Phase separation of a protein-water mixture in cold cataract in the young rat lens.

A remarkable resemblance between the appearance of opacity in lysozyme--salt water mixtures and the development of opacity in cold cataract in the young rat lens is strong evidence that cold cataract is fundamentally a phase separation of the "protein-water binary mixture" in the lens.

Nonenzymatic glycation of human lens crystallin. Effect of aging and diabetes mellitus.

We have examined the nonenzymatic glycation of human lens crystallin, an extremely long-lived protein, from 16 normal human ocular lenses 0.2-99 yr of age, and from 11 diabetic lenses 52-82-yr-old. The glucitol-lysine (Glc-Lys) content of soluble and insoluble crystallin was determined after reduction with H-borohydride followed by acid hydrolysis, boronic acid affinity chromatography, and high pressure cation exchange chromatography. Normal lens crystallin, soluble and insoluble, had 0.028 +/- 0.011 nanomoles Glc-Lys per nanomole crystallin monomer. Soluble and insoluble crystallins had equivalent levels of glycation. The content of Glc-Lys in normal lens crystallin increased with age in a linear fashion. Thus, the nonenzymatic glycation of nondiabetic lens crystallin may be regarded as a biological clock. The diabetic lens crystallin samples (n = 11) had a higher content of Glc-Lys (0.070 +/- 0.034 nmol/nmol monomer). Over an age range comparable to that of the control samples, the diabetic crystallin samples contained about twice as much Glc-Lys. The Glc-Lys content of the diabetic lens crystallin samples did not increase with lens age.

Spectroscopic studies on human lens crystallins.

Human lens crystallins were studied by absorption, circular dichroism and fluorescence spectroscopy. The absorption spectra in the near-ultraviolet region show some differences in intensity, but spectral features are similar, except for the alpha-crystallin, which gives a fine structure due to phenylalanine between 250 and 270 nm. Tryptophan fluorescence and near-ultraviolet circular dichroism indicate that tryptophan residues are more exposed in alpha-crystallin than in either beta- or gamma-crystallin, and that the degree of exposure decreases in the order of alpha less than beta 1 greater than beta 2 greater than beta 3 greater than gamma. The far ultraviolet CD suggests that these proteins exist mainly in a beta-sheet conformation and that the amount does not vary much among them. The greater exposure of the tryptophan residues in the high-molecular-weight crystallins may reflect greater unfolding in their protein domains. Spectroscopic measurements are thus useful in predicting protein tertiary structure in the absence of the complete sequence and X-ray data. The fact that the high-molecular-weight proteins exist in a more unfolded state may render them more vulnerable to exogeneous insults, and these effects may be studied by spectroscopic measurements.

The effect of oxidation on sorbitol pathway kinetics.

The rapid conversion of glucose to sorbitol by aldose reductase and the consequent hyperosmolarity of the cytoplasm has been shown to be the primary cause of the so-called "sugar" or "osmotic" cataract in many animal lenses. It is not as clear, however, that hyperosmolarity is the principal factor in the etiology of cataracts in human diabetic subjects. In fact, the comparatively low activity of aldose reductase in the human lens as compared with several animal lenses, and the osmotically insignificant levels of sorbitol pathway products (sorbitol and fructose), suggest that hyperosmolarity, per se, may not be as important a factor in human cataract formation as it is in animals. We present evidence that the flux of glucose and sorbitol through the rat lens is markedly reduced by oxidative stress (0.1 mM H2O2). Sorbitol accumulation is reduced by 114%, sorbitol turnover is reduced by 78%, sorbitol production is reduced by 90%, fructose accumulation is reduced by 60%, and fructose turnover is reduced by 76% in the presence of 36 mM glucose. H2O2 does not affect glucose turnover, the glucose rate constant, or the ATP level significantly at 36 mM glucose, but at 5.5 mM glucose, 0.2 mM H2O2 leads to a rapid loss of ATP that can be prevented by 0.04 mM sorbinil, an aldose reductase inhibitor. These results suggest that inhibition of aldose reductase by sorbinil renders rat lenses better able to cope with oxidative stress. In the absence of an aldose reductase inhibitor, elevating ambient glucose may render a lens less able to scavenge oxidants by diverting NADPH into sorbitol production.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct measurement of polyol pathway activity in the ocular lens.

