Epigenetic regulatory mechanisms in vertebrate eye development and disease.

Eukaryotic DNA is organized as a nucleoprotein polymer termed chromatin with nucleosomes serving as its repetitive architectural units. Cellular differentiation is a dynamic process driven by activation and repression of specific sets of genes, partitioning the genome into transcriptionally active and inactive chromatin domains. Chromatin architecture at individual genes/loci may remain stable through cell divisions, from a single mother cell to its progeny during mitosis, and represents an example of epigenetic phenomena. Epigenetics refers to heritable changes caused by mechanisms distinct from the primary DNA sequence. Recent studies have shown a number of links between chromatin structure, gene expression, extracellular signaling, and cellular differentiation during eye development. This review summarizes recent advances in this field, and the relationship between sequence-specific DNA-binding transcription factors and their roles in recruitment of chromatin remodeling enzymes. In addition, lens and retinal differentiation is accompanied by specific changes in the nucleolar organization, expression of non-coding RNAs, and DNA methylation. Epigenetic regulatory mechanisms in ocular tissues represent exciting areas of research that have opened new avenues for understanding normal eye development, inherited eye diseases and eye diseases related to aging and the environment.

Three distinct stages of lens opacification in transgenic mice expressing the HIV-1 protease.

Scanning electron microscopy of the lenses from transgenic mice (TG(72)) containing the HIV-1 protease linked to the lens alphaA-crystallin promoter showed structural changes around postnatal day 16. Frank opacification of the lens was observed at day 24. To relate the biochemical and biophysical changes that occur during the process of cataract development, high-resolution two-dimensional gel electrophoresis (2D), quantitative image analysis and ion measurements were carried out on lenses from postnatal day 10 and on days 15-24. The phase separation temperature (Tc), a measure of molecular interactions between proteins, was also determined for normal and transgenic lenses. A comparison of the transgenic and normal lenses on day 10 revealed no significant differences in any of the measured parameters. However, starting around day 16 or the first stage of observed structural changes, the TG(72)crystallin profiles of the alphaA- alphaB-, betaA3-, betaA4-, betaB3 and one gamma-crystallin began to deviate from the normal. By postnatal day 20, a second stage was initiated with an influx of calcium and sodium ions that was accompanied by modifications of betaB1- and betaB2-crystallin. In the third and final stage of the cataract process, a large increase in the proteolysis of crystallins was accompanied by the appearance of the frank cataract on day 24. The Tc initially increased in all of the mouse lenses until just prior to eyelid opening. After that time, the Tc decreased in all lenses. Although the Tc continued to decrease in the normal lenses with age, for the homozygous transgenic mice it exhibited a dramatic increase that began on day 20. Thus, in the TG(72)transgenic mouse, cataract formation occurs in a three-stage process. Tc and other biophysical parameters previously measured appeared to be insensitive to the modifications that occur during stage 1. However, during the second stage of cataract formation, there was a correspondence between abnormal Tc and the abnormal interactions between cellular constituents apparently resulting from lens hydration, the loss of ion homeostasis and continued proteolysis. The last stage of cataract formation results in a total loss of lens transparency and leakage of lens proteins.

The leucine zipper of NRL interacts with the CRX homeodomain. A possible mechanism of transcriptional synergy in rhodopsin regulation.

Photoreceptor-specific expression of rhodopsin is mediated by multiple cis-acting elements in the proximal promoter region. NRL (neural retina leucine zipper) and CRX (cone rod homeobox) proteins bind to the adjacent NRE and Ret-4 sites, respectively, within this region. Although NRL and CRX are each individually able to induce rhodopsin promoter activity, when expressed together they exhibit transcriptional synergy in rhodopsin promoter activation. Using the yeast two-hybrid method and glutathione S-transferase pull-down assays, we demonstrate that the leucine zipper of NRL can physically interact with CRX. Deletion analysis revealed that the CRX homeodomain (CRX-HD) plays an important role in the interaction with the NRL leucine zipper. Although binding with the CRX-HD alone was weak, a strong interaction was detected when flanking regions including the glutamine-rich and the basic regions that follow the HD were included. A reciprocal deletion analysis showed that the leucine zipper of NRL is required for interaction with CRX-HD. Two disease-causing mutations in CRX-HD (R41W and R90W) that exhibit reduced DNA binding and transcriptional synergy also decrease its interaction with NRL. These studies suggest novel possibilities for protein-protein interaction between two conserved DNA-binding motifs and imply that cross-talk among distinct regulatory pathways contributes to the establishment and maintenance of photoreceptor function.

