Biomedical subjects
S Zigman
Publications and source records attributed to S Zigman.
DNA damage and repair in rabbit lens epithelial cells following UVA radiation.
Since ultraviolet light may be a contributing factor to cataractogenesis, we investigated the response of the lens epithelium, a potential target for UV insult, to UVA radiation. Cell survival and the induction and repair of DNA single-strand breaks (SSBs) were measured in cultured rabbit lens epithelial cells following UVA exposure. The light was passed through a filter which eliminated wavelengths below 335 nm in order to ensure that the cells were exposed only to UVA. In order to study the effect of various fluences of UVA on cell survival, 2 x 10(6) cells suspended in Tyrode's buffer were exposed to UVA. During all irradiations the cells were maintained at 0.5 degrees C in order to minimize DNA repair. Following UVA treatment, 200 cells were cultured in minimal essential medium containing 10% rabbit serum, and a colony forming assay was used to quantify cell survival. UVA induced cell death in a dose-dependent manner. In additional experiments, confluent epithelial cells on glass slides immersed in Tyrode's buffer were irradiated and SSBs were quantified using the alkaline elution technique. A 30 min exposure to UVA (180 KJ/m2) induced measurable SSBs. An increase in UVA fluence brought about an increase in the number of DNA SSBs. Rejoining of SSBs was measured after the cells were irradiated in Tyrode's for 2 hrs and allowed to repair in the dark for 4 hrs at 36 degrees C in MEM containing 10% serum. Eighty percent of the DNA SSBs were repaired within 4 hrs as determined by analysis of the alkaline elution profile. The repair kinetics were biphasic with an initial fast and subsequently slower component. The results indicate that UVA can induce SSBs in lens epithelial cells, that the cells can repair most UVA-induced SSBs, and that UVA treatment can be toxic to the epithelium.
The effects of near-UV radiation on elasmobranch lens cytoskeletal actin.
The role of near-UV radiation as a cytoskeletal actin-damaging agent was investigated. Two procedures were used to analyse fresh smooth dogfish (Mustelus canis) eye lenses that were incubated for up to 22 hr in vitro, with elasmobranch Ringer's medium, and with or without exposure to a near-UV lamp (emission principally at 365 nm; irradiance of 2.5 mW cm-2). These were observed histologically using phalloidin-rhodamine specific staining and by transmission electron microscopy. In addition, solutions of purified polymerized rabbit muscle actin were exposed to the same UV conditions and depolymerization was assayed by ultracentrifugation and high-pressure liquid chromatography. While the two actins studied do differ very slightly in some amino acid sequences, they would react physically nearly identically. The results showed that dogfish lenses developed superficial opacities due to near-UV exposure. Whole mounts of lens epithelium exhibited breakdown of actin filaments in the basal region of the cells within 18 hr of UV exposure. TEM confirmed the breakdown of actin filaments due to UV exposure. SDS-PAGE and immunoblotting positively identified actin in these cells. Direct exposure of purified polymerized muscle actin in polymerizing buffer led to an increase in actin monomer of approximately 25% in the UV-exposed solutions within 3-18 hr, whether assayed by ultracentrifugation or HPLC. The above indicates that elasmobranch lens epithelial cells contain UV-labile actin filaments, and that near-UV radiation, as is present in the sunlit environment, can break down the actin structure in these cells. Furthermore, breakdown of purified polymerized muscle actin does occur due to near-UV light exposure.(ABSTRACT TRUNCATED AT 250 WORDS)
Light filters to improve vision.
Visual impairment not only derives from deficient processes within the ocular tissues, but also from the quality and quantity of environmental lighting. High levels of short wavelength light are not used efficiently in eyes that have cloudy media anterior to the retina or whose cones are deficient. Chromatic aberration, light scattering, and fluorescence are enhanced by short wavelength lighting. We show here that in human vision and in photography, elimination of environmental light with wavelengths shorter than 450 nm improves both contrast and visual clarity, especially through cloudy media. Cutoff filters with appropriate characteristics could be developed to improve vision in elderly and visually impaired people whose ocular media mishandle short wavelength light.
Relationships between human cataracts and environmental radiant energy. Cataract formation, light scattering and fluorescence.
This preliminary report has two parts. The first is based upon data obtained from a group of cataract patients in southern Florida (USA) with the object of relating the types of cataracts removed to their personal background and their protein biochemistry. Intra-capsular cataract surgery patients at the Venice Eye Clinic (Florida) were interviewed, and their extracted lenses were classified. The parameters were: age, place of residency, occupation, medical and family history and indoor/outdoor activity. Subcapsular cataracts were found mainly in the youngest patients and in those who were in Florida the least. Mixed cataracts predominated in the oldest patients, while non-nuclear cataracts were associated most with outdoor activity. Water-insoluble protein was elevated in nuclei of lenses with nuclear opacities. Soluble proteins in the nuclei of nuclear cataracts had increased levels of voided (heavy) protein, beta-crystallins, and less than 20 Kd peptides. The above changes were enhanced in brunescent cataracts. In lenses with cortical opacities, only increased size heterogeneity in the beta-crystallin region was observed. The second part of this report is based upon direct measurements of the optical properties of freshly extracted intra-capsular cataracts obtained in Rochester, New York (USA). The purpose was to attempt to learn the relative contributions that absorption, scattering, and fluorescence make toward obscuring vision. A general conclusion is that the shorter wavelengths of radiant energy in environmental lighting influence the above-stated optical properties the most, and thus appear to be the major contributors to obscured vision.
