Photobleaching of Melanosomes From Retinal Pigment Epithelium: I. Effects on Protein Oxidation

Aug 11, 04:00 AM

Current Headlines: By Burke, Janice M Henry, Michele M; Zareba, Mariusz; Sarna, Tadeusz

ABSTRACT Melanin in the long-lived melanosomes of the retinal pigment epithelium (RPE) may undergo photobleaching with aging, which appears to diminish the antioxidant function of melanin and could make photobleached melanosomes less efficient in protecting biomolecules from oxidative modification. Here we analyzed whether photobleaching of melanosomes affects their ability to modify the oxidation state of nearby protein. As conventional methods developed to study soluble antioxidants are not well suited for analysis of granules such as melanosomes, we developed a new analytic method to focus on particle surfaces that involves experimentally coating granules with the cytoskeletal protein beta-actin to serve as a reporter for local protein oxidation. Isolated porcine RPE melanosomes were photobleached with visible light to simulate aging, then photobleached melanosomes, untreated melanosomes and control particles (black latex beads) were actin coated and illuminated in a photosensitized cell free system. Protein was re-stripped from particles and analyzed for carbonylation by Western blotting. Quantitative densitometry showed no reproducible differences for protein associated with untreated melanosomes when compared with control particles. Melanin has both and- and pro-oxidant functions when light irradiated, but neither of these functions predominated in the protein oxidation assay when untreated melanosomes were used. However, protein extracted from photobleached melanosomes showed markedly increased carbonylation, both of associated actin and of endogenous melanosomal protein(s), and the effect increased with extent of granule photobleaching. Photobleaching of RPE melanosomes therefore changes the oxidation state of protein endogenous to the organelle and reduces the ability of the granule to modify the oxidation of exogenous protein near the particle surface. The results support the growing body of evidence that photobleaching of RPE melanosomes, which is believed to occur with aging, changes the physicochemical properties of the organelle and reduces the likelihood that the granules perform an antioxidant function.

INTRODUCTION

Pigment granules (melanosomes) of the retinal pigment epithelium (RPE) are long-lived organelles that show little turnover throughout life (1,2). Despite melanosome longevity, the melanin content of human RPE cells declines significantly with donor age, possibly due to photo-oxidative bleaching of melanin (3). Intact melanin has the potential to exhibit both anti- and pro-oxidant properties (4), but photobleaching of melanosomes has been shown to decrease their antioxidant potential (5,6). Taken together these observations raise the possibility that the pigment granules within RPE cells may lose antioxidant efficiency with aging as a consequence of photobleaching, thereby becoming less competent to protect cells from light-induced oxidative stress. Oxidative stress to the retina and RPE, including photic stress, is believed to contribute to diseases like age-related macular degeneration (7-9).

Unlike most factors that generate or scavenge short-lived reactive oxygen species, melanin is not soluble. Rather it is sequestered within granules, melanosomes, so its anti- or pro- oxidant functions are likely to operate in the near microenvironment of the organelle. In our previous studies to probe how photobleaching affects the properties of RPE melanosomes, we incubated the pigment granules in various solutions and analyzed the effects of the particles on bulk phase components (5,6,10). To more closely model how melanosomes may function within the cytoplasm of cells, here we developed new methods that focus on biomolecules, specifically proteins, that are associated with the particle surface rather than in solution. According to the protocol, control particles, untreated or photobleached melanosomes were coated with the cytoskeletal protein actin, then an oxidative environment was produced by illuminating the coated particles with visible light in the presence of a photosensitizer. Protein was then re-stripped from the particle surface for analysis of levels of carbonylation as a measure of local protein oxidation.

MATERIALS AND METHODS

Isolation and photobleaching of melanosomes. Melanosomes were purified from the RPE of porcine eyes as previously described (6). After purification, the granules were incubated in Laemmli (11) electrophoresis buffer containing 0.25% Triton X-100 and 2.0% sodium dodecyl sulfate (SDS) to remove proteins and membranes associated with the granule surface. The detergent buffer, and all other solutions used for granule treatments, also contained a protease inhibitor cocktail (Sigma, St. Louis, MO).

