Profiles of lens crystallins were analyzed and crystallin proteome maps of Rita rita were generated for the first time. A-crystallins, member of the -crystallin family, which are molecular chaperons and play crucial role in maintaining lens transparency were identified by 1-and 2-D immunoblot analysis with anti-A-crystallin antibody. Two protein bands of 19-20 kDa were identified as A-crystallins on 1-D immunoblots and these bands separated into 10 discrete spots on 2-D immunoblot. However, anti-B-crystallin and antiphospho-B-crystallin antibodies were not able to detect any immunoreactive bands on 1- and 2-D immunoblots, indicating B-crystallin was either absent or present in extremely low concentration in Rita rita lens. Thus, Rita rita -crystallins are more like that of the catfish Clarias batrachus and the mammal kangaroo in its A- and B-crystallin content (contain low amount from 5-9% of aB-crystallin) and unlike the dogfish, zebrafish, human, bovine and mouse -crystallins (contain higher amount of B-crystallin from 25% in mouse and bovine to 85% in dogfish). Results of the present study can be the baseline information for stimulating further investigation on Rita rita lens crystallins for comparative lens proteomics. Comparing and contrasting the -crystallins of the dogfish and Rita rita may provide valuable information on the functional attributes of A- and B-isoforms, as they are at the two extremes in terms of A-and B-crystallin content.

Differentiation and Transdifferentiation of Lens Epithelial Cells in Capsular Bag Model Culture

PURPOSE: To investigate the differentiation of lens epithelial cells (LECs) to lens fiber, and the transdifferentiation of LECs to fibroblast in capsular bag culture. METHODS: After observing the changes of LECs by using phase-contrast microscopy, we observed a cross section of capsular bag by using light microscope (LM) and electron microscope (EM). In addition, the expressions of alpha A-crystallin, a marker of differentiation of LEC to lens fiber, and of alpha-smooth muscle actin, a marker of LEC to fibroblast, were examined during the culture period by western blot. RESULTS: On phase-contrast microscopy, 7 to 14 days after culture, the portion of LECs was gradually elongated and cytoplasm became transparent, so that the differentiation resembled lens fiber. One to 7 days after culture, the portion of LECs changed to spindle shape and the transdifferentiation resembled fibroblast. LM and EM observations indicated that changes of each LEC were lens fiber, and fibroblast. According to Western blot, the expression of alpha A-crystallin was increased by 10 days after culture. The alpha-smooth muscle actin showed an increased expression 10 to 30 days after culture. CONCLUSIONS: From the capsular bag model, we observed the resemblances of the differentiation and transdifferentiation of LECs with lens fiber and fibroblast.

Transdifferentiation of Cultured Bovine Lens Epithelial Cells into Myofibroblast-like Cells by Serum Modulation

An after-cataract is caused by the proliferation of residual cells over the equator of the lens. These cells subsequently migrate to the posterior lens capsule, where they undergo aberrant differentiation into fiber-like cells or transdifferentiation into fibroblast-like cells. To study the precise molecular mechanisms of transdifferentiation, an attempt was made to establish an in vitro system, in which the lens epithelial cells (LECs) of the pre-equatorial zone could be transdifferentiated into fibroblast-like cells. The required conditions for culturing the LECs were identified as consisting of four phases; intact bovine explants, explant-cultured, serum-modulated and additionally modulated LECs. The LECs of each phase were compared by examining changes in the expression of the epithelial-mesenchymal transition (EMT) -related genes and changes in cellular morphology and adhesion. The explants that were cultured in a medium containing 10% fetal bovine serum (FBS) for 2 weeks, showed changes in the expression of the EMT-related genes, although the other explant-cultured cells maintained an epithelial morphology. To introduce a transition into mesenchymal cells, the explant cultures were subcultured in a medium containing 20% FBS for six passages. These cells displayed an elongated morphology and were able to grow and migrate in a similar way to fibroblast cells. The expression of the EMT-related genes, such as, extracellular matrix proteins and integrins, was altered. This was similar to the alteration of the 3-dimensional collagen gels model previously reported. During a further process of EMT by additional serum modulation, the inhibitory effect of disintegrin on cell adhesion was gradually decreased, integrin expression was differentially regulated and alpha-smooth muscle actin was post-translationally modified from the point of passage number six. Overall, it can be concluded that terminal transdifferentiation accompanies changes in the cytoskeletal proteins and cell surface molecules. These are modulated in systematic patterns of post-transcriptional and post-translational regulation and patterns of gene regulation, by the synergic effects of several transforming factors contained in serum. Therefore, posterior capsular opacification may also be accompanied by this molecular mechanism.

Accumulation and Aberrant Modifications of alpha-Crystallins in Anterior Polar Cataracts

Crystallins are the major proteins found in the lens, and the localization of specific crystallins is well known. Overexpression and accumulation of alphaB-crystallin has been observed in response to stress conditions or in certain diseases, such as brain tumors and neurodegenerative diseases. The purpose of this study was to examine whether alpha-crystallins are modified during pathological myofibroblastic changes in lens epithelial cells. Lens epithelial cells attached to the anterior capsules of patients with nuclear or anterior polar cataracts were analyzed quantitatively for alpha-crystallin proteins and mRNAs using Western blot and RT-PCR analysis., respectively. The degree of modification of alpha-crystallins was determined by 2-dimensional gel electrophoresis followed by Western blotting. Higher molecular weight protein bands that were immunoreactive to anti-alphaA- and anti-alphaB-crystallin antibodies around 45 kDa accumulated more in the anterior polar cataract samples than in those with the nuclear type of cataracts. Also monomeric alphaB-crystallins accumulated more in lens epithelial cells of patients with anterior polar cataracts. By comparison, no significant changes were found in the levels of the mRNAs encoding alphaA- and alphaB-crystallins in the different types of cataracts. Both alphaA- and alphaB-crystallin proteins seemed to undergo more extensive modification in anterior polar cataracts. Conclusion. In addition to fibrotic changes, which accompany increased levels of extracellular matrix molecules, accumulation and abnormal modification of alpha-crystallins might be implicated in the pathogenic mechanism of this type of cataract.