Modelling cortical cataractogenesis 21: in diabetic rat lenses taurine supplementation partially reduces damage resulting from osmotic compensation leading to osmolyte loss and antioxidant depletion.

The concentration of taurine and the amino acids, glutathione, cysteine, ascorbate and ATP were determined in the lenses of rats made diabetic with streptozotocin. In the clear lenses, prior to vacuole formation after 1 or 2 weeks of diabetes, the increase in concentration of sorbitol and the total decrease of all these osmolytes were not significantly different. The major components of the osmolytes lost were taurine and amino acids, which together accounted for over 75% of the total osmolyte loss. Since glutathione, ascorbate, taurine and cysteine have been reported to have antioxidant activity, it appears that their loss may potentiate damage occurring as a result of free radicals generated by nonenzymic glycation by the Maillard reaction. Amino acids also lost as a result of the osmotic compensation, are estimated to be responsible for almost half of the antioxidant activity lost. To test this hypothesis, normal and streptozotocin diabetic female Wistar rats were given taurine at 0.05% or 0.10% (w/w) in the diet. This treatment resulted in small only marginally significant increases in serum taurine levels. At the end of 6 weeks the rats were examined for weight gain or loss and at the time of killing, blood was collected for measurement of serum glucose. gamma-Crystallin levels were determined in vitreous and aqueous humours using a radioimmunoassay. A lens from each rat was homogenized in 8 m guanidinium chloride for adenosine triphosphate (ATP) analysis. In normal rats, a small amount of gamma-crystallin was found in the vitreous humour, and an even smaller amount in the aqueous humour. Diabetes caused a 4- to 5-fold increase in the vitreous humour and a 4-fold increase in gamma-crystallin in the aqueous humour. Diabetes also led to a significant worsening in general body condition, loss of body weight, formation of cataracts, and decrease in lens ATP levels. Addition of taurine to the diet of diabetic animals resulted in a significant decrease of gamma-crystallin leakage into the vitreous but not the aqueous humour. Taurine had no effect on the lens ATP levels. Neither streptozotocin diabetes nor taurine in the diet appeared to affect the weight of the lenses.

Uptake of vitamin E succinate by the skin, conversion to free vitamin E, and transport to internal organs.

The percent solubility at 34 degrees C (skin temperature) of radioactive tocopherol succinate was determined for a number of edible oils, and a semisynthetic oil, Myritol 318 (Henkel, Kankakee, IL, a medium chain triglyceride prepared from fractionated coconut oil). Its solubility in Myritol 318 was approximately 50% better than any of the other oils. 14C-tocopherol succinate was diluted (1) into pure Myritol 318, a cosmetic base or (2) 50% tocopherol succinate in Myritol 318. These preparations were applied topically to a 2 cm diameter circle of the back saddle skin of a hairless mouse (strain skh-1). After 24 hr, up to 65% of the label was absorbed by the skin and was also found in skin removed from areas of the back other than the application area, and internal organs such as liver and heart. Up to 6% was hydrolysed to free tocopherol. Topical treatment may be an alternative to oral administration in gastrointestinal malabsorption diseases.

Mechanical stretch alters the actin cytoskeletal network and signal transduction in human trabecular meshwork cells.

PURPOSE: Human trabecular meshwork (HTM) cells were mechanically stretched in vitro as a potential model for the distension of this tissue that can occur in vivo in response to increased pressure gradients. Cell morphology and certain components of the signal transduction pathways, including the mitogen-activated protein kinase (MAPK) and c-Jun N-terminal protein kinase (JNK) pathways, were evaluated for stretch-induced alterations. METHODS: Primary HTM cells grown in tissue culture were subjected to a mechanical stretch lasting from 10 seconds to 4 days. The actin cytoskeletal network was visualized by phalloidin staining. Proteins phosphorylated on their tyrosine residues were isolated using an immunoaffinity system and were analyzed by gel electrophoresis and immunostaining. Mitogen-activated protein kinase activity was evaluated using an in-gel assay system, and the mRNA levels of c-fos and c-jun were determined by quantitation of competitive reverse transcription-polymerase chain reaction. In addition, the amount of c-Fos protein was estimated by chemiluminescent immunoblot analysis. RESULTS: On stretching, the HTM cells elongated but regained their normal morphologic characteristics within 24 hours. Unstretched HTM cells displayed a diffuse F-actin microfilament network, whereas stretched cells exhibited complex geodesic patterns. Ten seconds after stretching began, the level of tyrosine phosphorylation on the six major phosphoproteins significantly decreased between 80% and 100%, whereas the level of paxillin tyrosine phosphorylation significantly increased 39%. Stretching caused MAPK activity and the amount of mRNA and protein of the immediate-early gene c-fos to decrease more than 60% within 2 minutes, but within 15 to 30 minutes they increased above or equivalent to normal levels. The level of c-jun mRNA was unchanged by stretching. CONCLUSIONS: In response to a mechanical stretch, major cytoskeletal alterations occur in HTM cells, which involve changes in the levels of tyrosine phosphorylation. Mechanotransduction (signal transduction by mechanical stimulation) through the MAPK signaling pathway was significantly depressed immediately after stretching; however, the JNK pathway appeared to be unaffected. The data suggest that HTM cells adapt to mechanical stress by altering the cytoskeletal network and signaling cascades.

