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No description available.

No description available.

No description available.

Cataract surgery is a common operation which involves removing the natural crystalline lens from the eye, when it has become opaque (cataractous), and replacing it with an artificial intraocular lens (IOL). A rare complication that has been associated with some IOLs is opacification.Hydroview (Bausch and Lomb) IOLs were widely implanted during cataract surgery throughout the UK between 1997 and 2001. These IOLs have been associated with symptomatic opacification of the optic necessitating IOL exchange to restore clarity of vision and redress the symptom of glare. As glare and misty vision have been purported as the most common presenting symptoms[1 -4], this thesis investigated the impact of IOL opacification on vision. To this end I interrogated the effect of IOL opacification on subjective visual symptoms, objective measurements of visual function and vision related quality of life, as well as measuring the beneficial effect or not of IOL exchange surgery on these variables. The study also investigated the possible aetiology of Hydroview opacification. I assessed the association of patient, surgical and lens related factors with the incidence of opacification. Explanted lenses were examined with a scanning electron microscope fitted with a wavelength dispersive x-ray probe to describe the ultrastructural findings and elemental analysis of opacified and clear hydroview IOLs to elucidate major contributing factors or underlying causes of opacification.

Posterior capsular opacification is the main complication of cataract surgery and results from the proliferation, migration and differentiation of lens epithelial cells remaining in the capsular bag. To better understand this pathological cell behaviour, 1 investigated the interactions between lens epithelial cells and the bovine lens capsule in vitro and their effect on cell viability. As determined by a colorimetric cell proliferation assay, in vitro culture of cells directly on the bovine lens capsule resulted in maintained cell viability in the presence of staurosporine in both lens epithelial cell lines tested, but in neither of the two non-lens cell lines tested. As determined by immunoblotting and reverse-transcriptase polymerase chain reaction (RT-PCR), cell viability on the bovine lens capsule could further be correlated to the presence of both ɑA-crystallin and αB-crystallin expression. A positive correlation of cell viability on the lens capsule with vimentin and HSP27 expression was also found in a smaller set of cell lines. As determined by gelatin zymography and immunoblotting, MMP-2 was expressed by lens epithelial cells, led to the release of FGF-2 and IGF-1 from the lens capsule and correlated with lens epithelial cell viability. Taken together, these results suggest that the lens capsule can act as a store of releasable growth factors available to the lens epithelial cells, with effects on their protein expression and cell viability.

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Age-related cataract is the commonest cause of treatable blindness in the world today. It is an ever-increasing problem with prolonged life-expectancy and a burgeoning elderly population. Although successfully treated by modem, sophisticated cataract surgery, the resources needed to provide a surgical set-up and the prolonged training time required to produce a competent surgeon can challenge the available finances of developed economies and result in unacceptable waiting times while they may simply be unaffordable in developing economies and result in high prevalence of blindness. Surgical treatment, although effective in dramatically reversing blindness, is associated with a small risk of sight-threatening complications. Against this background, it is worth considering an alternative, simpler, medical means of treating cataracts that is easily administered and equally effective. Calpains are Ca2+dependent intracellular proteases and several of these enzymes are believed to participate in cataractogenesis. Calpain 2 is the major isoform of calpain involved in human cataractogenesis and its activation appears to be investigate the mechanism of lipid activation of calpains as an essential link towards understanding the molecular and cellular dynamics of cataractogenesis in vivo. A clear picture of the enzymatic events in cataractogenesis will form the basis of drug development to cause selective enzyme inhibition. In chapter 2, atomic absorption spectroscopy shows that the progressive uptake of extra-lenticular Ca2+ by porcine lenses correlates with increases in levels of lens opacification with 8.0 umoles Ca2+ (gm wet lens weight)-1 corresponding to cataract occupying approximately 70% of the lens cell volume. This degree of cataract was reduced by approximately 40%, when a calpain inhibitor, SJA6017 (final concentration 0.8 uM), was included in the extra-lenticular medium, therefore suggesting that the observed porcine opacifications involve the Ca2+ mediated activation of calpains and that cataract could be retarded by the topical administration of calpain inhibitors. In chapter 3, DWIH (Depth weighted insertion hydrophobicity) analysis shows the small subunit of several calpain 2 isozymes to each posess a segment with the potential to form a lipid / membrane interactive a-helix (DWIH values circa 7.0). Extended hydrophobic moment analysis shows these segments to be potential oblique orientated a-helix formers (< uH > circa 0.5). This potential is confirmed by hydropathy plot and graphical analyses, which show each a-helix to possess a significant N -> C hydrophobicity gradient. It is suggested that the lipid / membrane activation of calpain 2 may involve oblique angled membrane penetration by an ahelical segment in domain V of the enzyme. In chapter 4, VP1, a peptide homologue of this domain V segment, is shown to be strongly haemolytic (half-maximal lyric dose = 1.45 mM). FTIR conformational analysis shows VP1 to adopt a-helical structure (20% - 65% of primary structure) in the presence of lipids. These levels are maximal in the presence of anionic lipid (65% of primary structure) and monolayer studies show the peptide to exhibit high levels of anionic lipid monolayer penetration with surface pressure changes (A SP) of 5-6 mN m-1 at 30 mN m-1, which are reduced by approximately 40% ± 15% in the presence of 100 mM NaCl. It is suggested that membrane penetration by the domain V a-helix of calpain 2 may proceed via electrostatic interactions and snorkelling, involving associations between an arginine residue located in the polar face of this a-helix and anionic membrane lipid. It has been suggested that lipid / membranes may modulate calpain 2 activity by lowering the enzyme's in vivo Ca2 requirements. In chapter 5, colorimetric assay of calpain 2 shows that the enzyme requires 4 mM Ca2+ for 100% proteolytic activity, as defined by this assay. In the presence of 1 mM Ca2+, negligible calpain 2 proteolysis is detected but at this level of Ca2+, in the presence of either Dimyristoyl phosphatidylinositol(DMPI), Dimyristoyl phosphatidylserine(DMPS), Dimyristoyl phosphatidylcholine(DMlPC) or Dimyristoyl phosphätidylethanolamine(DMPE), calpain 2 shows proteolytic activity which ranges between 37% and 77% of the protein's full enzymatic activity. The large subunit of calpain 2 (LS-calpain 2) is proteolytical]y active in the absence of the calpain 2 small subunit and it has been suggested that this latter subunit is dispensable for the lipid activation of calpain 2. LS-calpain 2 is assayed under conditions corresponding to those used here for calpain 2 assay and is shown to require the presence of 6 mM Ca 2 for 100% enzymatic activity. These Ca2+ levels are unaffected by the presence of either: DMPI, DMPS, DMPC or DMPE, and based on these combined results, it is suggested that the lipid activation of calpain 2 requires the presence of the small subunit. In addition, it is shown that when compared to zwitterionic lipid under corresponding conditions anionic lipid induces an approximate twofold enhancement of calpain 2 proteolytic activity (70% - 77% as compared to 37% - 49%) and a similar enhancement in its average rates of lipid monolayer interaction (ASP = 1.5 x 10 mN M-1 sec-1 at 10 mN M-1 as compared to ASP = 5.0 x 10-4 mN M-1 sec-1 at 10 mN M-1). It is suggested that calpain 2 may posess an electrostatically driven preference for anionic lipid, which contributes to lowering the enzyme's Ca2+ requirement for activation. In chapter 6, these combined data are discussed in relation to a model for the lipid activation of calpain 2 and proposals for future work are presented.