A method to measure the polyol pathway metabolic flux in the intact rabbit lens by 13C nuclear magnetic resonance spectroscopy is described. In the lens exposed to 35.5 mM glucose, the polyol pathway accounts for 1/3 of the total glucose turnover. The high metabolic activity of the pathway suggests a significant alteration in the reduced to oxidized pyridine nucleotide ratio in the lens exposed to high glucose.

Transactivation of involucrin, a marker of differentiation in keratinocytes, by lens epithelium-derived growth factor (LEDGF).

Human involucrin (hINV), first appears in the cytosol of keratinocytes and ultimately cross-linked to membrane proteins via transglutaminase and forms a protective barrier as an insoluble envelope beneath the plasma membrane. Although the function and evolution of involucrin is known, the regulation of its gene expression is not well understood. An analysis of the hINV gene sequence, upstream of the transcription start site (-534 to +1 nt) revealed the presence of potential sites for binding of lens epithelium-derived growth factor (LEDGF); stress response element (STRE; A/TGGGGA/T) and heat shock element (HSE; nGAAn). We reported earlier that LEDGF activates stress-associated genes by binding to these elements and elevates cellular resistance to various stresses. Here, gel-shift and super-shift assays confirm the binding of LEDGF to the DNA fragments containing HSEs and STREs that are present in the involucrin gene promoter. Furthermore, hINV promoter linked to CAT reporter gene, cotransfected in human corneal simian virus 40-transformed keratinocytes (HCK), was transactivated by LEDGF significantly. In contrast, the activity of hINV promoter bearing mutations at the WT1 (containing HSE and STRE), WT2 (containing STRE) and WT3 (containing STRE) binding sites was diminished. In addition, in HCK cell over-expressing LEDGF, the levels of hINV mRNA and hINV protein are increased by four to five-fold. LEDGF is inducible to oxidants. Cells treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA), known to stimulate production of H(2)O(2), showed higher levels of LEDGF mRNA. Furthermore, our immunohistochemical studies revealed that hINV protein is found in the cytoplasm of HCK cells over-expressing LEDGF, but not detectable in the normal HCK cells or HCK cells transfected with vector. This regulation appears to be physiologically important, as over-expression of HCK with LEDGF increases the expression of the endogenous hINV gene and may provide new insight to understand the molecular mechanism of transcriptional regulation of this gene. LEDGF may play an important role in establishing an important barrier in corneal keratinocytes by maintaining epidermal turn-over rate, and protecting HCKs against stress.

Effects of iron ions, protons and X rays on human lens cell differentiation.

We have investigated molecular changes in cultured differentiating human lens epithelial cells exposed to high-energy accelerated iron-ion beams as well as to protons and X rays. In this paper, we present results on the effects of radiation on gene families that include or are related to DNA damage, cell cycle regulators, cell adhesion molecules, and cell cytoskeletal function. A limited microarray survey with a panel of cell cycle-regulated genes illustrates that irradiation with protons altered the gene expression pattern of human lens epithelial cells. A focus of our work is CDKN1A (p21(CIP1/WAF1)), a protein that we demonstrate here has a role in several pathways functionally related to LET-responsive radiation damage. We quantitatively assessed RNA and protein expression in a time course before and after single 4-Gy radiation doses and demonstrated that transcription and translation of CDKN1A are both temporally regulated after exposure. Furthermore, we show qualitative differences in the distribution of CDKN1A immunofluorescence signals after exposure to X rays, protons or iron ions, suggesting that LET effects likely play a role in the misregulation of gene function in these cells. A model of molecular and cellular events is proposed to account for precataractous changes in the human lens after exposure to low- or high-LET radiations.

Should tonometry screening be done by technicians instead of physicians?

A random sample of 768 clinic patients 40 years or older were screened by a technician utilizing Schlotz tonometry. All patients with an intraocular pressure of 20 mm Hg or more were referred for ophthalmological evaluation. The prevalence of frank glaucoma and suspected glaucoma in this infirm population was 1.8% and 2.5%, respectively. The cost of detecting frank or suspected glaucoma decreased in the second half of the study due to a decrease in the number of false-positive tests. These findings suggest that all clinic patients 40 years or older should be screened for glaucoma. Because many internists are reluctant to screen for glaucoma and because there are large number of false-positive tests by those who do tonometry episodically, we suggest that technicians do glaucoma screening.

Dermatological and ocular examinations in rabbits chronically photosensitized with methoxsalen.