For photobleaching, melanosomes were suspended at 1 x 10^sup 9^ granules mL^sup -1^ in 20 mM phosphate buffer, pH 7.2, then irradiated at 190 mW cm^sup -2^ with visible light for intervals to 24 h as reported previously (6) using a ThermoOriel Solar Simulator outfitted with a 1000 W xenon lamp, a 420-630 nm dichroic mirror and an additional ultraviolet filter (cutoff 380 nm). Bleaching was monitored by measuring the absorbance of an aliquot of the melanosome suspension solubilized in Soluene350; samples showing decreases in absorbance of ~15-50% were used.

Actin coating of melanosomes. Melanosomes (untreated or photobleached) and control particles (1 [mu]m black latex beads; Interfacial Dynamics Microspheres & Nanospheres, Eugene, OR) were incubated for 1 h at room temperature with slight agitation in a solution containing 1 mg mL^sup -1^ human platelet beta-actin in General Actin Buffer supplemented with 0.2 mM ATP and 0.5 mM dithiothreitol (actin and buffer both from Cytoskeleton, Denver, CO). Unbound protein was removed by two washes with phosphate buffer. Actin-coated particles were resuspended in phosphate buffer containing the well known photosensitizer rose bengal (10 [mu]M) and either maintained in the dark or illuminated for intervals to 15 min with visible light. The solar simulator conditions described above for photobleaching were used for particle illumination except at an irradiance of 64 mW cm^sup -2^. The optical path of the sample was approximately 0.7 cm and samples were continuously stirred. After treatment, particles were pelleted and resuspended in Laemmli buffer to re-extract the surface-bound actin.

Figure 1. Protein blot of beta-actin (42 kDa). Actin was used to coat particles, then was re-extracted and blotted to illustrate equivalent protein coating of and stripping from different particle types. Lane 1: untreated melanosomes; lane 2: melanosomes photobleached for 24 h; lane 3: control particles (black latex beads).

Figure 2. (a) Protein blots of actin that was stripped from control particles or untreated melanosomes following illumination in the presence of a photosensitizer for intervals to 15 min, or after being held in the dark as a control (illumination time = 0). (b) Densitometric analysis of the blotting signal for monomeric actin protein (42 kDa) that was stripped from illuminated particles (control particles: dotted line; untreated melanosomes: solid line), expressed as a percentage of their respective dark controls. (c) Silver-stained gel of actin stripped from control particles that were illuminated for 15 min. The gel was heavily loaded to show higher mass species generated by illumination (bracket); the monomeric actin band is also indicated (42 kDa).

Protein carbonyl generation and analysis. Aliquots of protein extracts, prepared as described above, were derivatized using the OxyBlot Protein Oxidation Detection Kit (Chemicon International, Temacula, CA) for quantification of protein carbonyls by Western blotting using the manufacturer's recommended protocol. Extracts were electrophoresed using 10% SDS polyacrylamide gels. Aliquots of the protein extracts were also electrophoresed on paired gels for blotting with mouse monoclonal antibodies to beta-actin (Clone AC- 74; Sigma). Bands on both types of blots were visualized by chemiluminescence and subjected to densitometric analysis using an Alpha Innotech FluorChem 8900 densitometer and software (San Leandro, CA). Some gels were stained using the ProteoSilver Stain Kit (Sigma) to visualize total protein. Each type of experiment was performed a minimum of four times.

RESULTS

Analytic method and comparison of untreated melanosomes to control particles

As the ability of melanosomes to generate or scavenge reactive oxygen species is likely limited to the near domain of the organelle, detection of a melanosome effect on protein oxidation required methods to analyze molecules that are closely associated with particle surfaces rather than in bulk phase solution. A protocol was therefore developed that involved equivalently coating surfaces of both biological (melanosomes) and control particles with the protein actin (Fig. 1).

Illumination of protein-coated particles with visible light in the presence of a photosensitizer produced a light dose-dependent effect on the Western blotting signal for protein re-stripped from particle surfaces. As shown for samples obtained from control particles (black latex beads) and untreated melanosomes, light treatment modified the blotting signal for actin (Fig. 2). The amount of protein migrating at the 42 kDa position characteristic of the monomer shows a progressive decline (Fig. 2a,b). The decline in monomeric actin is due at least partly to the generation of higher mass species which can be detected on heavily loaded silver stained gels (Fig. 2c). The declining actin monomer signal therefore provides an indirect measure of the oxidation-induced production of protein aggregates. Figure 3. (a) Carbonyl blot of protein stripped from actin-coated control particles, actin-coated untreated melanosomes, or untreated melanosomes that were not actin coated. All particles were illuminated for intervals to 15 min in the presence of a photosensitizer, or held in the dark as a control (illumination time = 0). Indicated are the migration positions of monomeric actin (42 kDa) and two unidentified melanosome-derived proteins (27 and 60 kDa). (b) Silver-stained gel of protein stripped from illuminated melanosomes, with or without actin coating. Gels were heavily loaded to reveal the weak protein signal for the 27 and 60 kDa melanosome-derived species; the 42 kDa actin monomer is also indicated.

