Cataract is a leading cause of blindness throughout the world, yet the fundamental biochemical causes are unknown. A rodent model of the biochemical processes is selenite cataract. This cataract shows some of the features of human cataracts such as increased lens calcium, proteolysis of proteins, and insolubilization leading to lens opacity. The goals of the current experiments were: (1) To measure changes in transcript levels for calpains and caspase 3 and oxidation of epithelial proteins in selenite cataract. (2) To elucidate changes in calpain 10 and its interaction with other calpains in selenite cataract. (3) To investigate changes in stability of ��B1-crystallin caused by deamidation and truncation. These data would provide roles for apoptosis, protein insolubilization, proteolysis and deamidation observed in cataract. To induce cataract, 12-day old rats were injected with an overdose of Na���SeO���. Epithelium was analyzed by competitive RT-PCR, zymography, and thiol-blotting. Calpains were detected by western-blotting. For ��B1-crystallin stability studies, recombinant ��B1-crystallins were denatured by urea or heat. Urea stability was measured by circular dichroism and fluorescence spectrometry, and heat stability was measured by light scattering at 405 nm. During selenite cataract formation, calpains in epithelium were activated resulting in increased proteolysis of crystallins, but mRNA levels for calpains did not show appreciable changes. Oxidation of sulthydryls in epithelial proteins was minimal during cataract formation. These results suggested that calpain-induced proteolysis in the epithelium contribute to selenite cataract. In selenite cataract, calpain 10 proteins disappeared, which appeared to be due to degradation by calpain 2 and Lp82 calpain. Deamidated ��B1-crystallin was less stable in urea and heat, compared to wildtype. When the terminal extensions were removed, ��B1-crystallin was as stable as wild-type. However, without the extensions, truncated ��B1-crystallin caused accelerated precipitation in a complex with ��A-crystallin, suggesting that the extensions may contribute to proper association with other crystallins and to stability of the soluble complexes. In summary, proteolysis of proteins by calpains was more pronounced than protein oxidation in lens epithelium of selenite cataract. Deamidation and truncation caused instability of ��B1-crystallin and abnormal association with ��A-crystallin. Thus, proteolysis and deamidation may increase susceptibility of lenses to cataract. / Graduation date: 2003

Purpose: To test the viability of implanting the Artisan®/VerisyseTM lens in a low-income monocular aphakic African population with insufficient capsular support where contact lens wear is expensive and spectacle correction is not possible, by assessing the postoperative visual functions. To further assess whether adverse effects (e.g.: pigment dispersion with secondary glaucoma, prolonged uveitis) in patients with highly pigmented irises would be as low as with patients in European and American trials. Methods: A prospective, randomised, controlled clinical trial comparing outcomes in two groups of unilateral aphakic patients. The patients in the first group received an Artisan intra-ocular iris-claw lens as a secondary procedure while the second group remained aphakic (the current treatment status quo for public patients in KwaZulu-Natal province in South Africa). Follow-up was done for 1 year. Results: The study was terminated early due to ethical and statistical reasons. Nine treated and five control patients were included. Monocular uncorrected vision was significantly higher in the treatment group (P=0,012) and patient satisfaction was higher (p=0,002). Changes in other variables (intraocular pressure, angle pigmentation, change in cup-to-disc ratio, iris pigment changes and best spectacle corrected vision) were not significantly different between the two groups. Conclusion: The Artisan®/VerisyseTM lens is a feasible option for aphakic African patients with regard to visual outcome, safety and patient satisfaction. This form of refractive correction should be the standard for patients with no capsular support and where other options are too expensive or carry greater risk. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2007.