Four groups of female Dutch-belted rabbits (Oryctulagus cuniculus) were given methoxsalen (12 mg/kg) or placebo by oral intubation and 1 hr later were exposed to UVA for either 2 or 8 hr. This procedure was repeated 5 days each week for 18 mo. A fifth group received no drug and no UVA exposure. The skin of the animals given methoxsalen and UVA showed signs of acute and chronic phototoxicity. Multiple peripheral blood parameters of hepatic, renal and hematologic function were normal and were not different between groups. Complete ophthalmoscopic examinations were performed periodically. No cataracts were seen in any of the animals. This data provides the perspective that in one species the daily dose of methoxsalen and UVA required to induce chronic cutaneous photosensitization is lower than the daily dose required to induce cataracts. It is inadvisable to interpret this data as suggesting that no risk exists for patients being treated with oral methoxsalen photochemotherapy. The experimental evidence supporting photosensitization as a cause of cataracts and implicating a role of lens DNA in this cataractogenesis is reviewed. Because methoxsalen-UVA alterations of lens DNA or protein could lead to delayed onset of cataracts, and because of the serious nature and potential preventability of phototoxic lens opacification, appropriate protective eye wear is recommended for all patients receiving oral psoralen photochemotherapy.

Lens epithelium-derived growth factor (LEDGF/p75) and p52 are derived from a single gene by alternative splicing.

A human gene that encodes lens epithelium-derived growth factor (LEDGF) was isolated, and the DNA sequence and the exon/intron organization was determined. The gene contains at least 15 exons and 14 introns and encodes LEDGF mRNA and p52 mRNA. Exons 1-15 encode LEDGF mRNA, and exons 1-9, and a part of the ninth intron encode a splice variant (p52). Sequences of the exon/intron junctions of the gene have the highly conserved GT/AG rule. Most intron/exon junctions correspond to junctions of individual protein motifs. Almost equal amounts of LEDGF and p52 are expressed in lens epithelial cells in culture. The LEDGF gene is assigned to chromosome 9p22.2, which is adjacent to the major cell malignancy locus.

Epidemiologic evidence of a role for the antioxidant vitamins and carotenoids in cataract prevention.

The relationship between antioxidant nutrient status and senile cataract was examined in 77 subjects with cataracts and 35 control subjects with clear lenses. Subjects with low (below the 20th percentile) and moderate (20th-80th percentiles) plasma nutrient and nutrient intake levels of vitamin C, vitamin E, and carotenoids were compared with subjects with high levels (above the 80th percentile). The odds ratio (OR) of cortical (CX) cataract among subjects with low plasma carotenoid levels was 7.2 (P less than 0.05) and the OR of posterior subcapsular (PSC) cataract for persons with low plasma vitamin C was 11.3 (P less than 0.10). Low vitamin C intake was associated with an increased risk of CX (OR = 3.7, P less than 0.10) and PSC (OR = 11.0, P less than 0.05) cataract. Subjects who consumed fewer than 3.5 servings of fruit or vegetables per day had an increased risk of both CX (OR = 5.0, P less than 0.05) and PSC cataract (OR = 12.9, P less than 0.01).

Nutritional status in persons with and without senile cataract: blood vitamin and mineral levels.

As part of an exploratory study of nutrition and senile cataract relationships between biochemical markers of nutritional status and senile cataract were examined in 112 subjects aged 40-70 y. Seventy-seven subjects had a cataract in at least one lens. Blood levels were determined for total carotenoids, vitamin A, vitamin D, vitamin E, vitamin C, riboflavin, thiamin, vitamin B-6, zinc, copper, selenium, and magnesium. Subjects were grouped into quintiles for each nutrient. Logistic regression was used to estimate the odds ratios (ORs) for cataract among subjects in the highest quintile and the middle three quintiles relative to subjects in the lowest quintile. ORs were adjusted for age, sex, race, and presence of diabetes. Results suggest that risk of cortical cataract was reduced for subjects in the highest quintile of vitamin D and total carotenoids and that persons with cataract may have lower levels of vitamin C and higher levels of vitamin B-6 and Se.

Spectroscopic study on the effects of nonenzymatic glycation in human alpha-crystallin.

