Guinea Pig Lens Research Articles

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

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.

Distribution and characterization of dihydrodiol dehydrogenases in mammalian ocular tissues

The immunological relationship of two forms of dihydrodiol dehydrogenase (DD) in pig lens to pig muscle aldose reductase and kidney aldehyde reductase has been studied. Although the minor enzyme form, a monomer of Mr 35,000, was identical with aldose reductase, the major enzyme form, a dimer of Mr 65,000, was distinct from the two reductases. The two enzyme species, although their amounts were low, were distributed in the cornea, iris-ciliary body, retina and choroid of the pig eye. In other mammals, rabbit lens exhibited much higher DD activity than did lens of mice, rats, cats, hamsters, guinea pigs and monkeys, and contained large amounts of the Mr-65,000 enzyme form as well as the minor enzyme form of Mr 35,000. In contrast, only the Mr-35,000 form of the enzyme was found in the lens of other species, except that a small amount of the high-Mr enzyme was detected in mouse lens. The high-Mr enzyme, purified from rabbit lens, was functionally and immunologically similar to dimeric DD of pig lens. The low-Mr enzyme forms, isolated or partially purified from these animal lenses, showed several features in common with aldose reductases from mammalian tissues. The dimeric enzymes of pig and rabbit lenses were NADP(+)-specific, whereas the low-Mr enzymes exhibited dual cofactor specificity and their activities with NAD+ were more than 3-fold higher than those with NADP+.

Enzyme/crystallins and extremely high pyridine nucleotide levels in the eye lens 1

Taxon-specific crystallins are proteins present in high abundance in the lens of phylogenetically restricted groups of animals. Recently it has been found that these proteins are actually enzymes which the lens has apparently adopted to serve as structural proteins. Most of these proteins have been shown to be identical to, or related to, oxidoreductases. In guinea pig lens, which contains zeta-crystallin, a protein with an NADPH-dependent oxidoreductase activity, the levels of both NADPH and NADP+ are extremely high and correlate with the concentration of zeta-crystallin. We report here nucleotide assays on lenses from vertebrates containing other enzyme/crystallins. In each case where the enzyme/crystallin is a pyridine nucleotide-binding protein the level of that particular nucleotide is extremely high in the lens. The presence of an enzyme/crystallin does not affect the lenticular concentrations of those nucleotides which are not specifically bound. The possibility that nucleotide binding may be a factor in the selection of some enzymes to serve as enzyme/crystallins is considered.

ζ-Crystallin from guinea pig lens is capable of functioning catalytically as an oxidoreductase

ζ-Crystallin, a major taxon-specific protein of the guinea pig lens, has been shown to be distantly related to the alcohol/polyol dehydrogenase family and to specifically bind NADPH. The capacity of ζ-crystallin to function catalytically was investigated in the present study. ζ-Crystallin exhibited an NADPH-dependent oxidoreductase activity with 2,6-dichlorophenolindophenol (DCIP). The NADPH:DCIP oxidoreductase activity of ζ-crystallin exhibits a linear response with increasing protein concentration, and saturation kinetics with NADPH and DCIP. This activity was abolished by heat inactivation and immunoadsorption of the protein. Dicumarol, Cibacron blue, manganese, and sulfhydryl reagents were inhibitory.

Ascorbic acid uptake by young and aged guinea pig lenses

The dynamics of in vitro uptake of [ 14C]-ascorbic acid into intact lenses of young (50 days old) and old (730 days old) guinea pigs was evaluated in this study. Two-dimensional protein gel analysis of [ 35S]-methionine labeled proteins provided evidence that the isolated lenses were viable throughout the culture period. It was found that the in vitro uptake of ascorbate into these lenses follows similar saturation kinetics for lenses from both age groups. Moreover, the linear uptake rate was identical. Ascorbate transport was inhibited by phloretin, p-chloromercuribenzoate, insulin, glucose and ouabain; however, no inhibition was observed with 2,4-dinitrophenol or NaF. These results suggest that the lenticular ascorbate concentration is regulated by a facilitated diffusion process and it not energy-dependent. Data on ascorbate transport into lenses from aged guinea pigs provide strong evidence that the abnormally low ascorbic acid concentrations in lenses of old mammals is not likely the result of a decreased lenticular uptake ability.

