Proteases in the Emory mouse cataract.

Exopeptidases identified as dipeptidyl peptidase III and leucine aminopeptidase, and an endopeptidase, prolyl endopeptidase, were found in the Emory Mouse cataract and the Cataract Resistant mouse lens extracts. The specific activity measured on Arg-Arg-2-NNap for DPP III and the hydrolysis of Boc-Arg-Pro-2-NNap for prolyl endopeptidase were higher in the Emory Mouse cataractous lens extract. A relatively high rate of hydrolysis of the beta-naphthylamide of leucine aminopeptidase was present in both mouse categories; however, the Cataract Resistant mouse lens had approximately double the protease activity of the Emory Mouse cataract.

The Emory mouse cataract: accumulation of free leucine and incorporation into protein by cataractous and clear lenses.

Leucine metabolism has been studied in the Emory mouse lens to determine if there is a correlation between the rates of leucine accumulation and incorporation and the stage of cataractogenesis. Developing cataracts were graded in vitro according to severity and then incubated in tracer leucine. There was no significant difference between grades with respect to leucine incorporation or accumulation of free leucine, although there was a gradual decrease in CL/CM as the cataracts increased in severity. The results showed a high degree of variability, especially for incorporation, leading to the conclusion that the morphological appearance of these cataracts is not predictive of their metabolic behavior as revealed by leucine accumulation or incorporation.

The Emory mouse cataract: an animal model for human senile cataract.

The Emory mouse cataract is a late-appearing lens opacity which may serve as an animal model for some human senile cataracts. It is inherited as an autosomal dominant trait and has a typical course of development. Lens opacities may become readily apparent as early as 6-8 months in mice having a familial history of early cataracto-genesis. Many gross morphologic and microscopic features resemble findings in human senile cataract. As an animal model it has many desirable characteristics. Its slow development permits studies of the lens at the pre-cataractous stage and makes it a good assay system for drugs or other factors affecting cataractogenesis. In this paper are given some morphologic and histologic aspects of the developing cataract.

Glutathione metabolism in lenses of Emory and cataract-resistant mice: activity of five enzymes.

The activities of five enzymes of glutathione metabolism were determined in lenses from cataract-resistant and cataract-prone (Emory) mouse variants at three different ages (5 weeks, 10 weeks and 6 months). The enzymes included those required for glutathione synthesis, gamma-glutamylcysteine synthetase and glutathione synthetase, as well as glutathione-S-transferase, glutathione peroxidase and glutathione reductase. The differences in the activities of the five enzymes in the two mouse variants were not remarkable at any of the three ages. Activity of each enzyme was noted to be in excess of the preceding one in this integrated metabolic pathway, with the exception of glutathione reductase. gamma-Glutamylcysteine synthetase appears to be the pacesetting enzyme of this metabolic scheme in the mouse lens. The activity of each enzyme was compared with that earlier reported for human, rabbit and dog lenses.

The Emory mouse cataract: changes in the beta and gamma-crystallins during aging and cataractogenesis as revealed by isoelectric focusing of the native soluble proteins.

Isoelectric focusing on ultra-thin polyacrylamide gels was employed to study alterations in the distribution patterns of soluble beta and gamma-crystallins during the development of cataract in the Emory (EM) mouse. These alterations were compared with corresponding changes occurring in clear control lenses of the same age from the cataract-resistant (CR) strain. At two months of age both strains gave similar patterns. At four and fourteen months the EM lens showed depressed beta peaks compared to the CR lens. At fourteen months the EM lens had reduced gamma 2 and gamma 5 compared with the CR lens. At twenty-four months the EM lens had all gamma's markedly reduced, especially gamma 5, with the complete disappearance of gamma 4 which was however still present in the CR lens at twenty-six months. At twenty-four months the EM lens had a relatively large amount of beta 5 but no beta 5a or beta 5b; the latter two were present in the CR lens surrounding beta 5 which was less prominent than in the EM lens. The decrease in gamma's in the twenty-four months EM lens was approximately equalled by an apparent increase in beta's. Of the above changes only the decrease in beta's at four months can be considered precataractous and perhaps a primary change in cataractogenesis. The later changes in both beta and gamma may be secondary although they are certainly at least associated with cataractogenesis. The invariable decrease in gamma with age is noticeably accelerated in cataractogenesis.

Increased lipid peroxidation and altered membrane functions in Emory mouse cataract.

Lipid peroxidation has been shown to be involved in the pathogenesis of some types of cataract. The possibility of such a mechanism was investigated in Emory mouse cataract. Malondialdehyde, a breakdown product of lipid peroxides, increased 4-fold in advanced cataract. Studies on cation transport revealed that in early cataract there was no alteration in permeability and active transport of cations. However, these functions were significantly altered in advanced cataract as evidenced by about 300% increase in cellular influx of 22Na+ (140 mM) and 50% fall in cellular uptake of 86Rb+ (5 mM). At this stage of cataract, the ouabain- inhibitable component of uptake of Rb+ was drastically decreased, whereas the ouabain-resistant component was unchanged. The mannitol-space increased markedly with progression of cataract. Altered transport of cations in cataract was indicative of damaged membranes which may be due to peroxidation of unsaturated fatty acids in the lipid bilayers concomitant with oxidation of sulfhydryl groups of proteins of the plasma membrane. Superoxide dismutase, catalase and glutathione peroxidase, the defensive enzymes against reactive species of oxygen, were decreased 54%, 57% and 62% respectively in cataract, exposing the lens to oxidants such as 02(-), H202, 0H. and 1 delta 02, which can initiate lipid peroxidation and/or oxidation of protein.