Nonenzymatic glycated lens alpha-crystallin, isolated from human diabetic cataract lenses, does not differ in subunit size and secondary structure from nonglycated alpha-crystallin. The tertiary structure, however, has undergone a significant change as reflected in the changes of near-ultraviolet (UV) circular dichroism (CD) and fluorescence of intrinsic probes (tryptophan, nontryptophan) and extrinsic probes [(MIANS (6-(4'-maleimidylanilino)naphthalene-2-sulfonic acid) and TNS (6-(p-toluidinyl)naphthalene-2-sulfonate)]. A decrease in tryptophan fluorescence and an increase in nontryptophan fluorescence were observed for glycated alpha-crystallin. Decreased intensities of SH-specific fluorescent probes (MIANS) and hydrophobic probe (TNS) also were observed. The near UV CD and TNS polarization results suggest a more unfolded structure of the glycated crystallin. The significant decrease of sulfhydryl groups in glycated protein suggests that glycation facilitates a further secondary change.

Thiol oxidation in the crystalline lens. I. The rate-limiting role of hexokinase in aging rat and human lenses.

Reduction of nonprotein disulfides required both glucose or glucose-6-phosphate (G6P) and nicotinamide adenine dinucleotide phosphate (NADP). However, hexokinase (HK) was found to be the rate-limiting step: the glucose-supported reduction rate was only 50% of that of G6P-supported activity. This disulfide-reducing activity seemed to decline in the aging lens. Further, the glucose-supported activity dropped substantially if HK was deactivated with diamide; the deactivation was partially reversible. HK activity in aging clear and cataractous human lenses had greatly diminished. This might explain the disproportionate decrease in glucose-supported reduction in the aging lens. The components of the disulfide-reducing mechanism in human lens were reviewed and discussed.

Inexpensive stereoscopic CCRG camera for lens/cataract photography in vitro.

In 1978 the American Cooperative Cataract Research Group (CCRG) adopted a stereoscopic camera system for photographing human cataracts in vitro based upon a Zeiss OPMI 1 operating microscope (Carl Zeiss, Inc.; Oberkochen, West Germany). Photographs obtained with this system were used to classify human cataractous change according to the standardized CCRG protocol. Classification data correlated with laboratory data furthered the attempt to define the biochemical or biophysical basis for specific types of cataractous change. Presently, the high cost of this camera (exceeding $20,000) precludes its use by many laboratories wishing to do human lens research. This study describes an inexpensive (less than $2,200) alternative camera for this type of photography. Adjacent frames on the film strip constitute stereo pairs which can be viewed in a modified stereo viewer. In the original CCRG camera both members of a stereo pair were included in the same frame. The quality of the stereo images obtained with this new system nearly equals that with the original Zeiss system. It is hoped that this inexpensive system will allow more scientists to participate in CCRG-related research and increase the supply of intracapsularly extracted cataracts available to all collaborating CCRG scientists.

Molecular sieving in suprachoroidal fluid formation in man.

Suproachoroidal fluid (SCF) obtained at the time of the surgical evacuation of a clinically significant choroidal detachment (CD) was analyzed for its chemical and cellular components in four distinct subgroups: (1) CD following cataract surgery, (2) CD (nonhemorrhagic) following glaucoma surgery, (3) CD (hemorrhagic) following glaucoma surgery, and (4) intraoperative CD during glaucoma surgery in patients with elevated episcleral venous pressure. The fluid obtained in groups 1, 2, and 4 was clear and slightly xanthochromic and contained low-molecular-weight substances in concentrations essentially equal to serum. Proteins and other high-molecular-weight substances were present in lesser amounts than in serum. Albumin, alpha1-antitrypsin, and transferrin were present in amounts approximately equal to those in serum, whereas alpha2-macroglobulin, IgM and IgG were decreased. beta-Lipoprotein and beta-complement were absent. It is postulated that this distribution of serum proteins is a manifestation of molecular sieving and is consistent with the existence of an isoporous membrane between the intravascular and suprachoroidal space with a pore diameter of 144 A. In the intraoperative choroidal effusions, there was evidence for exclusion of more of the lower, as well as all of the higher, molecular weight proteins. This suggested that the degree of molecular sieving increased with increasing filtration rate. In the hemorrhagic SCF, the distinctive character of the fluid and the protein concentrations indicated that the integrity of the capillary membrane was mardkedly disrupted, thereby allowing higher-molecular-weight proteins and cellular elements to enter the space.

Classification of human senile cataractous change by the American Cooperative Cataract Research Group (CCRG) method: II. Staged simplification of cataract classification.