Free amino acids reflect impact of selenite-dependent stress on primary metabolism in rat lens.

PURPOSE: A decrease in phase separation temperature, prior to nuclear cataract, has been correlated with elevated free amino acid content. Hence, we determined how selenite-induced stress alters free amino acid pools in the rat lens, following a single subcutaneous dose of sodium selenite (30 nmol g-1 body weight) in 10- to 14-day-old Sprague Dawley rats. RESULT: Oxidative stress was evident in lenses 24 h after rats were treated with selenite. Glutathione content was decreased by 60% in the lens cortex and nucleus; the flux of glucose through the pentose phosphate pathway was increased; and glycerol-3-phosphate content was elevated. Amino acid transport, evaluated as 14C-cycloleucine uptake, was not altered, although 14C-glutamine was oxidized at a slower rate. Lenses from treated animals displayed, among the free amino acids, increased glutamine, proline, serine, glycine and the branched chain amino acids, while aspartate, glutamate, and taurine were less. CONCLUSIONS: A systemic delivery of sodium selenite caused oxidative stress in the rat lens. Direct effects on primary metabolism altered free amino acid pools that may contribute to transient and permanent changes in lens transparency.

Cysteine and ascorbate loss in the diabetic rat lens prior to hydration changes.

PURPOSE: Glutathione (GSH) loss precedes vacuole formation in the diabetic rat lens, but the cause of this loss is not known. Cysteine availability is a rate limiting factor to glutathione biosynthesis in rat and human lenses but its concentration is not known; therefore free cysteine was measured prior to lens hydration in the diabetic rat lens. GSH can regenerate ascorbate from dehydroascorbate within the lens and potentially modulate the ascorbate pool; therefore ascorbate loss is also a possibility that has not been examined previously. METHODS: Diabetes was induced in Wistar rats to provide a slowly progressing model of cortical cataract. Age-matched control rats were injected with buffer vehicle only. Lens condition was monitored by binocular slit-lamp microscope after pupil dilation. Lens cysteine and glutathione were measured in the same lens, while ascorbate and total ascorbate (ascorbate + dehydroascorbate) of the contralateral lens were quantified by high performance liquid chromatography electrochemical detection. The 1- and 2-week periods of diabetes were chosen as they both preceded lens hydration changes and Na+/K+ changes, to avoid leakage due to ruptured cell membranes. RESULTS: Lens weights were not significantly different compared to controls at either the 1- or 2-week periods, and lenses were completely free of initial vacuole formation. Lens GSH concentration was diminished by 72% compared with controls after 1 week of diabetes and 74% after 2 weeks of diabetes. Lens free cysteine was decreased by 62% and 78% compared with controls after 1 and 2 weeks of diabetes, respectively. Total lens ascorbate concentration was decreased by 34% after 1 week of diabetes and 48% after 2 weeks of diabetes. Dehydroascorbate levels represented less than 10% of the total lens ascorbate pool in all experimental groups. GSH and ascorbate concentration were correlated after 1 week of diabetes (p < 0.005) and after 2 weeks of diabetes (p < 0.001). GSH and cysteine concentration were also correlated after 1 week of diabetes (p < 0.001) and after 2 weeks of diabetes (p < 0.001). CONCLUSIONS: Decreased free cysteine, in the diabetic rat lens, precedes hydration changes and vacuole formation, contributing to decreased glutathione content. While cysteine was not abundant in the lens, its concentration is greater than previously supposed. The lens ascorbate pool was also diminished prior to lens hydration.

