Robert C. Augusteyn

Oxidative changes in human lens proteins during senile nuclear cataract formation.

1. Proteins from the cortex and nucleus of the human lens were studied to determine if any changes could be detected in their amino acids during senile cataract formation. 2. Senile nuclear cataract formation was found to be accompanied by a progressive oxidation of cysteine and methionine. The oxidation of methionine and changes in the distribution of the nuclear proteins did not appear to start until about 60% of the cysteine had been oxidized. 3. In the advanced nuclear cataractous lens, about 90% of the cysteine has been oxidized and 45% of the methionine is present as the sulphoxide in the nuclear proteins. The levels of other amino acids appeared to remain constant. 4. Similar, but smaller, changes were found in the cortical proteins in advanced nuclear cataractous lenses, suggesting that the oxidation spreads from the nucleus to the cortex. 5. These changes were discussed with regard to current views on cataract formation and it was concluded that they are probably the result of simple oxidation of the proteins with O2 or H2O2.

The state of sulphydryl groups in normal and cataractous human lenses

Abstract The levels of non-protein sulphydryls, protein sulphydryls and protein-bound sulphydryls were measured in the separated nuclei and cortices from individual normal and cataractous lenses. As the colour of the lens nucleus increases, there is a progressive decrease in the levels of non-protein sulphydryl and protein sulphydryl. In the nuclei of advanced cataractous lenses, these decrease to 10% and 3%, respectively, of the levels found in the normal nucleus. Similar, but smaller, changes take place in the cortex. The level of protein-bound sulphydryl increases in the early stages of cataract formation but thereafter remains constant in both the cortex and nucleus. In the advanced cataractous lens, the protein bound sulphydryl accounts for only about 2% of the loss of protein sulphydryl. There appear to be no intermolecular disulphide bonds in the urea-soluble proteins of the lens but some may be present in the urea-insoluble proteins.

Superoxide dismutase, catalase and glutathione peroxidase in the human cataractous lens☆

The activities of the protective enzymes, superoxide dismutase, catalase and glutathione peroxidase have been measured in the cortical and nuclear sections of 76 human cataractous lenses as well as in calf, rabbit and rat lenses. No changes was observed in the activity of catalase with the progressive development of cataract. However, a precipitous decrease (70%) in both superoxide dismutase and glutathione peroxidase in the nuclear region of the lens was found at the onset of nuclear cataract. Further decreases accompanied the progression of the cataract and similar, but less marked, decreases were observed in the cortical region of the lens. It is suggested that the inactivation of these enzymes may result in an elevation of the H2O2 and O2.- levels in the lens and that this may be responsible for the oxidative modification of lens proteins observed in nuclear cataracts.

Changes in human lens proteins during nuclear cataract formation

The increase in water-insoluble proteins associated with senile nuclear cataract formation is due primarily to the increase in a urea-insoluble yellow protein fraction which can be solubilized with urea containing a reducing agent. It is not an artefact produced by aerobic extraction since prevention of disulphide bond formation during extraction does not result in a decreased yield of this fraction. The yellow protein fraction is localized in the nucleus of the lens and appears to contain trimers and larger aggregates of normal lens polypeptides which are resistant to deaggregation by 8 m-urea, 6 m-guanidine hydrochloride and 1% SDS in the presence of reducing agents. The soluble and urea-soluble proteins of the lens do not contain significant amounts of these aggregates. The proportion of these aggregates increases until they represent over 50% of the yellow protein fraction in advanced cataractous lenses.

α‐crystallin: a review of its structure and function

α‐crystallin, the major protein of the mammalian lens in most species, is an aggregate assembled from two polypeptides, each with a molecular weight around 20,000 Da. It is polydisperse and can be isolated in a variety of forms, including spherical particles with molecular weights ranging upwards from about 200 kDa.

What causes steroid cataracts? A review of steroid-induced posterior subcapsular cataracts

Prolonged use of glucocorticoids is a significant risk factor for the development of posterior subcapsular cataract. This places restrictions on the use of glucocorticoids in the treatment of systemic and/or ocular inflammatory conditions as well as in organ transplantation.

In vitro dimensions and curvatures of human lenses

The purpose of this study was to determine dimensions and curvatures of excised human lenses using the technique of shadowphotogrammetry. A modified optical comparator and digital camera were used to photograph magnified sagittal and coronal lens profiles. Equatorial diameter, anterior and posterior sagittal thickness, anterior and posterior curvatures, and shape factors were obtained from these images. The data were used to calculate lens volumes, which were compared with the lens weights. Measurements were made on 37 human lenses ranging in age from 20 to 99 years. These showed that lens dimensions and the anterior radius of curvature increase linearly throughout adult life while posterior curvature remains constant. The relative shape (or aspect ratio) of the posterior lens is unchanged through adult life since both equatorial diameter and posterior thickness increase at the same rate. The ratio of anterior thickness to posterior thickness is constant at 0.70. It is suggested that in vivo forces alter the apparent location of the lens equator, that the in vitro lens shape corresponds to the maximally accommodated shape in vivo and that the shapes of the accommodated and unaccommodated lens progressively converge toward each other due to lens growth with age, with a convergence point located near the age of total loss of accommodation (55-60 years). Together, these observations provide additional support for the Helmholtz theory of accommodation.

