Alterations in human vitreous humour following cataract extraction.

Cataract extraction is associated with the risk of posterior vitreous detachment, macular edema and retinal detachment possibly as a result of a disturbance to the vitreous body during surgery. While it is common for lens cortical fiber debris to leak into the vitreous humour during cataract extraction, the extent to which the vitreous humour is altered post-surgery is unknown. The current study examines the integrity of the vitreous humour of pseudophakic and phakic human donor eyes by comparing the proteome, the viscosity and the size distribution of macromolecules in different regions of the vitreous humour from human pseudophakic and phakic donor eyes. Major differences between the proteomes of anterior and posterior vitreous humour were observed in phakic and pseudophakic donor eyes. Seventeen spots identified as complete, modified or cleaved forms of alphaA-, alphaB-, betaA4-, betaB2, and gammaS-crystallins were present in the anterior vitreous humour of all pseudophakic eyes studied. Crystallins were not detected in the posterior vitreous humour of the pseudophakic eye or the vitreous humour of the phakic eye. Significant alterations in abundance and/or modification of transthyretin, alpha antitrypsin, and retinoic acid binding protein were observed in all locations of pseudophakic vitreous humour as compared to phakic samples. In addition, a significant decrease in the number and intensity of protein spots was observed for the posterior vitreous humour of pseudophakic eyes when compared to posterior vitreous humour of phakic eyes. Proteins which were affected include antioxidant proteins and enzymes such as carbonic anhydrase and trisephosphate isomerase. A reversal of the viscosity gradient, anterior to posterior, in the vitreous humour of pseudophakic eyes was observed concomitant with alterations in the distribution of 50 nm particles. These particles are likely primarily composed of hyaluronan. While varying degrees of vitreous degradation may have existed prior to surgery and may have contributed to the cataract formation, in no case did the phakic donor eyes exhibit the same alterations in the vitreous humour proteome, viscosity or particle sizes as did the pseudophakic donor eyes. The examination of phakic/pseudophakic donor eye pairs confirmed that the vitreous humour proteome and structural integrity were very similar in the matched phakic donor eye to eyes from donors with no history of cataract. Even though the number of samples for this study was limited, the observed changes support the hypothesis that alterations in the vitreous humour proteome occur in psuedophakic eyes with concurrent alterations in the structure of the vitreous humor. These modifications of the microenvironment of the retina may contribute to the development of retinal complications following cataract surgery.

AlphaB-crystallin in lens development and muscle integrity: a gene knockout approach.

PURPOSE: To study the role of alphaB-crystallin (alphaB) in the developing lens and its importance in lens structure and function. METHODS: Gene targeting in embryonic stem cells was used to generate mouse lines in which the alphaB gene and its protein product were absent. Gene structure and expression were characterized by genomic Southern blot, immunoblot, and Northern blot analyses, and two-dimensional gel electrophoresis. The gene knockout mice were screened for cataract with slit lamp biomicroscopy, and dissected lenses were examined with dark-field microscopy. Lenses and other tissues were analyzed by standard histology and immunohistochemistry. Chaperone activity was determined by heating lens homogenate supernatants and measuring absorbance changes. RESULTS: In an unexpected result, lenses in the alphaB gene knockout mice developed normally and were remarkably similar to wild-type mouse lenses. All the other crystallins were present. The thermal stability of a lens homogenate supernatant was mildly compromised, and when oxidatively stressed in vivo with hyperbaric oxygen, the knockout lenses reacted similarly to wild type. In targeting the alphaB gene, the adjacent HSPB2 gene, which is not expressed in the lens, was also disrupted. Loss of alphaB and/or HSPB2 function leads to degeneration of some skeletal muscles. CONCLUSIONS: AlphaB is not essential for normal development of a transparent lens in the mouse, and therefore is more dispensable to the lens than the closely related alphaA-crystallin. It may play a small role in maintaining transparency throughout life. alphaB and/or the closely related HSPB2 is required to maintain muscle cell integrity in some skeletal muscles.

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.

Tracking pathology with proteomics: identification of in vivo degradation products of alphaB-crystallin.

Soemmerring's ring is one form of "after cataract" that can occur following cataract surgery. The ring structure is formed by adherence of the anterior lens capsule to the posterior lens capsule. Epithelial cells remaining after surgery differentiate into lens fiber cells but the resulting tissue mass does not remain transparent. The protein in normal lens and in Soemmerring's rings from four individuals was analyzed using two-dimensional (2-D) gel electrophoresis, matrix assisted laser desorption/ionization-time of-flight-mass spectrometry (MALDI-TOF-MS) and image analysis with Phoretix software. The 2-D protein patterns of the Soemmerring's rings were generally similar to that of cortical fiber cells of normal human lens with some notable exceptions. Several post-translationally modified forms of alphaB-crystallin(1-175) were identified. Two degradation products, alphaB-crystallin(1-170) and alphaB-crystallin(1-174), each make up 9.5-27% of the total alphaB-crystallin in the Soemmerring's rings and less than 1% in the normal lenses. Other modified forms of alphaB-crystallin are aberrant in the fiber cells of the Soemmerring rings relative to normal lens.

Identification of lutein and zeaxanthin oxidation products in human and monkey retinas.

