William E. Bruner

AGE-RELATED CHANGES OF GLYCOSIDASES IN HUMAN RETINAL PIGMENT EPITHELIUM

This study was undertaken to determine whether there are age-related changes in the specific activities of several glycosidases in fresh retinal pigment epithelial cells (RPE) isolated from the posterior pole of human donor eyes. One hundred and twenty-one pairs of eyes from human donors, between the ages of 43 and 95 years, were obtained from the National Disease Research Interchange (NDRI, Philadelphia, PA) and the Cleveland Ohio Eye Bank within 18 to 24 h of death. None had histories of diabetes, hepatitis, HIV infection, intraocular surgery, or documented age-related macular degeneration, although several older donors with evidence of drusen were included in the study. RPE cells were isolated from the posterior third of the retina using the conventional rush method and homogenized with a glass, Broeck tissue grinder. All post-nuclear supernatants were analyzed for glycosidase activity; a smaller number of nuclear pellets were assayed to verify that the majority of the enzyme activity was associated with the post-nuclear sypernatants. Glycosidase activity was quantitated fluorometrically by measuring the enzymatic release of umbelliferone from synthetic substrate preparations, specific for each enzyme. Total protein was determined by a micro BCA protein assay. Regression analysis revealed statistically significant age-related decreases for the specific activities of alpha-mannosidase (p = 0.0001), beta-galactosidase (p = 0.0001), N-acetyl-beta-glucosaminidase (p = 0.0001), and N-acetyl beta galactosaminidase (p = 0.0001) in fresh human donor RPE cells taken from the region of the posterior third of the retina that included the macula. Mannose and N-acetyl-glucosamine are major carbohydrate monomers of the oligosaccaride chains of human rhodopsin, and a relatively high percentage of the oligosaccharide chains are galactosylated. Defects in their degradation may lead to the accumulation of undigested residual material in the RPE.

Mannosyl transferases of the retina: Mannolipid and complex glycan biosynthesis II. Activity in different species; Subcellular distribution; During development☆

Abstract The activities of the mannosyl transferases which catalyze the transfer of mannose from the sugar nucleotide, GDP-[ 14 C]mannose to endogenous lipid and glycan acceptors were determined in homogenates of the retinas of the embryonic chick, dogfish, squid, cattle and adult chickens. The embryonic chick retina, in contrast to the others, incorporated relatively greater radioactivity into the complex glycan compared to that into the mannolipid. These mannosyl transferases were also examined during the development of the embryo. These reactions were unaffected by conditions of light or dark, or by exposure of the embryo, in ovo, to hydrocortisone. Most of the activity for both the lipid and complex glycan mannosyl transferases were located in the low-speed pellet (nuclei+cell debris) obtained after differential centrifugation.

Corneal alpha-galactosidase deficiency in macular corneal dystrophy.

Glycosidases, which cleave sugar molecules from complex glycopolymers, have been previously quantified in normal human cornea in our laboratory. Data quantifying glycosidases in macular corneal dystrophy are lacking. Tissue obtained at keratoplasty from patients with macular dystrophy and normal corneas obtained from eye bank eyes were used to determine levels of glycosidase activity. A fluorometric technique was employed using 4-methyl-umbelliferyl-glycosides as substrates. The corneal tissues were homogenized, centrifuged, and the supernatants assayed for enzyme activity. Specific activities (mumol/mg protein/hour) were determined and Km and Vmax values were obtained for all but one enzyme. Activity of alpha-galactosidase was significantly lower in cornea tissue and keratocytes from macular corneal dystrophy compared to normal.

Lysosomal Enzyme Levels in Corneal Storage Media

Evidence indicates that lysosomal enzymes can carry out corneal autolysis during corneal storage and that they are damaging to the corneal endothelium. The authors investigated the release of lysosomal enzymes into two corneal storage media (K-Sol and McCarey-Kaufman [M-K]) by paired human donor corneas during 4 degrees C storage. The authors also studied the interaction of these media with lysosomal enzymes from human cornea. K-Sol and M-K stimulated (P less than 0.01) both beta-glucuronidase and alpha-galactosidase about equally. beta-N-Acetyl-glucosaminidase, a major catabolic enzyme of the cornea, was inhibited by the chondroitin sulfate in K-Sol by over 90% (P less than 0.01). Corneas stored in M-K released more lysosomal enzymes than corneas stored in K-Sol. At 4 days, the values approached significance (P less than 0.06) and by day 10 significantly higher values were found in the M-K media (P less than 0.01). Both storage methods showed a linear release. Individual corneas were found to vary in their release rates. Whether corneas that release more enzyme will show higher endothelial cell loss or produce less successful penetrating keratoplasty grafts deserves further study.

Sex chromosome aneuploidy and Bardet-Biedl syndrome

The inheritance of Bardet-Biedl syndrome is thought to be autosomal recessive. Of the approximately 500 case reports in the literature, three patients were found to have sex chromosome aneuploidy. The authors describe two siblings with Bardet-Biedl syndrome, one of whom has a unique sex chromosome aneuploidy with mosaicism, including deletion of the short arm of the X chromosome (45,X/46,X,del(X)(p21)). The possible significance of sex chromosome aneuploidy and the Bardet-Biedl syndrome is discussed.