In addition to modifying the actin blotting signal, illumination of particles also increases protein carbonylation, which increases with longer illumination times (Fig. 3). Regarding the carbonyl blotting signal seen at the position of monomeric actin (42 kDa), there is some baseline carbonylation in the protein received from the vendor (notwithstanding particle coating), which produces a signal in samples that are not illuminated (time 0) (Fig. 3a). Carbonylation of the 42 kDa species increases on illumination; the blotting signal shown in Fig. 3 appears to decline, however, because the amount of monomeric actin declines dramatically with illumination (see Fig. 2a). Normalization of the 42 kDa carbonylation signal to the actin signal on paired blots by quantitative densitometry across multiple experiments indicates that carbonylation of monomeric actin increases as much as 19-fold by 15 min of illumination (data not shown).

Higher mass carbonylated actin species are also produced by illumination, which can be seen on analysis of actin stripped from control particles that have no endogenous protein to contribute to the carbonylation signal (Fig. 3a, control particles + actin). The higher mass material does not manifest as discrete carbonylated bands suggesting that protein aggregates in a broad range of sizes are carbonylated. Given that the amount of higher mass actin protein is low, the distinct carbonylation signal for this material suggests that it is more highly carbonylated than actin monomers.

Analysis of protein stripped from melanosomes demonstrates light- induced carbonylation of protein endogenous to the organelle in addition to actin (Fig. 3a, melanosome samples). Inspection of the carbonylation signal for extracts from melanosomes that were not actin coated indicates a discrete species at 27 kDa and a weaker band at approximately 60 kDa, as well as signal throughout the lane indicating carbonylated material of variable mass. The amount of melanosome-derived protein in the extracts is low, detectable only with overloading of silver stained gels (Fig. 3b). Very weak protein bands can be seen on the silver stained gels at the positions of the 27 and 60 kDa carbonyl bands indicating that these unidentified proteins are not abundant but are highly carbonylated.

To determine whether actin associated with melanosomes as compared to control particles is differentially affected by light- induced oxidation, two measures were compared: (1) the light- induced decline in the amount of the actin monomer, and (2) the levels of light-induced monomer carbonylation. Neither measure showed differences between the particle types. The similar declines in the actin monomer signal for control particles versus melanosomes are illustrated in Fig. 2a,b. For carbonylation levels of the actin monomer, quantitative densitometry was used to normalize the 42 kDa bands on protein carbonyl blots to paired actin blots across multiple experiments, but neither particle type was consistently higher than the other (data not shown).

Figure 4. (a) Carbonyl blot of protein stripped from actin- coated or uncoated melanosomes. The melanosomes were either untreated (time = 0) or photobleached for intervals to 18 h prior to actin coating. All granules were illuminated for 10 min in the presence of a photosensitizer. (b) Densitometry of the carbonylation signal across the photobleaching time course for the 27 kDa melanosomal protein extracted from uncoated granules, given in arbitrary densitometry units (AU).

Effects of melanosome photobleaching

Although the extent of actin carbonylation for protein associated with untreated melanosomes did not differ from protein associated with control particles, protein associated with melanosomes that were photobleached was markedly and consistently more susceptible to oxidative modification. The increased susceptibility was clearly demonstrated by analysis of protein carbonylation, which showed an increasing blotting signal as a function of photobleaching time for samples that were illuminated for a fixed interval (10 min) in the presence of rose bengal (Fig. 4). The carbonylation of endogenous protein is strikingly illustrated by the increase in signal intensity for the 27 kDa melanosome species (Fig. 4b).