PURPOSE: To characterize fully all the major and minor carotenoids and their metabolites in human retina and probe for the presence of the oxidative metabolites of lutein and zeaxanthin. METHODS: Carotenoids of a composite of 58 pairs of human retinas and a monkey retina were elucidated by comparing their high-performance liquid chromatography (HPLC)-ultraviolet/visible absorption spectrophotometry (UV/Vis)-mass spectrometry (MS) profile with those of authentic standards prepared by organic synthesis. RESULTS: In addition to lutein and zeaxanthin, several oxidation products of these compounds were present in the extracts from human retina. A major carotenoid resulting from direct oxidation of lutein was identified as 3-hydroxy-beta, epsilon-caroten-3'-one. Minor carotenoids were identified as: 3'-epilutein, epsilon,epsilon-carotene-3,3'-diol, epsilon,epsilon-carotene-3,3'-dione, 3'-hydroxy-epsilon,epsilon-caroten-3-one, and 2,6-cyclolycopene-1,5-diol. Several of the geometric isomers of lutein and zeaxanthin were also detected at low concentrations. These were as follows: 9-cis-lutein, 9'-cislutein, 13-cis-lutein, 13'-cis-lutein, 9-cis-zeaxanthin, and 13-cis-zeaxanthin. Similar results were also obtained from HPLC analysis of a freshly dissected monkey retina. CONCLUSIONS: Lutein, zeaxanthin, 3'-epilutein, and 3-hydroxy-beta,epsilon-caroten-3'-one in human retina may be interconverted through a series of oxidation-reduction reactions similar to our earlier proposed metabolic transformation of these compounds in humans. The presence of the direct oxidation product of lutein and 3'-epilutein (metabolite of lutein and zeaxanthin) in human retina suggests that lutein and zeaxanthin may act as antioxidants to protect the macula against short-wavelength visible light. The proposed oxidative-reductive pathways for lutein and zeaxanthin in human retina, may therefore play an important role in prevention of age-related macular degeneration and cataracts.

Hierarchy of lens proteins requiring protection against heat-induced precipitation by the alpha crystallin chaperone.

Gel filtration of the water-soluble extract from bovine lens yields a group of proteins, emerging between the peaks of beta H and beta L crystallins, which show a considerably greater sensitivity to heat-induced aggregation/precipitation than the far more abundant beta and gamma crystallins. However, the small heat shock protein: alpha crystallin was effective in protecting these trace constituents of the lens from precipitating out of solution at 55 degrees C (measured under the standard conditions in a pH 7.5 buffer containing 50 mM sodium phosphate, 100 mM NaCl, 1 mM EDTA and 0.05% NaN3). Prominent components of the precipitate, formed in the absence of a recombinant alpha B crystallin chaperone could be resolved by one- and two-dimensional electrophoresis. Identification by amino acid sequencing revealed that the heat-sensitive group of lens proteins comprised glyceraldehyde-3-phosphate dehydrogenase (M(r) approximately 39 kDa), enolase (approximately 48 kDa), leucine aminopeptidase (approximately 52 kDa) and aldehyde dehydrogenase (approximately 53 kDa). These findings indicate for the first time that the aggregation of such minor lens constituents could possibly contribute to initiating the process of opacification in the development of cataracts.

The nucleus of the human lens: demonstration of a highly characteristic protein pattern by two-dimensional electrophoresis and introduction of a new method of lens dissection.

A practical method for dissection of human lenses is described. The method utilizes the suture patterns as a guide to identify the developmental stage in which fiber cells were formed. Lenses were separated into cortex and adult, infantile, fetal and embryonic nuclear regions. Analysis of the proteins in each of these regions in adult lenses shows that the lens nucleus has a highly characteristic two-dimensional protein pattern distinct from that of the cortex. Each of the nuclear regions has essentially the same protein pattern. The data suggest that the conversion of cortical fibers to mature nuclear fibers involves well controlled processes.

Glyceraldehyde 3-phosphate dehydrogenase is an enzyme-crystallin in diurnal geckos of the genus Phelsuma.

Lenses from diurnal geckos, Phelsuma barbouri, Phelsuma madagascariensis grandis and Phelsuma serraticauda contain a prominent 37 kDa polypeptide (pi-crystallin) that is not present in lenses from the nocturnal geckos, Gekko gekko, Hemidactylus garnoti, Tarentola annularis, and Uroplatus henkeli. This protein was partially purified from P. serraticauda and was identified as glyceraldehyde 3-phosphate dehydrogenase (GAPD). The GAPDs, which constitute 14-24% of the lens soluble protein in the diurnal species, were highly active. The presence of this enzyme in the lenses of diurnal, but not nocturnal, geckos supports the hypothesis that the oxido-reductases found as enzyme-crystallins may function in providing protection against the increased oxidative stress to which diurnal species are exposed. These findings strongly support the concept that the recruitment of enzyme-crystallins is a selective response to changes in the visual environment.

Ascorbic acid and the eye.

In this report literature on transport and function of ascorbic acid in ocular tissues is reviewed. The role of ascorbic acid in various regions of the eye is not well understood. It appears one important function of this compound is protection against oxidative damage, particularly photoinduced damage. In contrast, data are also reviewed that suggest ascorbic acid may participate in the oxidative modification of lens proteins seen with aging.