Effect of chronic near-ultraviolet radiation on the gray squirrel lens in vivo.
The effects of ambient exposure to near-ultraviolet (near-UV) radiation (300-400 nm) on the ocular lens of the diurnal squirrel (Sciurus carolinensis) are reported. Gray squirrels lived in cages illuminated for 12 hr a day with near-UV light (6 mW/cm2, 365 nm) for 1 yr. The non-UV-exposed controls were housed separately. In the lenses of UV-exposed animals, anterior pole changes occurred. Central epithelial cells swelled, disappeared, or underwent proliferation. A band of disoriented degenerating fiber cells was seen in the midcortex, with a degree of liquefaction. When lens protein compartments were separated by centrifugation, water-insoluble but urea-soluble fractions were enhanced in the outer and inner cortex and the nucleus. Both high-performance liquid chromatography and polyacrylamide gel electrophoresis revealed that proteins mainly in the midcortex and nucleus were altered considerably. Evidence of a loss of sulfhydryl compounds (by chemical and Raman spectroscopic analyses) and an increase of protein-thiol mixed disulfides (chemically) was also observed. These data prove that repetitive ambient exposure of diurnal animals to near-UV radiation at subsolar levels damages the lens by interfering with the maintenance of epithelial cells and altering the structural proteins; some of this may be due to the conversion of sulfhydryls to mixed disulfides.
Comparative biochemistry and biophysics of elasmobranch lenses.
This presentation compares the features of the lenses of sharks and skates to those of land animals, including man. Of all the tissues of vertebrates, the chemistry and physical features of the ocular lens have been preserved intact. Functionally, while the transmission of light and images is common to both classes, the elasmobranch lens shares the added task of refraction with the teleosts (as the cornea does not refract in the sea), and the mammal lens is deformed to support accommodation, while the elasmobranch lens is not. Light filtration by lens pigments does occur mainly in diurnal or shallow-swimming species. The same group of lens crystallins is present in sharks (and skates) as is present in squirrels (and humans). The same structural arrangement of a mono-layer of epithelial cells anteriorly and concentric layers of differentiated and fiber cells from the periphery to the center apply to most vertebrates. Fiber cell ribbon shapes and interdigitating knobs are also very similar, as are the fine structures of the fiber cell membranes and the polymerized arrays of actin. Yet the elasmobranch lens is hard and spherical, while the mammalian lens is soft and disc-shaped. What the chemical basis is for the differing physical states of lenses of shark and man remains to be answered. This presentation will document the chemical and morphological bases for elasmobranch and mammal lens structure and function. It also will show that even though a shark is a primitive vertebrate, its lens is still useful as a model to assist in the understanding of lens aging, of lens swelling (or lack of it) and the classical basis of cataracts (or lack of them).
Comparative study of lens proteins of gray squirrel and human.
1. The four crystallins of the gray squirrel lens have been characterized using gel filtration chromatography, polyacrylamide gel electrophoresis, and immunoblotting. Alpha, beta-heavy, beta-light, and gamma crystallins of squirrel lenses have been identified immunologically, and they cross-react strongly with rabbit polyclonal antibodies. The gamma-24 crystallin of the squirrel lens also reacts strongly with monoclonal anti-human lens gamma-24, as shown by its inhibition of the ELISA reaction by 85%. 2. The water-insoluble urea soluble proteins represent non-covalently associated species of soluble crystallins and the lens cytoskeletal proteins. The membrane intrinsic protein in the urea insoluble pellet has a mol. wt of 27,000 but other lower and higher mol. wt components are also present, which were removed by washing with 0.1 NaOH. The N-terminal 30 amino acid of squirrel lens gamma crystallin was found to be identical to that of the bovine (and human) lens. 3. Measurements of the distribution and state of SH and SS compounds in the squirrel lens have shown greater similarities to those of primates than those of rodents. The findings show that on the basis of both protein and sulfur chemistry the squirrel lens is a representative model for studies of oxidative lens changes in diurnal animals, including man.
Vision enhancement using a short wavelength light-absorbing filter.
This report illustrates photographically the adverse influence of short wavelength-induced light-scattering and autofluorescence on image quality, and the improvement of image quality that results by filtering out light wavelengths shorter than 480 nm. It provides additional data on the improvement in human vision (under conditions of excessive intraocular light-scattering and fluorescence) by filters that prevent short wavelength radiant energy from entering the eye.
Special features of the lens relative to the environment.
