Exp. Eye Res.(1992) 55. 193-201

The Effects SEYMOUR

of Near-UV Radiation on Elasmobranch Cytoskeletal Actin

ZIGMAN”“,

NANCY

S. RAFFERTYb,

DIANE

L. SCHOLZbAND

Lens KRIS LOWE”

aOphthalmology Research Laboratory, Box 314, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 74642, bDepartment of Cell, Molecular and Structural Biology, Northwestern University School of Medicine and Dentistry, Chicago, IL, and “Marine Biological Laboratory, Woods Hole, MA 02543, U.S.A. (Received Bethesda November 1990 and accepted in revised form 29 August 1991) The role of near-UV radiation as a cytoskeletal actin-damaging agent was Investigated. Two procedures were used to analyse fresh smooth dogfish (Mustelus cunis) eye lenses that were incubated for up to 22 hr in vitro, with elasmobranch Ringer’s medium, andwith or without exposureto a near-UVlamp(emission

principally at 365 nm: irradiance of 2.5 mW cm-$). These were observed histologically using phalloidin-rhodamine specific staining and by transmission electron microscopy. In addition, solutions of purifiedpolymerizedrabbit muscleactin wereexposedto the sameUV conditionsand depolymerization

was assayedby ultracentrifugation and high-pressureliquid 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 LTV 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, While there appears to be a direct effect of this UV radiation on the isolated actin, the lens damage would be modified by the cellular constituents and could be at the actin binding sites relative to supporting cell architecture. Key words: actin: cytoskeleton; elasmobranch: epithelial cells: near UV radiation: ocular lens.

1. Introduction Environmental ultraviolet (UV) radiation has been implicated as a factor that enhances cytological damage to the ocular lens epithelium (Zigman, 1985). Only one previous research report has described changes in specific cytoskeletal elements that result from exposure of the lens to UV radiation (seeRafferty et al., 1988). A prominent component of lens epithelial cytoskeleton is actin (Rafferty, and Scholz, 1985 ; Scholz and Rafferty, 1988: Rafferty et al., 1990). In numerous species,much of the actin is arranged in filaments of polygonal arrays at the apical side of the cell nucleus. In some species, however, the actin is localized in bundles of stress-like fibers at the basal ends of lens epithelial cells (Rafferty and Scholz, 1989). In all cells, actin filaments are involved in both motile processesand in maintenance of cell shape. As actin is a protein and other lens proteins are altered by UV exposure (Zigman. 1985), it was hypothesized that if actin filaments were adversely effected by UV * For correspondence. 00144835/92/080193 13

+09

$08.00/O

exposure, cell architecture would suffer. This led to the research that is reported herein. It was convenient to use marine vertebrate lensesin vitro for this work, as such lenses are plentiful and resilient, so that they resist damage from handling and long-term incubations. They are also large so as to provide greater amounts of cytoskeletal proteins than do mammalian lenses. In a previous publication (Zigman, 199 1) we identified both actin and vimentin in dogfish lens epithelial cells using SDS-acrylamide gel electrophoresis and immunoblotting. Previous in vitro studies of near-UV effects on marine vertebrate ocular lenseshave shown that they are altered by UV exposure in long-time incubation experiments (Zigman, Lowe and Rafferty, 1989). A primary aim was to document the microanatomical changes induced in UV exposed elasmobranch lenses. We have observed immunocytochemically (Rafferty et al., 1988) that filamentous actin in dogfish lens epithelial cells, in association with other cytoskeletal elements, is degraded by in vitro exposure of whole lensesto near-UV (UVA wavelengths predominantly). The question was asked in the present work if muscle F actin is also directly degraded by this UV exposure 0 1992 Academic

Press Limited I;RK 55

194

S. ZIGMAN

ET AL.

FIG. 1. Opalescence in the dogfishlensdue to UV-A exposure(B) for 16 hr (approximately 166 J cm-?. A. Shieldedcontrol.

without any other cellular macromolecular components. While muscle actin (predominantly a) and cellular or filamentous actin (predominantly y) do differ slightly in some amino acid sequences,they are essentially the same in nearly all other known properties (see Pollard and Weihing, 19 74). Our intention in this portion of the work was not to compare the two actins but to establish if polymerized actin is sensitive to UV degradation even without attachments to other cellular constituents.