Transient loss of alphaB-crystallin: an early cellular response to mechanical stretch.

Human trabecular meshwork (HTM) is distended and stretched with increases in intraocular pressure. During this stretching, there is a rearrangement of actin filaments. The HTM cells express alpha B-crystallin, a small heat shock protein that may have a role in the stabilization and regulation of the cytoskeleton in mammalian cells. The levels of alpha B-crystallin were examined in trabecular meshwork cells after mechanical stretch. Human TM primary cell cultures, plated onto silicone sheets, were subjected to a single 10% linear stretch and samples were prepared at various times after stretch for immunoblotting or Northern blotting. Immunoblots of total protein extracts with antibody specific for alpha B-crystallin detected a 26% decrease of cellular alpha B-crystallin levels within 2 minutes. After 1 hour alpha B-crystallin levels had decreased 90% compared to control cells. The levels of alpha B-crystallin began to recover in cells stretched for 2 hours and returned to initial levels by 24 hours. Northern blots probed with alpha B-crystallin exon III cDNA detected a transcript of 0.65 kb in human TM cells and the levels of the alpha B mRNA remained constant during alpha B-crystallin protein decrease. Later, levels of the 0.65 kb transcript of alpha B-crystallin increased during the cellular recovery. These results suggest that decreased levels of alpha B-crystallin after mechanical stretch were probably not due to transcriptional changes but rather to increased degradation of alpha B-crystallin protein. An increase in mRNA levels may play a role in the recovery of alpha B-crystallin during reorganization of the cytoskeleton and attachment to the substratum. These data raise the possibility of a specific proteolysis of alpha B-crystallin protein in cells after a physiological challenge.

Cysteine protease activated by expression of HIV-1 protease in transgenic mice. MIP26 (aquaporin-0) cleavage and cataract formation in vivo and ex vivo.

Transgenic mice, homozygous for HIV-1 protease expression in the eye lens, display degradation of some lens crystallins and cytoskeletal proteins prior to cataract formation on postnatal days 23-25. Alterations to the internal lens hydration state also occur; therefore, the status of the aquaporin protein MIP26 was examined over postnatal days 16-25 to determine if it was altered during cataractogenesis. The MIP was identical in transgenic and control lenses until day 21. By postnatal day 25 (frank cataract), in the lenses obtained from transgenic animals, the 26-kDa band was absent and there was a concurrent increase in the proportion of MIP23. Immunoblotting demonstrated cleavage at the C terminus. Lenses were also maintained in an organ culture system to demonstrate that the cataractogenic process is inherent to the isolated lens and to determine the contribution of cysteine protease action. Organ culture experiments revealed a similar progression to nuclear cataract formation as seen in vivo. Two-dimensional gel analysis of the soluble lens crystallin fraction of organ cultured lenses revealed the same cleavage pattern as occurs in vivo. Organ culture of transgenic lenses with E64, a cysteine protease inhibitor, dramatically delayed cataractogenesis and prevented proteolytic cleavage of both MIP26 and crystallins. HIV-1 protease, while the trigger of cataract formation, does not appear to be the protease responsible for cleavage of MIP or lens crystallins. These results suggest that activation of endogenous cysteine protease activity is involved in the cleavage of these proteins and occurs downstream of HIV-1 protease action.

Precataractous changes affect lens transparency in the selenite cataract.

Selenite treatment of the preweanling rat stabilized the transparency of the lens nucleus to decreasing temperature. Hence, we compared properties of the cortex and nucleus from lenses of selenite-treated and age-matched control rats. A subcutaneous dose of 30 nmol Na2SeO3/g body weight was administered to 10- to 13-day-old Sprague-Dawley rats. Uninjected, age-matched littermates served as controls. As required, lenses were frozen in liquid N2 and separated into nuclear and cortical-epithelial fractions. Transparency of solutions of lens proteins (90-100 mg per ml) was monitored from 30 to 2 degrees C as percent transmittance (%T) at 490 nm. The critical phase separation temperature, Tc, was the temperature at 80%T. Protein associations were monitored with gel filtration chromatography. The nuclear 'cold cataract', in intact lenses, formed at similar temperatures at 14 and 15 days of age, but at a significantly lower temperature when the lenses were from a selenite-treated rat. The Tc, however, was greater by 1.5-2 degrees C for solutions of proteins isolated from whole lenses or lens nuclei from rats 24 and 48 h after treatment with selenite. Further, less gamma-crystallin was associated with the alpha-crystallin fraction in extracts from the nucleus of lenses from treated rats. Altered phase separation properties occurred as an early event in the etiology of selenite cataract. The different in vivo and in vitro responses to temperature indicated that properties of lens crystallins do not solely establish transparency in the intact lens.