DISCUSSION

The biological properties of melanin are complex and include the potential to act as both an antioxidant and a pro-oxidant. Which of these conflicting properties of melanin occurs within cells is not clear since the data on melanin's functions come largely from studies using synthetic melanin or dense suspensions of isolated melanosomes. These model systems may not adequately simulate how melanin behaves in the cytoplasm of pigmented cells where the polymer is confined to melanosomes that occupy only part of the cell's volume. On the theory that insoluble granules will have the greatest effect on reactions occurring near the particle surface, we developed an assay system to analyze oxidation of proteins closely associated with the melanosome. The protein actin was selected to coat particles to serve as an oxidation reporter because actin is highly susceptible to oxidative modification (12). Further, actin is a biologically relevant protein because it is known to localize to the cytosolic surface of melanosomes within cells where it participates in organelle motility (13).

When actin-coated particles were illuminated in a photosensitizer- containing milieu, the effects on the protein were predictably complex. Monomeric actin stripped from particle surfaces showed evidence of light-induced carbonylation, although the more prominent effect was the disappearance of monomers and an increase in higher mass species that were highly carbonylated. The higher mass material appears to arise from a combination of protein fragmentation and peptide aggregation since no discrete bands corresponding to actin multimers were seen on carbonyl blots that might suggest aggregates of intact protein. Further, the higher mass carbonylated material in extracts from actin-coated melanosomes appears to result from an interaction between oxidation products derived from actin and from the melanosome itself. Evidence for this possibility comes from the observation that the carbonylation signal in the higher mass region of the blots from actin-coated melanosomes was greater and had higher mass species than predicted from the signal for melanosomes alone plus actin alone (see Fig. 3a).

Given that an interaction between actin and endogenous melanosome protein appears to contribute to the higher mass carbonylated species seen on blots, this region of the blot was not analyzed to compare untreated melanosomes and control particles (which have no endogenous protein). Analyses of monomeric actin could be used, however, which showed no significant differences in oxidative modification for protein that was associated with these two particle types. One might predict that the antioxidant functions of intact melanin would produce protection of actin from oxidative modification. Melanin has multiple antioxidant properties including the ability to scavenge free radicals (14,15), to quench singlet oxygen and excited states of photosensitizers (16-18), and to sequester transition metal ions like iron (19,20), which reduces their availability to act as co-factors in Fenton-type reactions that yield highly reactive hydroxyl radicals (21). However, melanin can also act as a prooxidant by participating in reactions that photogenerate superoxide (10,22-24). The conditions of assay did not preferentially demonstrate the melanosome's ability to either scavenge or to photogenerate reactive species, perhaps because both processes occurred simultaneously and nearly equivalently.

Regardless of whether intact, untreated melanosomes affect the light-induced oxidation of nearby proteins, photobleached melanosome clearly exacerbate it. This effect could result from a photobleaching-induced loss of melanosome antioxidant potential and/ or an increase in its superoxide radical generating capacity. Several previous studies have indicated that photobleaching changes the physicochemical properties of melanosomes (3,5,6,10). Among the known properties of photobleached melanosome are reduced melanin content, an increased capacity to photogenerate superoxide anion, and a reduced ability to bind iron and to inhibit the iron ioncatalyzed free radical decomposition of hydrogen peroxide, or to inhibit the iron/ascorbate-induced peroxidation of lipids. As shown here, photobleached melanosomes also increase the light-induced oxidation of proteins associated with the particle surface, as well as the oxidation of proteins endogenous to the melanosome itself. Relatively little is known about endogenous proteins of the mature melanosome from which protein is not readily solubilized (25). A recent proteomic analysis of porcine RPE melanosomes demonstrated more than 100 proteins (26), although many of these may have originated from contaminating organelles or cytoplasmic proteins present in the melanosome preparation. The proteins identified in the melanosome proteome were largely absent from our samples, however. We pre-stripped our melanosomes by incubation in Laemmli sample buffer by a protocol similar to one of the procedures that was used to prepare extracts for proteomic analysis, and these extracts were discarded by us (see Materials and Methods). Very little protein was therefore secondarily extracted from melanosomes subjected to our protocol involving illumination in the presence of a photosensitizer. However, the protein that was removed was highly carbonylated, especially the protein that was extracted from melanosomes that had been photobleached. In addition to overall high levels of carbonylation, blots of photobleached melanosome extracts showed two discrete, unidentified species migrating at approximately 27 and 60 kDa. These highly carbonylated proteins are of low abundance, being faintly detectable only on overloaded silver- stained gels. Their high carbonylation state in more extensively photobleached granules indicates that endogenous melanosomal proteins are susceptible to oxidative modification, and further raises the possibility that age-related photobleaching changes the protein components as well as the melanin content of RPE melanosomes.