Thesis (PhD)--Stellenbosch University, 2001 / ENGLISH ABSTRACT: The eye is that biological instrument which conveys the light of the external world into the inner world of the mind, wherein we receive the miraculous gift of vision. So precious is this gift, that Science must search for ways to keep this portal clear for the flow of light. Indeed, Science is called upon to “make war upon the bloody tyrant, Time.” (Shakespeare W. Sonnet No. 16). For, in the course of ageing, the lens grows cloudy and cataractous. In this battle between Science and Time, we are fortunate to live in an era in which Science is uncovering the molecular basis for the various obstacles to vision. The question arises, whether or not, the ruinous hand of time can be stayed. Due to unrelenting, progressive lens opacification, most of the elderly are destined to be subjected to loss of vision and with passage of time, even blindness. Globally the cataract surgery rate is inadequate to keep pace with the ever growing demand on financial and human resources created by the cataract problem. An immense challenge therefore is directed to primary eye care: “Can cataract be prevented or can its onset at least be postponed?” This laudable ultimate aim can only be achieved once the etiology of cataractogenesis is well understood. This dissertation seeks to examine two previously unrecognized etiological aspects that, if correctly understood and managed, have the potential to achieve preventive ophthalmological goals that may indeed help to stay the ‘ruinous hand of time’. The first aspect deals with the role of lipids and was examined using a study group of dyslipidemic subjects. The first part of the study concluded that dyslipidemic patients develop cortical lens opacities more frequently and at an earlier age than the normal population, and that cortical lens opacities should be regarded as one of the most reliable clinical signs of dyslipidemia. Furthermore, an extremely strong correlation was found to exist between low HDL Cholesterol levels and the development of opacities. Below a HDL-Cholesterol level of 1,5mmol/l, subjects had more than seven-fold higher risk of falling in the lens opacity subgroup than those with HDLCholesterol levels above 1,5mmol/l. An equally strong correlation was demonstrated between high (>5) LDLHDL ratios and the development of lens opacities. Subjects with a LDL:HDL-C ratio below 5 possessed a 2.35 times greater risk of having lenticular opacities than the group with a LDL:HDL-C ratio greater than 5. The prevention or retardation of dyslipidemia associated lens opacities is therefore possible, provided patients with a genetic predisposition are detected early and their blood lipids managed adequately. The second aspect deals with the relationship between age related cataracts and the acetylation status of the individual. This study compellingly submits that the slow acetylator pheno- and genotype may be regarded as a genetic indicator of risk for age related cataract. The ability accurately to classify a patient genotypically and phenotypically, may henceforth be useful in health counseling since, if an individual is identified as being a slow acetylator, additional preventative and precautionary measures may be taken, i.e. the prevention of UVexposure to the eye and caution with the ingestion of xenobiotics like caffeine, commercial dyes, food preservatives, and drugs. Furthermore, such a finding should be taken into account in the long term therapeutic management of glaucoma, with special regard to carbonic anhydrase inhibitors which are sulphonamide-related drugs and totally dependent on the N-acetyltransferase pathway for metabolism. These drugs may accumulate in the slow acetylator over time and exert toxic effects intra-ocularly, conceivably including cataractogenesis. The search for genetic and metabolic mechanisms that may contribute to human cataractogenesis should be pursued with great enthusiasm. This endeavour may help Science to achieve its primary objective, ablate the effects of Time and really aid in preventing cataracts in man. / AFRIKAANSE OPSOMMING: Die wondergawe van visie word vir ons moontlik gemaak deur die oog wat as biologiese instrument die lig van die buitewereld inlaat na die binnewereld van die brein. So kosbaar is hierdie gawe dat die Wetenskap deurgaans moet poog om die poort oop te hou. Inteendeel, die Wetenskap word gemaan deur Shakespeare in sy Sonnet nommer 16 om “oorlog te maak teen die bloeddorstige tiran, Tyd”. Soos ‘n mens ouer word, word die lens dof en ‘n katarak mag vorm. Ten spyte van hierdie stryd tussen ‘Wetenskap’ en ‘Tyd’ leef ons in die gelukkige era waarin die Wetenskap meer en meer leer van die verskeie obstruksies tot visie. Die vraag