John F.R. Kuck

Induction, Acceleration and Prevention (in vitro) of an Aging Parameter in the Ocular Lens

A fluorogen (444-nm emission) is present in mature mammalian ocular lenses (human, rat and mouse) and can serve as an aging parameter in this organ. This fluorogen can be induced in very young human, rat and mouse lenses by incubating them in a media containing 3-aminotriazole and exposing them to ultraviolet (UV) light (300–380 nm). Such in vitro incubations can also accelerate the rate at which this fluorogen normally increases with age; similar results were obtained with in vivo experiments in the mouse. It is proposed that UV-induced free radicals could play a role in this particular aging process. D-Penicillamine is capable of preventing this phenomenon in vitro.

Late onset hereditary cataract of the Emory mouse. A model for human senile cataract

The late onset cataract of the Emory mouse has appealed to many investigators as a useful animal model for human senile cataract. It has been the subject of about 15 publications, beginning in 1982. These have explored many features, including histology, chromatography and isoelectric focusing of the crystallins, enzyme profiles, amino acid and ion transport, membrane studies including changes in MP24 and MP26, analysis for a number of biochemical constituents, and its use as an assay system for testing the effect of anticataractogenic drugs and dietary restriction. These investigations have not uncovered a single metabolic lesion marked enough to be considered an important cause of this cataract. There is some evidence that the effect of oxidation may be a major factor; likewise there is evidence for faulty protein synthesis. In many cases, the changes in cataractogenesis appear to be accelerated aging changes. For this reason, any study of this cataract must employ age-matched controls of the cataract-resistant strain. A recent discovery is the finding that many earlier studies used a mixture of both early and late-onset forms, accounting for the wide variability in analytical results. The two substrains may have somewhat different applications in cataract research. Thus, the availability of these two substrains should extend the usefulness of this animal model.

Near-infrared Fourier transform Raman and conventional Raman studies of calf gamma-crystallins in the lyophilized state and in solution

We present in this report a detailed structural study of calf gamma-crystallins both in the solid state and in solution by the newly developed technique of near-infrared (IR) Fourier transform (FT)-Raman spectroscopy as well as by the conventional Raman method. In comparison with conventional laser Raman spectroscopy, the near-IR FT-Raman approach exhibits several attractive features such as fluorescence rejection capability, frequency accuracy, and the FTs multiplex and throughput advantages. These distinct characteristics combined form the basis for the particular suitability of FT-Raman in crystallin structural analysis and elucidation. We have thus obtained evidence in support of the view that native calf gamma-II crystallin does not contain a disulfide bond either in the lyophilized state or in solution. In addition, conventional Raman spectra are examined for all four gamma-crystallin fractions. gamma-S, gamma-II, gamma-III, and gamma-IV, and the results indicate a high degree of structural similarities among them. It is also found that the sulfhydryl groups in all four gamma-crystallins are highly resistant to air oxidation and are capable of maintaining their reduced state during isolation in the absence of added reductants or such chelating agents as EDTA.

Surface-Enhanced Raman Spectra of Eye Lens Pigments

Surface-enhanced Raman spectroscopy (SERS) has been applied to study lenticular pigments that are present in the eyes of certain diurnally active animals. Using Ag hydrosols pre-aggregated with NaClO4, we have obtained SERS spectra from dilute solutions of various model pigment compounds, including kynurenine, N-formylkynurenine, 3-hydroxykynurenine, β-carboline, bityrosine, anthranilic acid, 3-hydroxyanthranilic acid, and oxindole. The results obtained from these model compounds show that SERS is a particularly sensitive technique for the identification of lens pigments. We also report a procedure that enables high-quality SERS data to be obtained for the yellow pigments in the lens homogenates of grey squirrels, ground squirrels, and chipmunks. The surface Raman results confirm the identity of the low-molecular-weight, water-soluble pigment in the grey squirrel lens as a derivative of 3-hydroxykynurenine, but reveal that lens pigmentation in ground squirrels and chipmunks involves new chromophores. The significance of this finding in relation to the metabolic/photochemical generation of lens pigments is discussed.

Resonance Raman detection of a carotenoid in the lens of the deep-sea hatchetfish

A laser scanning micro-probe has been used to elicit resonance Raman signals from frozen sections of the lens of the deep-sea hatchetfish, Argyropelecus affinis. The signals demonstrate with certainty the presence of a carotenoid and its distribution in the lens. The carotenoid exhibits characteristic resonance Raman vibrational modes at 1551 cm-1 (C = C stretch, v1), 1147 cm-1 (C-C stretch with C-H bend, v2), 2285 cm-1 (2v2) and 2681 cm-1 (v1 + v2), upon excitation at 441.6 nm. Unlike glycogen in the nucleus of dove lens, the carotenoid in the lens of A. affinis occurs at a higher concentration in the cortex, although its presence in the nucleus is established. A study of lenses of varying age showed that carotenoid incorporation is accelerated as the fish grows older and hence its concentration is highest in the cortex. Because of the extremely low concentration of the carotenoid in the nucleus, it was detectable only by the very sensitive resonance Raman technique.

Nature and localization of avian lens glycogen by electron microscopy and Raman spectroscopy.

Electron microscopy confirms the presence of a high concentration of glycogen particles in the lens nuclear region of birds of flying habit such as the ring-neck dove and pigeon. This observation is consistent with Raman spectroscopy. The glycogen particles in the dove lens, which are approximately 35 nm in diameter, are classified as beta type particles. Although this type has been previously characterized by high rates of glycogen turnover in other tissues, its localization in the lens nucleus indicates that it may serve a structural function rather than as a storage depot of carbohydrate in the lens. In a comparative electron microscopy study, glycogen particles were not observed in the chicken lens.

Use of a UV-Blocking contact lens in evaluation of UV-induced damage to the guinea pig lens☆

Abstract Much evidence has been accumulated to suggest that UV-A and UA-B of sunlight, which penetrate the cornea and are partly absorbed by the lens, are important factors in cataractogenesis. We have examined the efficacy of a UV-absorbing contact lens in reducing UV-induced damage to the guinea pig lens in vivo. Each of the animals was fitted with a UV-blocking hydrogel contact lens, possessing a monolayer of UV-absorbing chromophore, on the control eye and a regular hydrogel contact lens on the contralateral eye. After 12–19 months of continuous exposure to UV from a blacklight source, the unprotected lens showed increased (a) opacification, (b) pigmentation, (c) fluorescence, and (d) disulfide formation. Such changes also occur in human lenses during cataractogenesis. Therefore, this study strongly supports the idea that increased exposure to LTV light is an important causative factor in human cataract formation.