Our intent in developing the assay used here was to probe reactions occurring in the microdomain of the melanosome surface where an insoluble granule is most likely to exert its effects within cells. Nonetheless, care must be taken in interpreting the biological ramifications of what we have observed. In our model, reactive oxygen species (ROS) photoinduced by rose bengal are generated acutely on illumination and produced homogeneously in a protein-free aqueous solution surrounding the melanosomes. In contrast, within cells endogenous photosensitizers are presumably inhomogeneously distributed with reference to the location of melanosomes, and ROS are generated in a complex, protein-rich cytosol. How this microenvironment affects access of photically generated ROS to the melanosome surface is not known. Melanosomes may be competent to protect against local protein oxidation if reactive species are generated less acutely than by the short-term photosensitized reaction that was used here. Regardless, melanosome photobleaching appears to increase the likelihood that the granules perform a phototoxic rather than photoprotective role in modulating protein oxidation.

Should increased protein oxidation occur in aged RPE ceils in the domain of photobleached melanosomes, one would not expect profound effects on overall cell survival. Rather, chronically diminished protein function and/or increased protein turnover might occur. It is the accumulated effects of small changes such as these that are believed to contribute to cellular aging and to the functional declines associated with age-related diseases. In this regard, the type of analysis performed here that involves assessment of an oxidative stress effect short of overt cell death may have particular relevance for aging. The results suggest that in aged RPE cells, photobleached melanosomes could contribute to the chronic low levels of photic stress that compromises tissue function over time. A consequence may be reduced capacity of the RPE to support adjacent retinal photoreceptors and a greater likelihood of photoreceptor degeneration.

Acknowledgements-This work was supported by National Eye Institute grants RO1 EY013722 and P30 EY01931 (J.M.B.), Poland Ministry of National Education and Science grant 3 P04A 009 25 (T.S.), the Posner Foundation and the Coleman Charitable Foundation (Milwaukee, WI), and by an unrestricted grant from Research to Prevent Blindness, Inc.

REFERENCES

1. Boulton, M. (1998) Melanin and the retinal pigment epithelium. In The Retinal Pigment Epithelium (Edited by M. F. Marmor and T. J. Wolfensberger), pp. 68-85. Oxford University Press, New York.

2. Schraermeyer, U. and K. Heimann (1999) Current understanding on the role of retinal pigment epithelium and its pigmentation. Pigment Cell Res. 12, 219-236.

3. Sarna, T., J. M. Burke, W. Korytowski, M. Rozanowska, C. M. Skumatz, A. Zareba and M. Zareba (2003) Loss of melanin from human RPE with aging: Possible role of melanin photooxidation. Exp. Eye Res. 76, 89-98.

4. Sarna, T. and H. M. Swartz (2006) The physical properties of melanins. In The Pigmentary Systems: Physiology and Pathophysiology (Edited by J. J. Nordlund, R. E. Boissy, V. J. Hearing, R. A. King, W. S. Oetting and J. P. Ortonne), pp. 311-341. Blackwell Publishing Ltd, Oxford.

5. Zadlo, A., M. B. Rozanowska, J. M. Burke and T. J. Sarna (2007) Photobleaching of retinal pigment epithelium melanosomes reduces their ability to inhibit iron-induced peroxidation of lipids. Pigment Cell Res. 20, 52-60.

6. Zareba, M., G. Szewczyk, T. Sarna, L. Hong, J. D. Simon, M. M. Henry and J. M. Burke (2006) Effects of photodegradation on the physical and antioxidant properties of melanosomes isolated from retinal pigment epithelium. Photochem. Photobiol. 82, 1024-1029.

7. Beatty, S., H. Koh, M. Phil, D. Henson and M. Boulton (2000) The role of oxidative stress in the pathogenesis of age-related macular degeneration. Surw Ophthalmol. 45, 115-134.

8. Cai, J., K. C. Nelson, M. Wu, P. Sternberg Jr and D. P. Jones (2000) Oxidative damage and protection of the RPE. Prog. Retin. Eye Res. 19, 205-221.

9. Zarbin, M. A. (2004) Current concepts in the pathogenesis of age-related macular degeneration. Arch. Ophthalmol. 122, 598-614.

