J.S. Zigler

Cataracts in the Royal College of Surgeons rat: evidence for initiation by lipid peroxidation products.

The Royal College of Surgeons (RCS) rat has been extensively studied as a model system for inherited retinal degeneration. As in a number of human retinal degenerative diseases, posterior subcapsular cataracts (PSC) are associated with the retinal changes. It has been hypothesized recently that such cataracts may be initiated by toxic products generated by the peroxidation of polyunsaturated lipid components from degenerating photoreceptor outer segments. In the present study, the possibility that such a mechanism might be responsible for cataract initiation in the RCS rat has been investigated. The degeneration of the rod outer segments (ROS) occurs rapidly in these animals, beginning a few weeks after birth. Due to the failure of the retinal pigmented epithelium to phagocytize normally, ROS degeneration is accompanied by an accumulation of debris in the eye. During the brief period of maximal debris accumulation there is a marked increase in lipid peroxidation products in the vitreous. Cataract formation is correlated temporally with these events, becoming evident immediately following the time during which peroxidation products are present in the vitreous. In addition, the primary damage detected in the RCS lenses is an increase in the passive permeability of the lens membranes. Similar lens damage has been found in studies in which normal rat lenses were exposed to degenerating ROS in vitro. These findings are consistent with the hypothesis that cataracts in the RCS rat may be initiated by toxic lipid peroxidation products.

Zeta-crystallin, a novel lens protein from the guinea pig.

Lens proteins from the guinea pig (Cavia porcellus) were found to be similar to those of other mammals with the exception of the presence of a previously undescribed constituent comprising about 10% of the total soluble lens proteins. This oligomeric protein is composed of polypeptides with apparent molecular weight of 38,000 and elutes from gel exclusion chromatography columns in the beta H-crystallin fraction. Following purification by ion exchange chromatography an antibody was raised against the protein. Using that antibody and antibodies specific for other crystallins we could detect no cross-reactivity between the guinea pig protein and any other reported lens crystallin. This protein, which we have named zeta (zeta)-crystallin, is the first reported mammalian lens crystallin which is not part of the alpha- or beta-gamma families of crystallins. Unlike all other known mammalian crystallins, which have little or no alpha-helical structure, zeta-crystallin is estimated to be approximately 30-40% alpha-helix.

Crystallins and their synthesis in human lens epithelial cells in tissue culture.

Explants of epithelial cells from young human lenses of 5-12 months of age, obtained from patients who underwent surgery for retinopathy of prematurity, were cultured in Dulbeccos modified Eagles medium supplemented with 20% fetal calf serum. Without exception, every piece of the anterior capsule explant showed cell outgrowth within 48-72 h and resulted in confluent monolayer culture within 2 weeks. From these monolayer cultures, two to three passages of subcultures were obtained by routinely seeding cells in a ratio of 1:4. The doubling times for these human lens epithelium (HLE) cultures during the first 4 weeks of two passages were found to be 24-36 h. In a majority of cultures through the first three passages, more than 12 population doublings were attained. However, no lentoid bodies were formed during this period. These cells were studied for the presence of crystallins and their synthesis. Using SDS-polyacrylamide gel electrophoresis, the presence of alpha- and beta-crystallins was demonstrated in HLE cells through three passages. The amount of alpha-crystallin in the first two passages amounted to nearly 13% of the total protein, but decreased significantly in the third passage. The presence of crystallins was corroborated by antibody reaction to the specific crystallins. Indirect immunofluorescence revealed the presence of actin and vimentin in these cell cultures. The synthesis of crystallins in HLE cultures was shown by the incorporation of [35S]methionine which was time dependent. The crystallin synthesis was found to decrease in third passage when the cell growth slowed down without consistent formation of confluent monolayer. These studies have demonstrated that primary cultures of HLE cells can be successfully grown from young lenses through several passages which continue to express the characteristic crystallins of the epithelial cells.