2. Materials and Methods Experimental Design Dogfish (Mustelus canis) were killed by concussion. Eyes were enucleated and placed on ice: lenseswere removed, weighed to approximate the animal’s age, and placed in Elasmobranch Ringer’s solutions with pH at 6.8870 in 50 ml Pyrex beakers (Zigman et al., 1989). All beakers were set under an inverted beaker. Circulating sea water maintained the temperature at 18-22°C and 5 % CO,/9 5 % air flowed into the system to maintain pH. Incubation times were up to 22 hr of exposure. UV-radiation was delivered through Pyrex glass beakers, by a Woods lamp, which provided 25 mW cm-” at 365 nm through the Pyrex glass to the tissue being exposed. While the lamp emission wavelengths ranged from 400 to 250 nm, only < 1 Y0 of the energy below 320 nm penetrated the Pyrex glass beakers, Thus the lenses received 3 1, 83, 166 and 2 19 J cm-2 of 36 5 nm energy when exposedfor 3, 8, 16 and 21 hr, respectively. Microanatomical and Electrophoretic Procedures After termination of the UV light exposure, the lenseswere placed on glassslidesand the epithelium/

capsule peeledoff as describedpreviously (Rafferty and Scholz, 1989) to make whole-mount preparations. The preparations were flattened and dried at the edges, then fixed in 4% formaldehyde in Elasmobranch Ringer’s solution for 10 min at room temperature. After washing in the same Ringer’s solution at 5 min intervals, the slideswere put into absolute acetone at - 20°C for 10 min to permeabilize the membranes. N,-dried rhodamine phalloidin was diluted ten-fold with phosphate-buffered saline (PBS) to give a 0.32 ,UM solution, placed on the tissue for 20 min. and then rinsed off briefly in PBS. The slides were coverslipped in 50150 glycerol/PBS and viewed and photographed with a ZeissAxiophot fluorescence microscope. For examination with TEM, whole lenseswere fixed in glutaraldehyde in 0.05 M phosphate buffer, pH 7.2 for 1 hr, washed in the same buffer, dehydrated through 100% ethanol and embedded in L. R. White resin. Sections (1 ,UM thick and 80 nm thin) were cut on a Porter-Blum ultramicrotome, tangential to the anterior face of the lens. Thin sections were stained for routine microscopy or immunogold labeling with an anti-actin antibody and with a second antibody conjugated with 5-nm gold particles (Janssen,Inc.) as described in detail previously (Scholz and Rafferty, 1988). In these studies, vimentin was also labeled, but with lo-nm gold particles. Thin sections were viewed with a JEOL 100 CX electron microscope at accelerating voltages of 60 or 80 keV. For PAGE, samplesfrom the supernatant portion of the spins required concentration as the sampleswere too dilute to obtain protein bands on the gels: they were concentrated by evaporation. Discontinuous gels were prepared according to Laemmli (19 70) as described above. The gels were run between 45 and 60 min at 150 and 200 V: they were stained with Coomassie Brilliant Blue R-250 for 1-2 hr and destained in 9% acetic acid (EM Science, Cherry Hill,

UV

EFFECTS

ON

LENS

ACTIN

195

FIG. 2. Whole-mount preparations of dogfish lens epitheliai cells (original magnification x 313). These were stained with phailoidin-rhodamine and photographed with a fluorescence microscope. A, Shielded control ; B, exposed to near-UV radiation for 17 hr: C, exposed to near-UV radiation for 22 hr.

FIG. 3. Transmission electron microscopy of dogfish lens epithelial cells (original magnification x 10000). A. Shielded controls: B. near-UV irradiated lenses for 22 hr. Actin filaments are observed in bundles in the subcapsular area of epithelial ceils in the controls. but are nearly absent in the UV-exposed lens.

NJ): 46% methanol (VWR Scientific, San Francisco, CA). The position of the actin polypeptide band was compared with rainbow protein molecular weight standards (Amersham, Arlington Heights, IL). In each run, one gel was prepared for Western blot analysis in a BioRad mini apparatus using nitrocellulose paper ~Schleicher and Schuell, Keen, NH). Transfer took

place for 1 hr at 100 V. The papers were assayed for actin using a poiyclonal antibody made in rabbits against chicken breast muscle actin. After blocking the papers for 1 hr in 2% bovine serum albumin the papers were agitated overnight in the actin antibody (1: 1000). The secondary antibody was biotinylated goat anti-rabbit IgG (1: 400) (Amersham) for 2 hr and 13-2

196

S. ZIGMAN

ET AL

FIG. 4. ~ansmission eIectron microscopy of dogfish lens epitheiial cells labeled with ~ti-act (5 nm goid-labe~ed~and antivimentin (10 nm gold) antibodies (magni~ca~on x 80000) : A, Control; B, UV exposed. The very small dots are the anti-actin reaction, while the large dots are the anti-vimentin reaction. Arrows identify the gold labeled actin filaments.

this was followed by incubation in streptavidin peroxidase (1: 100). Development was in 4-chloro-lnaphthol and H,O, until the color appeared on the papers.