The transcripts of zeta-crystallin, a lens protein related to the alcohol dehydrogenase family, are altered in a guinea-pig hereditary cataract

Zeta-Crystallin, a major component of the guinea-pig lens proteins, is distantly related to the enzymes of the zinc-containing alcohol dehydrogenase family (ADH). Analysis of the structural similarities between ζ-crystallin and ADH reveals that while characteristics important in maintaining the tertiary structure of the molecule appear conserved, the amino acids binding the catalytic zinc atom are absent in ζ-crystallin. Significantly, ζ-crystallin does not have ADH activity. Previous studies showed that the ζ-crystallin protein is modified in the lens of guinea-pigs affected with an autosomal dominant hereditary cataract. We have further investigated the molecular origin of the lens defect by examining the steady-state levels of ζ-crystallin transcripts in normal and mutant eyes. Our data indicate that no normal ζ-crystallin mRNA is present in the lens of the homozygous animals; instead, a cross-hybridizing lower molecular weight mRNA is detected at significantly reduced concentrations. Heterozygous lenses exhibit both mRNA species.

Extremely high levels of NADPH in guinea pig lens: Correlation with zeta-crystallin concentration

Zeta-crystallin, a major “taxon-specific” protein of the guinea pig lens, specifically binds NADPH. Analysis of pyridine nucleotide levels in guinea pig lens revealed values for NADPH approximately 50-fold higher than in other lenses. Indeed to our knowledge the values reported are higher than have been observed in any tissue. A clear correlation exists between NADPH and zeta-crystallin contents of the lens both in normal guinea pigs during development and in a line of guinea pigs with a mutation in the gene for zeta-crystallin. Heterozygotes for this mutation had a 50% reduction in NADPH, while homozygotes have only about 6% of the normal level. NADP+ levels were also markedly elevated suggesting that redox cycling of the NADPH is occurring.

Zeta-crystallin, a novel protein from the guinea pig lens is related to alcohol dehydrogenases

ζ-Crystallin is a major component of the water-soluble proteins of the guinea pig lens. We have constructed a lens cDNA library from one- to seven-day-old guinea pigs in the plasmid Bluescript KS + and used the 16 amino acid (aa) sequence of a CNBr peptide to design an oligodeoxyribonucleotide probe. Analysis of two positive clones and direct sequence of the 5′ end of the RNA resulted in the completion of a most probably full-length mRNA comprising 1842 nucleotides (nt). The ATG start codon occurs 83 nt downstream from the 5′ end. The open reading frame, ending with a stop codon at nt position 1070, predicts a protein of 328 aa with a calculated M r of 35071. Comparison of the amino acid sequence with the National Biomedical Research Foundation protein data base reveals a significant similarity of ζ-crystallin with the enzyme of the alcohol dehydrogenase family.

Effects of age and dietary vitamin C on the contents of ascorbic acid and acid-soluble thiol in lens and aqueous humour of guinea-pigs

Variations in ascorbate and thiol concentration in lens and aqueous humour, with age and vitamin C nutrition, are of potential biological importance. To study these relationships, Dunkin-Hartley guinea-pigs were maintained for periods of up to 1.3 yr on diets containing either high or low (marginal) vitamin C. Ascorbate contents of liver, spleen, adrenals, lens and aqueous humour, and acid-soluble thiol of lens and aqueous humour were measured. High vitamin C intake maintained ascorbate levels in the internal organs between five and 30 times the level attained by the low vitamin C intake and aqueous humour vitamin C was 10-20 times higher at high vitamin C intake. Lens ascorbate, however, was only about twice as high at high vitamin C intake than at low intake, and at both intake levels it declined steadily to about half its initial value, after 1.3 yr. Thus an animal aged 1.3 yr on the low intake had about one-quarter to one-fifth of the lens ascorbate level of a young animal receiving a generous intake. Acid-soluble thiol in the lens, in contrast to ascorbic acid, increased significantly with age but was not significantly affected by dietary vitamin C intake. Acid-soluble thiol in the aqueous humour was only 0.5-2% of the concentration found in lens, and unlike the lens thiol level, it declined with age. No sex differences were observed for ascorbate or thiol levels either in lens or in aqueous humour.

Raman spectroscopic evaluation of aging and long-wave UV exposure in the guinea pig lens: A possible model for human aging

The laser Raman optical dissection technique makes it possible to study individual points of minute volumes (2 X 10(-3) microliter) in the intact living lens in vitro. This technique was used to measure the sulfhydryl and disulfide content of 21 distinct points along the visual axis of the guinea-pig lens after aging and long-wave ultraviolet exposure (9-month duration in vivo). To facilitate comparison between different lenses, data was compiled as the intensity ratio of sulfhydryl (2580 cm-1) to a protein reference signal (2731 cm-1) or disulfide (508 cm-1) to phenylalanine (622 cm-1). These 21 ratios for each experiment were plotted as a function of the distance of the point from the nuclear center of the lens to give a visual axis profile. From these profiles we have found that the loss of sulfhydryl can be accelerated in the guinea-pig lens by in vivo ultraviolet exposure (353 nm peak from an incoherent source) for nine months. There is also a subsequent uniform increase in the disulfide content across the visual axis after UV exposure suggesting a direct sulfhydryl to disulfide conversion in the guinea-pig lens.