The Oxidative Modification of Lens Proteins

In summary, in vitro oxidation of lens crystallins mimics many of the post-translational modifications observed with age and in cataracts These results lend further support to earlier proposals that oxidation is a key factor in cataract formation. The extent to which ascorbate contributes to the oxidation reactions in vivo is not known. In addition to the data presented here that ascorbate can produce these effects in vitro, other observations support the possibility that under certain conditions ascorbate may be involved in the generation of reactive oxygen species in the lens. Using electron spin resonance the ascorbyl radical can be detected early in nuclear cataract formation, and there appears to be a decrease in total ascorbic acid (reduced and oxidized), suggesting further oxidation of ascorbate. Iron and copper are both present in mammalian lenses, and there are reports of increased copper levels in the lens with age and in cataracts. Increased metal ion concentrations would facilitate these oxidative processes.

Changes in Lens Crystalling during Cataract Development in the Philly Mouse

Laurell ‘rocket’ immunoelectrophoresis has been applied to the quantitation of lens crystallins during development of hereditary cataract in the Philly mouse. A marked decrease in γ crystallin and sma

Further studies on low molecular weight crystalline: Relationship between the bovine βS the human 24kD protein and the γ-crystallins

Three classes of monomeric crystalline separable by SDS gel electrophoresis or by gel filtration have been demonstrated in human lens. In previous studies we have concluded from physico-chemical and immunological data that all three polypeptides are related and should be classified as γ-crystallins. The present paper presents further evidence supporting this conclusion, but also demonstrates that the 24,000 dalton (24kD) polypeptide corresponds to the βS-crystallin. βS-crystallin was purified by classical techniques from bovine lens and was shown to cross-react with a monoclonal antibody specific for the human 24kD polypeptide. This antibody exhibited no reactivity to other crystallin fractions from either bovine or human lenses. The identification of the 24kD polypeptide as βS was further supported by analysis of the tertiary structures of the molecules by near UV-circular dichroism and by the finding of a blocked amino terminus on the 24kD polypeptide. Our finding that the human βS (24kD polypeptide) sh...

Characterization of polypeptides from human nuclear cataracts by Western blot analysis

Water-soluble and water-insoluble polypeptides from nuclei of clear vs. opaque and brunescent human lenses were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose paper. Treatment of the nitrocellulose blots with monospecific antisera to human alpha and beta crystallin and to antisera against the Major Intrinsic Polypeptide (MIP26) of lens membrane demonstrated no difference in binding between microdissected sections of clear vs. opaque (and brunescent) nuclei. In contrast, treatment of nitrocellulose blots with monospecific antisera to human gamma crystallin demonstrated little or no binding to polypeptides from opaque (and brunescent) nuclei as compared with age-matched clear nuclei. These results demonstrate the selective involvement of gamma crystallins in opacification (and brunescence) in the human lens nucleus, and strongly suggest the presence of covalent changes of the gamma crystallin molecule during development of the human nuclear cataract.

Analysis of some immunochemical properties of human β-crystallin by radioimmunoassay

While radioimmunoassays for α- and γ-crystallin have been reported, this technology has not previously been applied successfully to the β-crystallins because of their instability to iodination by oxidative methods. We have succeeded in iodinating the human β-crystallins with the Bolton-Hunter reagent, a non-oxidative technique, and have developed radioimmunoassays for these proteins. Antisera to each of the three major classes of human β-crystallins were prepared and the feasibility of assaying the β1, β2 and β3-crystallins individually by radioimmunoassay was investigated. Antisera to β1 and β3-crystallins were found to react equally well with all three β-crystallin antigens, suggesting that specific antigenic determinants may not be present in these fractions. In contrast β2-crystallin was found to contain at least one highly specific antigen which can be detected as a minor precipitin band in immunodiffusion, but which appears to confer a high degree of specificity in the competitive binding situation of the radioimmunoassay. This apparent discrepancy between results from the two methods is discussed in terms of the marked differences in antibody concentration present.