Photochemical Studies with Purijied Muscle Actin

Polymerized actin prepared from rabbit muscle was obtained from Dr Tom Pollard (marine Biological Laboratory, Woods Hole, MA). The actin at a concentration of 1 mg ml-’ was polymerized in the

UV

EFFECTS

ON

LENS

197

ACTIN

MOleCUlar weight (kD0)

200-

9369-

46-

3022-

Lanes:

I

2

3

4

5

FIG. 5. Polyacrylamide gel electrophoretic immunoblotting patterns of dogfish epithelial cell total soluble proteins, Gels were loaded with 100 pg of protein, runs were for 1.2 hr at 150 V and 70-30 amps. Lane 1, Amersham rainbow standards; 2, polyacrylamide gel of dogfish lens epithelium total soluble protein: 3, immunoblot vs. anti-a&n antibody of dogfish lens epithelium total soluble protein: 4, immunoblot of purified actin standard; 5. immunoblot of rainbow standard.

following formulation : 50 mM KCI, 2 mM Mg Cl,. 6H, (Mallinckrodt. Paris, KY), 2 mM ATP (ICN Biochemicals, Cleveland, OH), in 0.05 M, pH 6.8, Tris buffer. Samples (100 ~1) of the polymerized actin gel-like solutions were placed in 1.5 ml quartz cuvettes which were cooled by circulating sea water at 20-22’C and irradiated through the clear sides for 3-18 hr using a Woods lamp, which provided 2.5 mW cmm2 at 365 nm; the range was from 400 to 300 nm, but only < 1% of the energy below 300 nm reached the sample. Actin degradation was measured by the sedimentation method of Pardee and Spudich (1982) with s~ctrophotometric analyses, and by gel filtration HPLC. Subsequent to irradiation for the appropriate times, 50 ~1 of actin sample was spun through a 100 ,ul bed of 30 % sucrose in a Beckman centrifuge for 15 min at 100000 rpm (3SOOOOg) and 20°C. This procedure sediments the polymerized actin (F-actin) to the bottom and suspends the non-polymerized form (G-actin) near the top of the tube. Fifty microliters of the supernatant was removed from the top layer (above the sucrose bed) and diluted 1: 100 with the polymerization buffer to a final volume of 0.5 5 ml. The samples were scanned with a Beckman DU-6 Spectrophotometer from 320 to 190 nm. The diluted supernatant values at 290 nm were used as a measure of the actin concentration in the presence of nucleotide,

as nucleotide does not absorb at 290 nm. The 290 to 280 nm ratio was constant. The procedure followed was as previously described (Lehrer and Kerwar, 1972). HPLC analyses of actin samples was carried out using a gel filtration column (TSK 3000, 75 x 0.5 mm with guard column, 50 x 0.5 mm) at a pressure of 100-200 psi, flow rate of O-4 ml min-’ and with an elution buffer made up of 0.1 M Na,PO, 0.1 M Na,SO, and 0.05 % sodium azide. The monitoring wavelength was at 280 nm, the time constant was 2, and the range was O+Ol. The areas under the 15 plus 16 min peaks from the densitometer were considered to represent polymerized actin, while the areas under the 28 min peaks were considered to be monomeric actin. 3. Results Figure 1 displays the appearance of dogfish lenses in which mild opalescence appears only in the UVexposed lenses (B). In Fig. 2 the fluorescence staining of filamentous actin in whole mounts of dogfish lens epithelial cells can be seen. Figure 2(A) shows the control or shielded epithelial cells after 22 hr of incubation. Bundles of actin filaments and the membranes into which these filaments insert (via other protein intermediaries) are brightly lit up. The UV-irradiated [Fig. 2(C)] epithelial