Sodium-potassium-dependent-ATPase activity in Emory mouse lens.

Previous morphological and biochemical studies indicate that a late appearing hereditary Emory mouse cataract may be a good model for certain human senile cataracts. The development of lenticular opacity in the Emory mouse is a slow process which provides an opportunity to conduct analysis of the progression of alterations that lead to cataract development. Biochemical investigations have not yet demonstrated any specific correlation between alterations in the lens and the extent of opacity. We have conducted studies to determine the role of Na+K+-ATPase in the development of cataract in the Emory mouse. In this report we present results obtained on the site and level of activity of Na+K+-ATPase in six- and twelve-month-old Emory mouse lenses in which visible cataractous changes are beginning to appear. CFW mice (the parent strain) were used for controls in this study. Ultrastructural cytochemistry for the localization of Na+-K+-ATPase exhibited the enzyme reaction product for this enzyme to be present mainly between the lateral epithelial cell membranes and between the apical epithelial cell membranes and superficial cortical fiber membranes. In cortical fibers the reaction product was localized between fiber membranes. Although there was very little or no significant differences in the extent of reaction product in epithelial cells, the reaction product in the cortical fibers of six-month-old Emory mouse was less extensively distributed as compared to lenses from control CFW mice of the same age.(ABSTRACT TRUNCATED AT 250 WORDS)

The Emory mouse cataract: the effects on cataractogenesis of alpha-tocopherol, penicillamine, triethylenetetramine, and mercaptopropionylglycine.

The Emory mouse develops a late-onset hereditary cataract bearing some resemblances to human senile cataract. It was used as a model system for testing the effects of several drugs expected to have anticataractogenic potential. A low level of added dietary alpha-tocopherol had only a marginal effect. Penicillamine increased lens soluble protein, a good index of lens viability. Triethylenetetramine was too toxic to permit satisfactory treatment. Mercaptopropionylglycine produced several positive effects including a retardation of cataract at 6 months of age; parameters which increased under drug treatment were lens weight, soluble protein content and protein sulfhydryl, but not glutathione. There was no effect on the total calcium concentration.

The Emory mouse cataract: increased accumulation of calcium during cataractogenesis.

Total calcium and total protein contents have been determined in Emory mouse cataracts from 6 to 19 months of age and also in age-matched Cataract-resistant control lenses. The normal lens calcium is about 0.006 micrograms/mg fresh lens weight; in cataracts the value increases from about 0.02 in beginning cataracts to 0.4 in severe cataracts. Calcium does not increase with age in control lenses. Elevation of calcium levels never precedes initial cataractous changes and is most probably a secondary change. This mouse cataract, unlike human senile cataracts, invariably accumulates calcium and the magnitude is roughly proportional to the severity of cataract. It appears to be a simpler and more homogenous type than human cataract and is therefore more suitable for biochemical studies.

The Emory mouse cataract: loss of soluble protein, glutathione, protein sulfhydryl and other changes.

The Emory mouse develops a late-appearing hereditary cataract having many characteristics which suggest its usefulness as an animal model for human senile cataract. This paper presents some results of analyses designed to determine biochemical changes associated with initiation and development of the cataract. The measurements carried out include water-soluble and water-insoluble protein, glutathione, protein sulfhydryl, non-protein disulfide, sodium, potassium, calcium and free phosphate. The most useful and consistent index of cataract progression seems to be the conversion of soluble to insoluble protein, a change which in the normal aging mouse lens is accompanied by some conversion of total sulfhydryl to disulfide. Cataract development also produces a significant reduction in the glutathione concentration.

Establishment of lens epithelial cell lines from Emory and cataract resistant mice and their response to hydrogen peroxide.

We determined the conditions required for the establishment of lens epithelial cell lines from individual Emory and age matched cataract-resistant (CR) mice, and investigated the response of these cells to hydrogen peroxide. The technique described here permits the establishment of mouse lens epithelial cell lines from individual animals and provides an opportunity to study changes in epithelial function that precede and accompany cataract formation and aging. Capsules with lens epithelial cells were isolated from 1, 6 month, and 1.5-2 year old Emory and CR mice and cultured in minimal essential medium (MEM) containing 4% heat inactivated fetal bovine serum and 4% heat inactivated rabbit serum. Seeding efficiency at 3 hours was approximately 83% for all lines, doubling time was 22-24 hours, and the shape of the growth curves was comparable for Emory and CR mice from each age group. A three hour exposure of Emory and CR mouse lens epithelial cells from older animals to a constant level of 0.02 mM hydrogen peroxide or to an initial concentration of 0.01, 0.03, or 0.05 mM hydrogen peroxide resulted in a delay in growth. The delay in cell proliferation was decidedly more pronounced in lens epithelial cells from Emory mice. Lens epithelial cells from cataractous mice appear to be more sensitive to oxidative insult than their CR counterparts.