Biochemical and histochemical evidence for lysosomal proteases in rodent lenses

The lysosomal proteases cathepsin B and dipeptidyl peptidase II (DPP II) were identified biochemically in the supernatant fraction of rat and guinea-pig lens homogenates, using as substrates CBZ-Arg-Arg-4-methylcoumarin-7-amide (AMC) (5 microM, pH 6.0, 37 degrees C for cathepsin B, and Lys-Ala-AMC or Lys-Pro-AMC (5 microM, pH 5.5, 37 degrees C) for DPP II. Cathepsin B-like activity was totally inhibited by the specific inhibitor lepeptin (0.1 mM); while DPP-II-like activity was partially inhibited by Tris (10 mM) or puromycin (0.1 mM), which are specific for DPP II, and by phenylmethylsulphonyl fluoride (PMS-F), an inhibitor of serine class proteases. DPP-II-like activity also was demonstrated histochemically. The histochemical preparations consisted of cryostat sections (6 micron) of glutaraldehyde-fixed rat lenses, incubated in medium containing cacodylic acid-NaOH (0.05 M), Lys-Pro-4-methoxy-2-naphthylamide (MNA) or Lys-Ala-MNA (1 mM), and 0.1% w:v Fast Blue B salt (pH 5.5, 37 degrees C). Control sections were treated with medium in which the substrates were replaced by Lys-Ser-MNA (1 mM), which is not acted upon by DPP II; or medium containing PMS-F (1 mM). Sections treated with experimental medium containing Lys-Pro-MNA showed intense enzymatic activity throughout the epithelium and bow region. In the bow region and superficial cortex activity was mostly granular, suggesting a lysosomal localization. Histochemical activity was reduced partially by inclusion of PMS-F, and totally by substituting Lys-Ser-MNA for Lys-Pro-MNA.

Pulse techniques for the suppression of individual components in multiexponential relaxation curves.

NMR pulse sequences have been investigated which selectively suppress undesired signal components from multiple-exponential spin-spin and spin-lattice relaxation curves. Suppression is achieved by exploiting differences in the relaxation times of sample components. Special attention is devoted to discussion of practical details necessary for implementation of these methods, as well as their relative advantages and disadvantages. The techniques are illustrated using multiple-compartment aqueous phantoms and are then employed in measurements of proton T1 and T2 values for freshly isolated guinea pig lenses immersed in aqueous buffer solutions to prevent dehydration and maintain viability. These methods obviate many of the difficulties inherent in obtaining accurate relaxation times for minor components of multiple-exponential curves and should be useful in many applications of NMR to the study of intact tissue, both in vivo and in vitro.

Finding of a galactose-oxidation-product in lens of galactose-fed guinea pig

From studies on polyols in lens of galactose-fed guinea pigs, r-galactono-1,4-lactone was found, which proves the presence of galactonic acid as a product of galactose oxidation, by gas liquid chromatography and mass spectrometry. The content of this component was one tenth of that of galactitol. In vitro culture of rat lens in 30 mM galactose-loaded media demonstrated the formation of the lactone. The significance of the lactone was discussed with respect to the galactose metabolism in lens.

Elemental analysis of cataractous lenses by PIXE

Elemental analysis of the lenses of hereditary and galactose cataracts were carried out by the particle induced X-ray emission technique (PIXE) using 28 MeV α-particle beam from a cyclotron accelerator. The lenses of the hereditarily cataractous mice (ca mouse) showed marked increases with age in the concentrations of Cl and Ca. The spatial distributions of elements in the lenses of guinea pigs, galactose-fed and normal diet-fed were measured by scanning with a small beam spot of 200 μm diameter. It was commonly found that K and Cl were concentrated in the polar regions and S was predominantly located in the central region. In opaque lenses tremendous increases in the concentrations of Cl and Ca accompanied by a decrease in that of K were generally observed.

In vivo Action of Corticosteroids on the Guinea Pig Lens

The influence of intravitreal injection of corticosteroids in guinea pigs on lens cation concentration, lens density and leucine-l-[14C] incorporation into the lens protein was examined. 6-