Causes of decreased phase transition temperature in selenite cataract model.

PURPOSE: Lenses from selenite-treated animals develop reversible "cold cataract" at a lower temperature than is required for lenses from age-matched control animals. This unexplained, stabilized phase transition is readily observed in intact lenses 36 to 48 hours after treatment and occurs in lenses before the appearance of the irreversible nuclear opacity observed 72 to 96 hours after treatment. The objective of this study was to investigate factors that may be responsible for this difference. METHODS: Preweanling rats were injected with sodium selenite. Lens extracellular water volume was measured using 14C-inulin. Free amino acids were analyzed using precolumn derivatization and high performance liquid chromatography. Soluble protein was isolated from lenses of control, and treated animals and temperature-dependent changes in light scattering were measured at 490 nm. RESULTS: Lens extracellular water was increased by the selenite treatment, with a concurrent 10% decrease in intracellular volume. Solutions of soluble protein from lenses of selenite-treated animals after postinjection hours 24 and 48 had higher critical phase transition temperatures (Tc) compared to similar proteins from control lenses. From 24 to 72 hours after injection, the free amino acid content of the lens increased 42%. Taurine levels were unchanged over the same period. The addition of 7 mM glycine and 7 mM proline to solutions of soluble protein (96 mg ml-1) decreased the phase transition temperature. Taurine (14 mM) had a similar effect. Combining taurine and the glycine plus proline solutions had an additive effect in lowering the Tc. CONCLUSIONS: Increases in free amino acid concentration occur in lenses in response to the stress imposed by a systemic dose of selenite. The altered polyion content in lenses from selenite-treated animals, before nuclear cataract formation, contributes to the greater thermal stability of transparency in these lenses, thus lowering the temperature at which "cold cataract" forms.

High-performance liquid chromatography-electrochemical detection of antioxidants in vertebrate lens: glutathione, tocopherol, and ascorbate.

The HPLC-EC method has good specificity for the analysis of glutathione, tocopherol, and ascorbate. The same HPLC system can be used for all three analysis with changes of mobile phase and the electrode cell to match the procedure required. The same C18 reversed-phase column has been used with a refillable guard column for 3 years with no noticeable loss of resolving power. The main advantage of the glutathione procedure was the ability to monitor both GSH and GSSG, which allowed us to confirm that loss of GSH in the diabetic rat lens does not result in the appearance of GSSG. The main benefit of the tocopherol procedure was the ability to measure the tocopherol content of a single rat lens. Our previous experience with UV or fluorescence detection showed those methods to be not sensitive enough for a single lens determination. The mammalian lens has the lowest tocopherol content of the tissues of the eye, 10 to 40 times less than most body tissues as measured by gas chromatography-mass spectrometry (GC-MS). The better sensitivity of electrochemical detection has allowed for a single lens determination, keeping the number of experimental animals to a minimum. An advantage of the ASC analysis procedure was the extra specificity imparted by both the chromatography and the detector as well as the ability to estimate the total ascorbate (ASC plus DHAA) and DHAA content. Other reducing agents such as GSH and uric acid can interfere in colorimetric methods that rely on the reducing action of ASC. The very high GSH content of the mammalian lens was a concern when choosing a procedure. GSH levels exceeding 10 times the level of lens samples were found to yield no response using the HPLC-EC procedure for ASC. The only disadvantage with electrochemical detection was that the electrode response could drift with time, requiring more frequent calibration with standards. We continue to utilize these methods to examine the prevacuole loss of ASC and GSH in the diabetic rat lens model of cataract.

Topical application and uptake of vitamin E acetate by the skin and conversion to free vitamin E.

Radioactive tocopherol acetate was diluted with either (1) unlabelled tocopherol acetate or (2) Delios S (Henkel, a medium chain triglyceride prepared from fractionated coconut oil), a cosmetic base. These preparations were applied topically to a 2 cm diameter circle of skin. After 24 hours the percent of label which was still removable by swabbing the skin surface was 1.7% for (1) and 11.5% for (2). The central circles contained 2.86% of the label applied in 259 mg skin for (1) and 24.2% of the label applied in 226 mg skin for Delios S for (2). Surprisingly, combined samples of approximately one third of the side skin contained 0.7% of the label applied in 460 mg for (1) and 13.2% of the applied label in one third of the side skin in 523 mg for (2). The percent conversion to tocopherol in the skin central areas was 4.52% by HPLC and 4.13% by TLC for (1) and 5.97% for (2). In the side skin the percent conversion to tocopherol was 5.0% for (1) and 6.01% for (2).