10. Rozanowska, M., W. Korytowski, B. Rozanowski, C. Skumatz, M. E. Boulton, J. M. Burke and T. Sarna (2002) Photoreactivity of aged human RPE melanosomes: A comparison with lipofuscin. Invest Ophthalmol. Vis. Sci. 43, 2088-2096.

11. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.

12. Dalle-Donne, I., R. Rossi, D. Giustarini, N. Gagliano, L. Lusini, A. Milzani, S. P. Di and R. Colombo (2001) Actin carbonylation: From a simple marker of protein oxidation to relevant signs of severe functional impairment. Free Radic. Biol. Med. 31, 1075-1083.

13. Barral, D. C. and M. C. Seabra (2004) The melanosome as a model to study organelle motility in mammals. Pigment Cell Res. 17, 111-118.

14. Rozanowska, M., T. Sarna, E. J. Land and T. G. Truscott (1999) Free radical scavenging properties of melanin interaction of eu- and pheo-melanin models with reducing and oxidising radicals. Free Radic. Biol. Med. 26, 518-525.

15. Sarna, T., B. Pilas, E. J. Land and T. G. Truscott (1986) Interaction of radicals from water radiolysis with melanin. Biochim. Biophys. Acta 883, 162-167.

16. Bielec, J., B. Pilas, T. Sarna and T. G. Truscott (1986) Photochemical studies of porphyrin melanin interaction. J. Chem. Soc. 82, 1469-1474.

17. Sealy, R. C., T. Sarna, E. J. Wanner and K. Reszka (1984) Photosensitization of melanin: An electron spin resonance study of sensitized radical production and oxygen consumption. Photochem. Photobiol. 40, 453-459.

18. Ye, T., J. D. Simon and T. Sarna (2003) Ultrafast energy transfer from bound tetra(4-N,N,N,N-trimethylanilinium)porphyrin to synthetic dopa and cysteinyldopa melanins. Photochem. Photobiol. 77, 1-4.

19. Pilas, B., T. Sarna, B. Kalyanaraman and H. M. Swartz (1988) The effect of melanin on iron associated decomposition of hydrogen peroxide. Free Radic. Biol. Med. 4, 285-293.

20. Zareba, M., A. Bober, W. Korytowski, L. Zecca and T. Sarna (1995) The effect of a synthetic neuromelanin on yield of free hydroxyl radicals generated in model systems. Biochim. Biophys. Acta 1271, 343-348.

21. Halliwell, B. and J. M. C. Gutteridge (1999) Free Radicals in Biology and Medicine, 3rd edn. pp. 48-60. Oxford University Press. New York.

22. Felix, C. C., J. S. Hyde, T. Sarna and R. C. Sealy (1978) Melanin photoreactions in aerated media: Electron spin resonance evidence for production of superoxide and hydrogen peroxide. Biochem. Biophys. Res. Commun. 84, 335-341.

23. Korytowski, W., B. Pilas, T. Sarna and B. Kalyanaraman (1987) Photoinduced generation of hydrogen peroxide and hydroxyl radicals in melanins. Photochem. Photobiol. 45, 185-190.

24. Rozanowska, M., J. Jarvis-Evans, W. Korytowski, M. E. Boulton, J. M. Burke and T. Sarna (1995) Blue light-induced reactivity of retinal age pigment. In vitro generation of oxygen- reactive species. J. Biol. Chem. 270, 18825-18830.

25. Bratosin, S. (1973) Disassembly of melanosomes in detergents. J. Invest Dermatol. 60, 224-230.

26. Azarian, S. M., I. McLeod, C. Lillo, D. Gibbs, J. R. Yates and D. S. Williams (2006) Proteomic analysis of mature melanosomes from the retinal pigmented epithelium. J. Proteome Res. 5, 521-529.

Janice M. Burke*1, Michele M. Henry1, Mariusz Zareba1,2 and Tadeusz Sarna2

1 Department of Ophthalmology, Medical College of Wisconsin, Milwaukee, WI

2 Department of Biophysics, Jagiellonian University, Krakow, Poland

Received 10 January 2007; accepted 20 January 2007; DOI: 10.1111/ j.1751-1097.2007.00081.x

* Corresponding author email: jburke@mcw.edu (Janice M. Burke)

(c) 2007 The Authors. Journal Compilation. The American Society of Photobiology 0031-8655/07

Copyright American Society for Photobiology Jul/Aug 2007

(c) 2007 Photochemistry and Photobiology. Provided by ProQuest Information and Learning. All rights Reserved.