S. ZIGMAN

198 cell bundles of actin filaments are not present after 22 hr of irradiation, although the plasma membranes are still fluorescent. In Fig. 2(B) interrupted bright spots are discernible in the cytoplasm after 17 hr of irradiation. As shown by electron microscopy [Fig. 3(A)] actin filaments in control, shielded lenses were arranged in thick masses under the basal epithelial cell membrane. The filament bundles appeared to originate from one or two focal points on the membrane, as seen also in Fig. 2(A), the phalloidin-stained fluorescence micrograph. By electron microscopy or light microscopy, the ‘free ’ ends could not be followed to an insertion. Only a few filament bundles were found in the UV-i~adiated lenses [Fig. 3(B)]. When S-nm gold-labeled anti-rabbit IgG was applied to electron microscopic sections and then viewed by EM, the labeled, densely dot-stained areas were associated with the filaments, both in the capsule and epithelial cells [Fig. 4(A)]. The controls showed a greater degree of actin labeling than the UV exposed Iens epithelium, indicating a decrease in polymerized actin due to UV [Fig. 4(B)]. The vimentin antibody gave sparse labeling in the region of heavy actin filaments, near basal cell membranes. Vimentin filaments were labeled heavily deeper within the cell, especially around the nucleus. Figure 5 represents the electrophoretic data for dogfish lens total protein (lane 2). and Western blots of the same gel (in lane 3). as compared with a blot of the purified actin used in our photochemical studies reported herein (lane 4). A clearly defined 44 to 4 5 kDa band is apparent in the stained gel (lane 21 and the two blots. The results of physical studies of the response of polymerized actin to direct exposure to near-UV radiation follow. Figure 6 shows the absorption spectra of actin and ATP demonstrating that at 290 nm no ATP absorption occurs. A suspension of highly polymerized actin (F-a&n) was degraded by near-UV exposure for 3 and 18 hr. The data are expressed in the form of 290 nm-320 nm absorption of the upper phase (i.e. above the sucrose) solutions, after centrifugation for IS min. The increase in absorbance in the upper layers of centrifuged UV-exposed samples indicates an increase in the amount of monomer released (see Table I). This increase is approximately 2 5 % in most experiments. Figure 7 ilIustrates the eiution profile for 18 hr IJV exposed and unexposed polymerized actin when examined by HPLC analysis. The areas under the void volume peaks and under the 4 S 000 Da peaks were assessed from their 280 nm absorptions. The areas under the 2%min peaks were used as a measure of actin degradation (see Table II). Under the conditions of the experiment the highly pol~erized actin was eluted in two close peaks, at 15 min (V,f and at 16 min. Monomeric actin eluted at 28 min, as compared with a known monomeric actin standard (Sigma Chem. Co., Inc.). There are lower molecular

ET AL.

1A i.5

I

209 nm 260 nm

290 Wavelength

inm)

FIG. 6. tlltraviolet spectra of concentrated actin (A) in pofymerizing buffer (2 mM MgCl,, 50 rnM KCl, 2 mM ATP) at O-5 mg ml-‘. Note that the sample is read with the ATPcontaining buffer in the sample and reference compartments. B. Tris-based actin polymerizing buffer vs. tris buffer : ATP is present at 20 PM. TABLE

I

Degradation of actin polymer to actin monomer by exposure to near-TV rad~ut~o~z~ asi~g the centrifugal-spectroyhotametric method Concentration of monomeric actin in top layer (pg)* ~-.

Condition

3 hr

18hr

Increase f%)

Dark lJV exposed

6.3 9.45

9.19 12.6

25 25

* Theconcentration of monomeric actin in the upper layer of the tube was calculatedfrom the absorption at 290 nm minus that at 320 nm. where ATP does not absorb, and using the actin extinction value for 1 mg ml-’ of 0.63 (see Materials and Methods),

weight actin polymers in between (20-26 min) which do not provide sharp peaks. The 36 min peak is a buffer component, while the 40 min peak is ATP: it was identified by eluting ATP (Sigma Chem. Co., Inc.) through the same column and by its absorption spectrum. The significant changes due to UV exposure are: an increase in the 16-min lighter polymer: a decrease in the species eluting between 20 and 26 min ; and an increase in the 2%min monomer (44 kDa from a standard curve). The increase of monomer in 18 hr of IJV exposure was approximately 30%. Table I1 summarizes the observed changes.