Reduction of sunburn damage to skin by topical application of vitamin E acetate following exposure to ultraviolet B radiation: effect of delaying application or of reducing concentration of vitamin E acetate applied.

The skin of the skh-1 mouse after ultraviolet B (280-320 nm, UVB) irradiation shows the pathological changes typical of sunburn damage: spongiosis (edematous spaces) around some cells, necrosis of keratinocytes, giving rise to sunburn cells, inflammatory infiltration of polymorphonuclear leucocytes, etc. In our previous study, these were accompanied by erythema, increased skin sensitivity, and edematous swelling. The topical application of tocopherol acetate (TA) immediately after the UVB exposure decreased these changes. In this paper, multiple measurements of the skin thickness were made at different locations along the magnetic resonance imaging (MRI) cross-sectional image of the skin. This permits effects to be quantified with (if desired) the contralateral half of the back serving as an internal control, either exposed (positive control) or unexposed (negative control). Topical application of TA resulted in an increase in the concentration of free tocopherol in the skin. No qualitative differences in ultrastructural appearance of the UVB-irradiated, TA-treated skin could be discerned by careful examination. In vivo high resolution video microscopy of blood flow in venules of the irradiated mouse ear revealed a large (tenfold) but not statistically significant decrease in stationary lymphocytes adhering to the venule walls. The delaying of the application of TA up to 8 hours after the termination of UVB irradiation still offered statistically significant protection as did immediate application of 5% TA in diluent Myritol 318 (Delios S, Henkel).

Modelling cortical cataractogenesis. 13. Early effects on lens ATP/ADP and glutathione in the streptozotocin rat model of the diabetic cataract.

A possible contribution to cell toxicity in the diabetic lens due to early ATP loss is not well characterized prior to the appearance of vacuoles in the lens. Changes in lens ATP levels at longer periods of hyperglycaemia (6-8 weeks) have been reported. We used [31P]NMR analysis of lens extracts at three time periods, comparing diabetic to concurrent control groups at 1, 2 and 4 weeks of hyperglycaemia. With this design, significant alterations (> 10%) in the ATP/ADP ratio can be monitored. NMR analysis revealed a decreased ATP/ADP ratio at all time periods, averaging a 38% decrease. Luminescent determination of ATP levels indicates that this decrease is mainly caused by a decrease of 25% in ATP concentration. The early loss of GSH was large and not accompanied by an appearance of GSSG, as monitored by HPLC electrochemical detection. A 1-week experiment with animals receiving daily insulin treatment was carried out to control for effects of STZ on the lens. This treatment resulted in normal lens GSH levels and a near normal [31P]NMR profile.

Acetate assimilation by the fission yeast, Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe utilizes acetate at subinhibitory concentrations in the presence of D-glucose. The nonionized form of acetate is preferentially utilized, oxidized to 14CO2, and assimilated into lipids and proteins. Acetyl CoA synthetase activity greatly increases in the yeast cells grown in media containing acetate. However, glyoxylate cycle enzymes are not detectable in Schizosaccharomyces pombe. [1-14C]Acetate is incorporated into stereols, sterol esters, neutral lipids, and phospholipids. Assimilation of [1-14C]acetate into the peptide structure of proteins was confirmed by a proteolytic digestion experiment.

Modelling cortical cataractogenesis. X. Evaluation of lens optical function by computer based image analysis using in vitro rat lens elevated glucose model.

As a model system rat lenses (Wistar) were isolated and cultured intact in medium M199 or M199 with 55 mM glucose. Glucose-stressed lenses developed opacities within 20 hours while control lenses remained clear. The comparative functionality of lenses was examined by two different video/computer-based imaging systems: one based on image analyses of laser light after transmission through the lens (Scanning Lens Monitor), and the other based on image analyses of the projection of white light transmitted through the lens (Kevex Image Analysis System). The combination of both methods detect changes in focal length and light scattering. On the basis of these results, these techniques can be used in animal studies for grading lenses by functional properties to complement morphologically based grading (slit-lamp) of cataracts provided by a veterinary pathologist.