The lenses of certain shallow-swimming marine vertebrates contain near-UV absorbing pigments. These pigments minimize chromatic aberration and maximize contrast sensitivity. Such pigments are chemically similar to those found in diurnal terrestrial vertebrate (ie: gray squirrel) lenses. Deep-swimming marine vertebrate lenses generally lack these pigments, so they must utilize all light available for vision. Two near-UV absorbing pigments were observed in the lenses of assorted teleosts and elasmobranchs: pigment I absorbs maximally at 320, while pigment II does so at 360 nm. They are both dialyzable, water extractable compounds; and I is more polar than II. Studies of the properties of extracted and purified teleost lens pigment using absorption spectroscopy, HPLC, TLC, and mass spectrometry suggest that these pigments are kynurenines with differing side groups. The absorption spectrum of I resembles N-formyl kynurenine, while that of II resembles L-kynurenine. Preliminary assays of tryptophan pyrrolase (TP) in teleost lenses showed a much greater enzyme activity in shallow-swimmer's lenses. Light in the environment may stimulate lens pigment production. The use of short wavelength light cutoff filters may be useful in improving vision by minimizing light-scattering and fluorescence.
Comparison of retinal photochemical lesions after exposure to near-UV or short-wavelength visible radiation.
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Temporal sequence of changes to the gray squirrel retina after near-UV exposure.
Monocularly aphakic gray squirrels (Sciurus carolinensis) were exposed for 10 min to monochromatic near-ultraviolet radiation (lambda = 366 nm, radiant exposure = 4.3 J/cm2) to determine if their yellow pigmented lens protected retinal tissue from photochemical damage. Eyes were examined from 1 to 30 days after exposure to determine the temporal sequence of retinal damage and the extent of recovery from such exposures. Light microscopy of exposed aphakic retinas revealed irreversible lesions to the photoreceptors. Swelling of inner segments, accumulation of heavy pigment deposits in the PE, presence of macrophages in the subretinal space, and pyknosis of photoreceptor nuclei were observed in the exposed region of the aphakic eye. Eyes exposed to ultraviolet radiation with their lenses intact were devoid of significant retinal lesions. This study represents a model system for studying the potential damaging effects of near-UV radiation to the aphakic eyes of humans.
The nature and properties of squirrel lens yellow pigment.
The low molecular weight yellow pigment in the gray squirrel lens is confirmed to be n-acetyl-3-OH-kynurenine (NAK). This conclusion is based upon the results of studies of the compound's optical, chromatographic, and mass spectroscopic properties. The original tentative identification of this compound was reported by Van Heyningen (1971, 1973). The pigment is distributed equivalently throughout the lens. Its absorbance increases with age, but the increase is related to the growth of the lens and not to an increase in pigment concentration. The function of NAK in the squirrel lens is to enhance vision by eliminating short-wavelength light that causes scattering and chromatic aberration. The pigmented lens also protects the retina from near-UV radiation-associated damage to the photoreceptors (Collier and Zigman, 1987).
Effects of near-UV radiation on the protein of the grey squirrel lens.
In vivo exposure of grey squirrels to 40W BLB illumination resulted in alterations in the state of the lens crystallins, mainly in the outer layer of the lens. HPLC revealed an increase of the void volume or crosslinked crystallins and an increase in peptides with molecular weights lower than 20,000 d. In vitro exposure of squirrel lens aqueous extracts to Woods lamp radiation (predominantly 365 nm) led to similar but more exaggerated changes as viewed by high performance liquid chromatography. When viewed by polyacrylamide gel electrophoresis (PAGE), soluble protein crosslinking was also observed. The near-UV absorbing chromophores of low molecular weight present in the lens served as photosensitizers that enhanced the protein changes. Sodium azide inhibited the changes, indicating a role for singlet oxygen in the crosslinking.
The effect of near-UV light on Na-K-ATPase of the rat lens.
The influence of in vitro near-UV radiation exposure on the physical state of the rat lens and on its membrane-bound Na-K-ATPase activity was investigated. Lens swelling was correlated to the appearance of opacities and the inactivation of the enzyme. The results show a significant decrease in the Na-K-ATPase activity which may be an early change leading to osmotic type cataracts. The dose-effect curves obtained for cortical and epithelial enzymes were different. Since the data do not follow a monoexponential function, the existence of two forms of Na-K-ATPase in the lens is discussed.
Lactate dehydrogenase activity and its isoenzymes in concentric layers of adult bovine and calf lenses.
The activity of lactate dehydrogenase (LDH) and its isoenzyme pattern were studied in four concentric layers of adult bovine and calf lenses. In both groups the specific activity of the total LDH diminished progressively toward the internal nuclear layer; the decrease was greater in the adult lenses. The enzyme activities in the cortical layers of the calf lens were lower than in the adult lens, but in the inner nuclear layers, the opposite was found. All of the 5 LDH isoenzymes were found in each layer. In both groups of animals the LDH1 isoenzyme prevailed, followed by LDH2. No differences were found in the percentage of each isoenzyme in the different lens layers. The differences in the activitie(s) of LDH found may be due to post-translational or post-synthetic modifications which may occur during the aging process.
The gray squirrel lens protects the retina from near-UV radiation damage.
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Age-related changes in the proteins of individual skate lenses.
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