ontstaan of die rinnewerende hand van ‘Tyd’ gestuit sal kan word. Vanwee ongenaakbare, progressiewe lensvertroebeling is die meeste bejaardes bestem om aan visie verlies, en met verloop van tyd selfs blindheid, te ly. Die wereldwye katarakchirurgie tempo is nie voldoende om by te hou by die immergroeiende finansiele en mannekrag eise wat deur die katarak probleem gestel word nie. Daar word dus ‘n reuse uitdaging aan primere oogsorg gestel naamlik: “Kan katarakte nie eerder voorkom of die aanvang daarvan ten minste uitgestel word nie?” Hierdie prysenswaardige doelwit kan nie bereik word alvorens die etiologie van kataraktogenese goed verstaan word nie. Hierdie tesis ondersoek twee voorheen onerkende etiologiese aspekte wat, indien hulle korrek verstaan en hanteer word, beslis die potensiaal het om die gemelde voorkomende doelwitte te bereik en sekerlik te kan bydrae om die rinnewerende hand van Tyd te stuit. Die eerste aspek spreek die rol van lipiede aan deur te kyk na 'n studiegroep van dislipidemiese persone. Die eerste deel van die studie kom tot die gevolgtrekking dat dislipidemiese pasiente kortikale lens opasiteite meer dikwels en op ‘n vroeer ouderdom ontwikkel as die normale populasie en dat sulke opasiteite beskou moet word as een van die mees betroubare kliniese tekens van dislipidemie. Daar is ook ‘n baie sterk korrelasie gevind tussen lae HDL cholesterol vlakke en die voorkoms van opasiteite. Persone in die studie met ‘n HDL cholesterol vlak laer as 1,5mmol/l het ‘n sewe keer hoer kans gehad om in die lensopasiteit subgroep te val as die met ‘n HDL cholesterol vlak laer as 1,5mmol/l. ‘n Sterk korrelasie tussen ‘n hoe (>5) LDLHDL verhouding en die voorkoms van lens opasiteite is ook gevind. Persone met ‘n LDLHDL verhouding >5 het ‘n 2.35 maal groter risiko gehad om lensopasiteite te he as die met ‘n LDL:HDL verhouding van <5. Die voorkoming of vertraging van dislipiedemie geassosieerde lens opasiteite is dus moontlik, solank persone met ‘n genetiese geneigdheid daartoe vroeg ontdek en hulle bloedlipiede voldoende beheer word. Die tweede deel van die tesis handel oor die verhouding tussen ouderdoms verwante katarakte en die asetilasie status van die individu. Met oortuiging kom hierdie studie tot die gevolgtrekking dat die stadige asetilator fenoen genotipe as 'n genetiese merker vir ouderdoms verwante katarakte beskou moet word. Die vermoe om ‘n individu beide genotipies en fenotipies akkuraat te klassifiseer mag voorts bruikbaar wees in gesondheidsraadgewing. Indien ‘n individu geTdentifiseer is as ‘n stadige asetileerder, kan addisionele voorsorg maatreels getref word soos bv. die voorkoming van blootstelling van die oog aan UV lig sowel as omsigtigheid met die inname van xenobiotika soos kaffei'ene, kleurstowwe, voedsel preserveermiddels en geneesmiddels. Hierdie bevinding moet ook in berekening gebring word in die langtermyn terapeutiese hantering van gloukoom. Die koolsuuranhidrase inhibitore, dikwels gebruik in die behandeling van gloukoom, is sulfonamied-agtige middels en dus totaal afhanklik van die N-asetieltransferase pad vir hulle metabolisme. Hierdie middels kan ophoop in die stadige asetileerder en gegewe genoeg tyd, bes moontlik toksiese intra-okulere effekte tot gevolg he. Die soeke na genetiese en metaboliese meganismes wat mag bydra tot menslike kataraktogenese behoort nagestreef te word met groot entoesiasme. Hierdie strewe mag dalk net vir die 'Wetenskap' bystaan om sy primere mikpunt te bereik, die effek van ‘Tyd’ te neutraliseer en te help om katarakte werklik te voorkom.

A 3- to 5-fold increase in Ca2+ accompanies cataract formation induced by selenite. The mechanism of selenite cataractogenesis involves calcium activation of calpain with subsequent proteolysis within the lens nucleus. This study was undertaken to investigate the biochemical mechanisms that lead to calcium accumulation in these circumstances. The components responsible for rat lens calcium regulation were defined by using either lens membrane vesicle preparations or intact lenses. Both Na+ gradient-dependent Ca2+ uptake and efflux occurred in lens membrane vesicles. Experiments with intact lenses showed that Na + ICa2 + exchange plays an important role in lens calcium regulation. ATP-dependent Ca2+ uptake and Ca2+ -dependent ATP hydrolytic activity have been characterized in lens membrane vesicles. Therefore, both Ca2+ -ATPase and Na + ICa2+ exchange participate in rat lens calcium regulation. Calcium accumulation in lenses treated by selenite may result from either increased influx (via non-selective cation channel), decreased efflux (via Ca2 +-ATPase and Na+ ICa 2+ exchange) or both. The selenite effects on the different components involved in lens calcium regulation were tested. / Ph. D.