UV EFFECTS

ON

LENS

ACTIN

199

(A)

I 0

I

I

6.7

13.4

v

44 kDo

t

I

I

28. I Elution

1

n

I

-26.7 time

33.4 imtn)

I

40, I

I

46.7

I

53.4

FIG. 7. HPLC analysis of the breakdown of F-actin due to I8 hr of IJV-A exposure (A). B, Control. The voided peak represents F-a&m, and the peak at 2 8 min is G-a&in. Standards : thyroglobulin (670 kDa) ; -globulin f 15 8 kDa) : ovalbumin (44 kDa) ; myoglobin ( 17 kDa) : vitamin B 12 f 1.3 5 kDa) : ATP (disodium 551 Da).

TABLE II

Actin degra~uti~n by near-W ~a~~~~~o~,Percentage of total peak areas of ~o~gmeric and ~o~~~e~ic nctin representedby the peak at 28 min

-_-_~-

Exposure times fhr) -_.-.0

monomeric actin (%) ____I-Dark control UV-exposed .-~~ __-. 14 -

3 8 18

19 16 16

30 20 32

4. Di~ussion LetIs Epit~el~al Cell Effects Cytoskeletai elements are universai. In dogfish lens epithelium the actin is clearly arranged in stress-lie fibers at the basal end of the cells but not as polygonal arrays as shown previously for numerous other lens epithelial celis (Rafferty and Scholz, 198 9). It has been

suggested that the arrangement of actin filament bundles in elasmobranch lenses may be ‘reIated to differences in the stress on the epithelial cells resulting from a different mode of accommodation as compared with that in birds, reptiles and mammals (Sivak, 1980; Rafferty and Scholz, 1989) ; still the structural basic elements of the cytoskeleton are quite similar as

judged by their immunological cross-reactivity. Clearly and s~prisingly, the structure of actin filaments in the epithelial cells is disrupted by the UV exposure (essentially UV-A) provided. This is at subsolar h-radiances and due to relatively small amounts of total UV energy provided. The UV radiation is mainly at 365 nm, with small components ( < I %) at 300 and 400 nm. This is the wavelength range that has been implicated in former findings of lens crystallin alterations (Zigman, Paxhia and Waldron, 1988), of DNA damage {Sidjanin, 1990) and as an inhibitor of the NaK-ATPase pump (Torriglia and Zigman, 1988). The above ocular lens defects occur in mammals (Rafferty and Zigman, submitted) as well as in marine vertebrates. However, mamma~an lens epitheliai cells have polygonal arrays of actin filaments which the

200

marine animal lenses do not. The new cytological data obtained show a definitive breakdown of actin stress tibers due to in vitro near-UV irradiation of the whole lens.

It is worthy to discuss that the cytotoxic changes in actin were predominantly to y-actin, while the photochemical data were obtained for predominantly ctactin. Pollard and Weihing (1974) have compared cellular vs. muscle actin and found them to be very similar in most chemical and physical properties. Pollard offered the opinion that the UV effects would be quite similar in both a- and y-actin (pers. commun.), and Estes also gave this opinion (pers. commun.). The greater degradative effect on actin observed in the lens epithelial cells may reflect the presence of natural UV sensitizers such as riboflavin and tryptophan. Another possibility is that the ATP level was diminished so as to destabilize the actin polymer. This possibility is supported by the recent ilndings by Thomas et al. (1991) that the ATP level of squirrel lenses exposed to the same near-UV wavelengths led to a drop in lens ATP as measured by 13’P]NMR procedures. If actin and cytoskeleton breakdown result in accommodating lenses from UV environmental exposure. a role in loss of lens accommodation with aging and in cataractogenesis could be attributed to epithelial cell disruption. With regard to the photochemical experiments, it. has been demonstrated that F-actin is partly degraded by UV exposure so that the monomeric form of actin is elevated both in HPLC and in centrifugal studies. The degree of monomeric actin formed is only about 2 5 %. There are differences in methodology between the centrifugal and HPLC results that would account for the discrepancy of degree of degradation observed. This finding can be compared to that for near-UV radiation effects on the lens crystallins, in which crosslinking to form aggregates has been observed by many researchers, (Goosey, Zigler and Kinoshita, 1980: Dillon, 1984: Zigman, 1988). However, Zigman et al. (1988) have also shown that small degrees of crystallin breakdown to smaller than 20 kDa peptides occur both in squirrel lens epithellal cells in vitro and in vivo. A conclusion of these data is that while crystallins are usually aggregated by UV exposure, some protein breakdown also occurs in the epithelium in response to near-UV exposure of the lens. Polymerized actin does not aggregate, but does degrade to monomeric actin at about 20-30%. This paper demonstrates the near-UV induced breakdown of polymerized actin by cytochemical data and the release of monomeric actin in solutions by photochemical methods. It provides evidence that epithelial cells, which are the most anteriorly placed