Lysosomal enzymes in corneal storage media and corneal graft outcome.

Purpose The purpose of this study was to relate lysosomal enzyme activities in corneal storage media to the outcome of the transplanted corneas. Methods Corneal storage media from 358 transplanted corneas were frozen at −70°C and kept for enzyme analysis. Corneas were stored in K-Sol (28), CSM (35), Dexsol (80), Index medium (five), Optisol (158), and Optisol GS (52). Activities of α-D-mannosidase, β-glucuronidase, α-glucosidase, and N-acetyl-β-glucosaminidase were assayed fluorometrically. Mayo Clinic records were examined for donor information, including cause of death and 2-month graft follow-up data. Results For all corneas, there was a low but significant correlation between activities of each enzyme and storage time (r-s = 0.13–0.35; p = 0.02–0.0001), and donor age (rs = −0.14 to −0.23; p = 0.009–0.0001). There was no significant correlation of enzyme activity with 2-month endothelial cell density, structure, cell loss, or corneal thickness. Enzyme activities for four primary donor failures and six grafts with <65% 2-month endothelial cell loss were not significantly different from those for the rest of the transplanted corneas. Enzyme activities were higher for corneas from donors with renal failure but not from those with diabetes mellitus. There was no significant difference in graft outcome for different cause-of-death groups. Conclusions The activities of lysosomal enzymes released into corneal storage media are not useful as predictors of graft outcome.

The Effect of hEGF and Insulin on Corneal Metabolism During Optisol Storage

We examined the effect of the growth factors, human epidermal growth factor (hEGF) and insulin, on corneal metabolism during storage in Optisol, a chondroitin–sulfate-(CS)-based storage medium. Paired cat corneas, in either Optisol only or Optisol with growth factor(s), were analyzed using ex vivo 31P nuclear magnetic resonance, after storage for 1 week at 4°C. Lysosomal enzyme release into the media at the end of the storage period also was measured fluorometrically. Both epithelial-intact and epithelial-denuded corneal pairs were examined for all conditions. Considering corneas having either intact epithelia or epithelium-denuded corneas, the addition of either growth factor alone to Optisol did not alter the relative corneal concentrations of five of the six phosphatic metabolite spectral bands measured or two metabolic ratios calculated from these bands. Phosphodiesters, however, were significantly lower in corneas stored in Optisol containing both hEGF and insulin (23%) than in corneas stored in Optisol alone (30%). Intracorneal pH was unaffected by the addition of growth factors). A significantly higher release of α-glucosidase and α-mannosidase was noted in those corneas stored in Optisol containing both hEGF and insulin. Optisol maintains high-energy phosphate corneal metabolism similar to other CS-based media, K-Sol and Chondroitin Sulfate Corneal Storage Medium (CSM). The addition of the growth factors hEGF and insulin to Optisol alters corneal metabolic activity during storage in a manner indicative of conserving corneal phospholipids.

Beta-N-acetyl-glucosaminidase: possible role in ocular neovascularization

The possibility that lysosomal enzymes might be involved as angiogenic factors in ocular neovascularization (NV) was investigated. Beta-N-acetyl-glucosaminidase (NAGase) activity, and that of two other glycosidases, were present in the retinal derived protein fraction (RDPF) reported by others (10) to be angiogenic. NAGase, but not the other glycosidases, was inhibited by vitreous. NAGase exhibited the same stability characteristics as RDPF. In diabetic rats there was a significant rise in vitreous but a fall in retinal NAGase activity. The sera of these animals, however, showed elevation in the activities of all five glycosidases. Preliminary experiments indicate that only the intermediate isoenzyme of NAGase, putatively insulin dependent, is elevated in the eyes of these diabetic rats. NAGase was also specifically elevated in the intraocular fluid from monkey eyes with retinal vein occlusion (RVO), and markedly so if NV was present. These results suggest the involvement of NAGase in the neovascular process in the eye.

Effect of scleral buckling on unsutured cataract wound strength.

BACKGROUND AND OBJECTIVE Modern cataract surgery has evolved to include small, unsutured wounds with rapid visual rehabilitation and fewer complications. Properly constructed, these unsutured wounds can withstand increased intraocular pressures without leakage, rupture, or incarceration of intraocular contents. However, scleral buckling surgery may alter the architecture of these wounds and thus their strength. The authors wanted to study the effect of scleral buckling on the integrity of these unsutured cataract wounds. MATERIALS AND METHODS Eighteen fresh human globes underwent creation of scleral and limbal corneal incisions so as to create self-sealing wounds. Scleral buckles were then placed. Intraocular pressures were elevated to 400 mm Hg before and after placement of the scleral buckles and evidence for wound leakage was sought. RESULTS Two globes with clear corneal incisions and no scleral buckles leaked slightly at 300 mm Hg, but no globe with a scleral buckle, regardless of incision type or silicone element style, leaked at pressures to 400 mm Hg. CONCLUSION Sutureless cataract incisions, if properly constructed, provide a strong, pressure-resistant wound. Scleral buckling does not appear to affect the strength of these wounds.