One thousand nine hundred and seventy-six cataracts extracted intracapsularly were classified according to the system adopted by the Cooperative Cataract Research Group (CCRG) Consortium. A nine-stage protocol for simplifying the classification data is presented. The method of simplifying the basic CCRG classification emphasizes anatomic similarities among lenses. Simplification is indicated when small numbers of cataracts in any class provide insufficient statistical power to allow detection of scientifically or clinically important differences in rates between comparison groups, if such differences exist; however, simplification unavoidably obscures the anatomic, biochemical, and biophysical features of individual cataracts. Age-specific characteristics of this population of 1976 cataracts are used to demonstrate the effects of the simplification process. The preponderance of mixed cataracts and the relative scarcity of pure cataracts are documented, and the implications of these numbers for cataract research are presented.

Classification of human senile cataractous change by the American Cooperative Cataract Research Group (CCRG) method: III. The association of nuclear color (sclerosis) with extent of cataract formation, age, and visual acuity.

Nineteen hundred and seventy-six immature human cataracts extracted intracapsularly were classified according to the Cooperative Cataract Research Group (CCRG) method of cataract classification. Data on cataract location and extent, nuclear color, preoperative visual acuity, age, and sex were organized and stored in the PROPHET system. The data were examined for relationships between nuclear color (sclerosis) and the age of the cataractous lens, the extent of opacification in seven anatomical regions including the degree of nuclear opacification and the preoperative visual acuity. Nuclear color correlates with age in a curvilinear manner. Nuclear yellowing increases gradually with increasing nuclear opacification, but the color change is so slight as to be useless for the purposes of inferring the intensity of nuclear opacification from the color of the nucleus. There is no association between the extent of anterior cortical, equatorial cortical, posterior cortical, subcapsular or supranuclear opacification, and nuclear color. Nuclear color impairs vision only for the range dark yellow through black. These data justify the recommendation that nuclear color be abandoned as the single index of the severity of any type of senile cataractous change.

Age and the control of glycolysis in the rat lens.

Previous studies with lens dispersions indicated that the rate-limiting step in glycolysis shifts from hexokinase (HK) in the young lens to phosphofructokinase (PFK) in older lenses. Because the concentrations of the complex controlling factor for these enzymes could not be reproduced reliably in homogenates, the question of age-related control of glycolysis was re-examined in intact lenses. Toward this end, the levels of several metabolites of glucose were measured in fresh and incubated clear lenses. Of the substrates measured per fresh lens, only one changed significantly with age; fructose diphosphate was increased. When lenses were incubated in 2 to 12 mM glucose, the lactate production per lens was not significantly different with age. Together these results suggested that the glycolytic mass of the lens was constant with age. In both young and older lenses, increases in glucose in the medium led to increases in both glucose and glucose-6-phosphate in the lens. The lack of corresponding increase in lactate production suggested that the regulatory step lay downstream from HK, probably at PFK. This finding was corroborated by evidence that the initial acceleration of lactate production by the addition of cyanide (the Pasteur effect) was accompanied by decreases in the substrates of PFK, glucose-6-phosphate and fructose-6-phosphate. A secondary disinhibition of HK, as indicated by decreased lens glucose, became apparent after longer incubation with cyanide. This suggested that after disinhibition of PFK, HK became rate-limiting until the level of glucose-6-phosphate fell enough to allow the disinhibition of the latter enzyme as well. Thus PFK seemed to be the primary regulatory step in aerobic glycolysis in lenses of rats from 1 to 12 months of age.

Supplementing glucose metabolism in human senile cataracts.

Assay of the activities of hexokinase, phosphofructokinase, and pyruvate kinase showed that the first two declined in aging human lens cortex and all three enzymes retained constant activities in the epithelium throughout life. Moreover, both clear and cataractous aging lenses contained the same enzyme activities. ATP contents in cataracts, however, were lower than in clear lenses; in fact, after incubation at 37.5 degrees C in isotonic (290 to 300 mOsm), glucose-containing media, ATP was rapidly lost from cataracts (but not from clear lenses), suggesting excessive ATP expenditure in cataracts for osmotic balance. Cataracts incubated in media containing either glucose-6-phosphate or fructose-1, 6-diphosphate produced significantly higher ATP than with glucose in the media, indicating that glucose metabolism in human senile cataracts could be supplemented with hexose phosphates. Fructose-1, 6-diphosphate appeared to be more efficient than glucose-6-phosphate in preventing lens swelling during incubation.