On the growth and internal structure of the human lens

Growth of the human lens and the development of its internal features are examined using in vivo and in vitro observations on dimensions, weights, cell sizes, protein gradients and other properties. In vitro studies have shown that human lens growth is biphasic, asymptotic until just after birth and linear for most of postnatal life. This generates two distinct compartments, the prenatal and the postnatal. The prenatal growth mode leads to the formation of an adult nuclear core of fixed dimensions and the postnatal, to an ever-expanding cortex. The nuclear core and the cortex have different properties and can readily be physically separated. Communication and adhesion between the compartments is poor in older lenses. In vivo slit lamp examination reveals several zones of optical discontinuity in the lens. Different nomenclatures have been used to describe these, with the most common recognizing the embryonic, foetal, juvenile and adult nuclei as well as the cortex and outer cortex. Implicit in this nomenclature is the idea that the nuclear zones were generated at defined periods of development and growth. This review examines the relationship between the two compartments observed in vitro and the internal structures revealed by slit lamp photography. Defining the relationship is not as simple as it might seem because of remodeling and cell compaction which take place, mostly in the first 20 years of postnatal life. In addition, different investigators use different nomenclatures when describing the same regions of the lens. From a consideration of the dimensions, the dry mass contents and the protein distributions in the lens and in the various zones, it can be concluded that the juvenile nucleus and the layers contained within it, as well as most of the adult nucleus, were actually produced during prenatal life and the adult nucleus was completed within 3 months after birth, in the final stages of the prenatal growth mode. Further postnatal growth takes place entirely within the cortex. It can also be demonstrated that the in vitro nuclear core corresponds to the combined slit lamp nuclear zones. In view of the information presented in this review, the use of the terms foetal, juvenile and adult nucleus seems inappropriate and should be abandoned.

Ontogeny of human lens crystallins.

The soluble proteins from prenatal and neonatal human lenses were fractionated by gel filtration into four distinct size classes viz. high molecular weight alpha-crystallin (HM-alpha), alpha-crystallin, intermediate molecular weight (IMW) proteins and low molecular weight (LMW) proteins. Extinction coefficients of the isolated proteins were determined and used to calculate the proportions of each fraction on a weight basis. The distributions of polypeptides within each of these fractions were analyzed by SDS gel electrophoresis and isoelectric focussing, followed by densitometric scanning of the gels. HM-alpha is detectable as early as the 14th week of gestation and its proportions increase rapidly, to about 9% of the total protein in the 1 year postnatal lens. The alpha-crystallin, IMW and LMW fractions show concomitant decreases and by 1 year they represent about 34, 35 and 18%, respectively. However, the proportions of IMW and LMW proteins do not accurately reflect those of the beta- and gamma-crystallins, as is often assumed. Substantial levels of non-crystallin polypeptides were found in the IMW protein fractions, including a group of very basic polypeptides (VBP) which comprised up to one-third of this material in the youngest lenses. Moreover, in postnatal lenses beta s-crystallin accounted for almost half of the LMW proteins. These points considered, alpha-crystallin is the major protein in the neonatal lens (approximately 42%, including HM-alpha), followed by the beta-crystallin (approximately 36% at most and probably less), the gamma-crystallins (approximately 11%) and beta s-crystallin (approximately 9%). Substantial changes in the proportions of specific polypeptides were observed throughout early development. These appear to result from changes at the level of protein synthesis and from postsynthetic modification. The A:B subunit ratio of alpha-crystallin drops from about 12 to below 3 during early development. This coincides with increasing levels of various deamidated and degraded subunits. The major beta-crystallin polypeptide also undergoes rapid deamidation and evidence is presented suggesting that the gamma-crystallins are subject to similar modification. The most dramatic changes were observed in the constituents of the LMW proteins. The synthesis of gamma-crystallins virtually ceases at some time around birth. At the same time, the levels of beta s-crystallin undergo an explosive increase. These and other changes are discussed in terms of their possible functional significance. They are also related to the complex protein status found in old lenses.

αm-Crystallin: the native form of the protein?

Evidence is presented that alpha-crystallin isolated at 37 degrees C exists as a species, alpha m, which has a minimum molecular weight of about 320000 and a sedimentation coefficient of about 12 S. The amino acid composition, subunit distribution, near- and far-UV CD spectra and immunochemical properties were identical to those of the previously studied, 19 S protein, alpha c-crystallin (minimum molecular weight, 635000). It was demonstrated that only alpha m-crystallin was present in 37 degrees C lens extracts and that cooling of lenses or extracts resulted in a conversion of alpha m- to alpha c-crystallin. This conversion appears to be a general phenomenon, independent of age or species. It was concluded that alpha c-crystallin is an aggregate, produced by cooling, and that alpha m-crystallin is more likely to represent the in vivo form of the protein.