S. ZlGMAN

ET AL.

cells of the ocular lens, and which differentiate into fiber cells that maintain the physical state and optical functions of the lens, are vulnerable to cytoskeletal alterations by exposure to environmental ultraviolet energy. It also appears that the type of damage observed is related to partial degradation of actin polymer either directly or due to effects on its binding sites, Acknowledgements Support for this research was provided by grants from the National Institutes of Health (EY 00459: EY 00698) and Research to Prevent Blin~ess, Inc.

References Dillon. J. (1984). Photolytic changes in lens proteins.

Cum.

Eye Res. 3, 145-50.

Goosey. J. D.. Zigler. S. J. Jr and Kinoshita, J. H. (1980). ~ross~inking of lens crysta~~ins in a photodyuanlic system. A process mediated by singlet oxygen. Science 208. 1278-80. Laemmli, U. K. (1970). Cleavage of structural proteins during the assemblyof the head of ba~te~ophageT,. Nature 227. 680-5.

Lehrer.S.S.and Kerwar, G.(1972). Intrinsic fluorescenceof actin. Biochem11, 1211-7. Pardee, J. D. and Spudich, J. A. (1982). Purification of muscleactin. ~e~~o~sEnz~mffl. 85. 164-233. Pollard. T. D. and Weihing. R. R. (1974). Actin and myosin and cell movement. CRCCrit. Rev.Biochem.2. l-65. Rafferty, N. S.,Lowe,K., Rafferty, K. and Zigman,S.(1988). Initial studiesof vertebratelenscytoskeleton.Biol. Buli. 175. 302. Rafferty, N. S. and Scholz,D. L. (1985). Actin in polygonal arrays and sequesteredactin bundles (SABs) in lens epithelial cells of rabbits and mice. Curr. Eye Res. 4, 713-18.

Rafferty, N. S. and Scholz, D. L. (1989). Comparativestudy of actin filament patterns in lens epithelial cells. Are these determinedby the mechanismsof lens accommodation?Curr. Eye Res. 8, 569-79. Rafferty, N. S., Scholz. D. L.. Goldberg,M. and Lewyckyz, M. J. (1990). Immunocytochemical evidence for an actin-myosin systemin lens epithelial-cells.Exp. Eye Res. 51, 591-60. Scholz. D. I,. and Rafferty. N. S. (1988). Immunogoid-~ localizationof actin and vimentin filamentsin relation to polygonal arrays in lensepitheliumin situ. Curr. Eye Res.7, 705-19. Sidjanin. D. (1990). Simple Strunk Breaks in DNA of’ Lens Ep~~~Ie~ial Cellslndli~ed bg W-A Radlatio~.MS. thesis, University of Rochester,Rochester,NY. Sivak, J. G. (1980). Accommodation in vertebrates: a contemporarysurvey. In CurrentTopics in Eye Research, Vol. 3. (Eds Zadunaisky, J. A. and Davson, A.). Pp. 28 1-3 30. AcademicPress.New York. Thomas, D. M.. Papadopoulou,O., Mahendroo, P. P. and Zigman, S. (1991). Phosphorus-31NMR study of the effectsof UV on squirrellenses.Invast.Op~~fh~~rno~. Vis. Sci. 32 ~Supp~.), 748. Torriglia, A. and Zigman. S. (1988). The effect of near UV light on NaK-ATPaseof the rat lens.Curr. Eye Res. 7, 539-48.

Zigman, S. (1985). ~hotobiology of the fens.In The OLulur

UV

EFFECTS

ON

LENS

201

ACTIN

Lens. (ed. Maisel,H.). Pp. 301-47. Marcel Decker: New

York, Zgman, S.(1991). Comparativebiochemistryandbiophysics of elasmobranchlenses.I. Exp. Zool. 5 (Suppl.),29-40. Zigman. S., Lowe. K, and Rafferty. N. S. (1989). Near UV

effectsonthethymidineincorporation intodogfishlens,

BioI, BulI,177,320,

Zigman, S.,Paxhia,T. and Waldron,W. (1988). Effectsof near UV radiation on the protein of the grey squirrel lens. Curr. Eye Res. 7, 531-7.