Effects of Freezing on Weights of Cottontail Lenses

Decline in cottontail (Sylvilagus floridanus) Iens weight due to freezing was dramatic and quite variable, indicating use of frozen lenses would be of little value for determining age. The presence of a black ciliary ring around the equator of frozen lenses was not a suitable criterion for separating frozen from normal lenses. Since the development of e eye-lens technique for aging wildlife (Lord 1959), much time and effort have been involved in delineating possible variables that would alter the lens weight and thus the estimated age of an animal (Friend 1967a, 1967b ) . Friend (1967b ) emphasized that techniques in handling, preserving, and weighing of lenses may alter results. Montgomery (1963) and Rongstad (1966) reported that decomposition caused changes in lens weight. Montgomery (1963) also presented evidence of a reduction in weight of lenses due to freezing. Longhurst (1964) and Friend (1967b) found no evidence of this phenomenon in domestic sheep or whitetailed deer ( Odoicoi1Mus virgtnianus ) . However, Friend (1967a ) noted a slight decrease in weight of frozen lenses of guinea pigs (Cavgporcellus). This paper discusses the effect of freezing on lens weight as a variable in estimating the age of the cottontail rabbit. lThis study was supported through a research contract from the Federal Aid Division of the Georgia Game and Fish Commission. Certain equipment, supplies, and other funds were also made available from McIntire-Stennis Project No. 11 of the College Experiment Stations, Athens, in cooperation with the Georgia Forest Research Council. 2Present address: Department of Forestry, University of Tennessee, Knoxville. MATERIALS AND METHODS One eye from each of 70 rabbits was removed immediately after collection and placed in 10 percent formalin. The rabbit was then frozen (-15C) for 2S30 days prior to dissection. Upon dissection, the other eye was removed and placed in formalin. Eyes remained in formalin for a minimum of 30 days. Lenses were dried in a forced air oven for 6 days at 85 C and weighed. Loss in weight due to freezing was expressed as the percent difference between the weights of the frozen and unfrozen lenses of each pair. This method of handling these data is based on previous findings that the weights of both lenses of a given individual cottontail are essentially the same (Lord 1959). An additional 70 pairs of normally preserved lenses in the present study revealed a mean difference of 1.6 mg between the right and left lenses (SD = + 3.08, SE = 0.3S, range 0-15.6 mg). RESULTS AND DISCUSSION The mean difference between frozen and unfrozen lenses ( n = 70 ) was 41.4 mg (SD = + 23.6, SE = 2.81, range 2.0-100.0 mg). Distribution of percentage change between the 70 pairs of cottontail lenses is presented in Fig. 1. These data indicate 205 BRIEFER ARTICLES This content downloaded from 157.55.39.17 on Thu, 01 Sep 2016 05:13:35 UTC All use subject to http://about.jstor.org/terms 206 Journal of Wildlife Management, Vol. 34, No. 1, January 1970

GLUTATHIONE PEROXIDASE IN LENS AND A SOURCE OF HYDROGEN PEROXIDE IN AQUEOUS HUMOUR.

1. Glutathione peroxidase has been demonstrated in cattle, rabbit and guineapig lenses. 2. The enzyme will oxidize GSH either with hydrogen peroxide added at the start of the reaction or with hydrogen peroxide generated enzymically with glucose oxidase. 3. No product other than GSSG was detected. 4. Oxidation of GSH can be coupled with oxidation of malate through the intermediate reaction of glutathione reductase and NADPH(2). 5. Traces of hydrogen peroxide are present in aqueous humour: it is formed when the ascorbic acid of aqueous humour is oxidized. 6. Hydrogen peroxide will diffuse into the explanted intact lens and oxidize the contained GSH. The addition of glucose to the medium together with hydrogen peroxide maintains the concentration of lens GSH. 7. Glutathione peroxidase in lens extracts will couple with the oxidation of ascorbic acid. 8. It is suggested that, as there is only weak catalase activity in lens, glutathione peroxidase may act as one link between the oxygen of the aqueous humour and NADPH(2).

Pyridine nucleotides in the lens of different species

The levels of reduced and oxidized pyridine nucleotides were measured in the lens of several species. Both guinea pig and rabbit lens had high levels of NADH, measured in two different ways. Pyridine nucleotide levels in the lenses of two diabetic rabbits were about the same as in normal rabbits, except that of NAD, which was lower in diabetic rabbits. Human cataractous lens had no reduced pyridine nucleotides.

Light absorption in the crystalline lens of the cat.

DURING a series of experiments in which the lenses were removed from many cats' eyes1, the extracted material appeared colourless. The lenses remained in this condition for at least three days when stored in saline. The lenses of guinea pigs were also found to be colourless; human, cattle and pig lenses, however, are yellow in colour. A large amount of work has recently been done on the physiology of vision of the cat. In view of the filtering effect which the pre-retinal media in general, and the lens in particular, have on the visibility curve measured with the intact eye, their absorption characteristics must be known if visibility data are to be interpreted correctly. Since the cornea, aqueous and vitreous have always been observed to be colourless, attention may be focused on the lens. Accordingly, the spectral absorption characteristics of two cat lenses were measured in the range of 430–650 mµ in the same manner as described elsewhere for two human lenses2. The averages of both sets of data, expressed as densities, are compared in Fig. 1, where they are plotted against the wave-length. The difference in the subjective appearances of human and cat lenses is borne out by these results: the spectral variation of the absorption of the cat's lens is negligible as compared with that of man. This emphasizes inter-species differences, and throws some light on recent work.