Hemoprotein(s) mediate blue light damage in the retinal pigment epithelium

In order to elucidate the mechanisms of blue light damage on ocular tissues, the transepithelial transport, electrical characteristics and ultrastructural properties of irradiated isolated bovine retinal pigment epithelium (RPE) were investigated. Blue light (430 nm) irradiation at 20 mW/cm2 significantly reduced the transepithelial potential and short circuit current of RPE. During blue light exposure, a decrease in chloride transport was observed, and this decrease appeared to be closely coupled to changes in the electrical properties of the pigment epithelium. A decrease in leucine transport was also noted, but the effect required 10-30 min of exposure to be manifested on some occasions. Utilizing the observed depolarizing effect of blue light, an action spectrum was determined which encompasses the absorption spectrum of the oxidized and reduced forms of cytochrome c oxidase. O2 uptake studies on isolated pigment epithelial cells verified the reduction of respiration by exposure to blue light, which is observed in other cells. Ultrastructural studies revealed that the major cytopathology observed up to 60 min after blue light exposure was a blistering of the mitochondria which progressed to a swollen, disrupted state within the post irradiation period of 1 h. Comparison of these results with those of other studies suggests that the mechanism of UV-A damage differs substantially from that of blue light.     

Blue light irradiation inhibits the production of HGF by human retinal pigment epithelium cells in vitro

Blue visible light damage to retinal pigment epithelial cells occurs through a photooxidative mechanism and the resultant damage is hypothesized to induce or exacerbate age-related macular degeneration. The purpose of the present study was to identify changes in the cell growth and the expression of hepatocyte growth factor (HGF) in cultured human retinal pigment epithelium (RPE) cells as a result of both blue and red light irradiation. HGF is a growth factor and neurotrophic factor that stimulates growth of various ocular cells and promotes the survival of RPE and retinal neurons. Early passages of human RPE cells were exposed to blue light (460 nm) and red light (640 nm). Nonirradiated cells were used as controls. After 24 and 48 h, conditioned medium was collected and the amount of HGF was measured by ELISA. Cells were detached from the well and counted. Cell viability was evaluated by trypan-blue exclusion study. Blue light at dosage of 63 J/cm(2) significantly inhibited the growth of RPE cells without affecting of cell viability. Amounts of HGF in the culture medium were significantly inhibited by blue-light irradiation at the dosage from 32 to 63 J/cm(2). Red light at a dose of 174 J/cm(2) causes a nonsignificant inhibition of growth of RPE cells and a slight decrease of secretion of HGF. As HGF promotes survival of RPE cells and retinal neurons, the inhibition of production of HGF by visible light, especially by blue light, may enhance the phototoxic effects of visible light on the RPE and retinal neurons.     

Photobleaching of melanosomes from retinal pigment epithelium: I. Effects on protein oxidation

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 anti- 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.     

The role of A2E in prevention or enhancement of light damage in human retinal pigment epithelial cells

The process of sight (photostasis) produces, as a by-product, a chromophore called 2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E, 5E,7E-octatetraenyl]-1-(2-hydroxyethyl)-4-[4-methyl-6-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E, 3E, 5E-hexatrienyl]-pyridinium (A2E), whose function in the eye has not been defined as yet. In youth and adulthood, A2E is removed from human retinal pigment epithelial (h-RPE) cells as it is made, and so it is present in very low concentrations, but with advanced age, it accumulates to concentrations reaching 20 microM. In the present study we have used photophysical techniques and in vitro cellular measurements to explore the role of A2E in h-RPE cells. We have found that A2E has both pro- and antioxidant properties. It generated singlet oxygen (phiso = 0.004) much less efficiently than its precursor trans-retinal (phiso = 0.24). It also quenched singlet oxygen at a rate (10(8) M(-1) s(-1)) equivalent to two other endogenous quenchers of reactive oxygen species in the eye: alpha-tocopherol (vitamin E) and ascorbic acid (vitamin C). The endogenous singlet oxygen quencher lutein, whose quenching rate is two orders of magnitude greater than that of A2E, completely prevented light damage in vitro, suggesting that singlet oxygen does indeed play a role in light-induced damage to aged human retinas. We have used multiphoton confocal microscopy and the comet assay to measure the toxic, phototoxic and protective capacity of A2E in h-RPE cells. At 1-5 microM, A2E protected these cells from UV-induced breaks in DNA; at 20 microM, A2E no longer exerted this protective effect. These results imply that the role of A2E is not simple and may change over the course of a lifetime. A2E itself may play a protective role in the young eye but a toxic role in older eyes.     

Photobleaching of melanosomes from retinal pigment epithelium: II. Effects on the response of living cells to photic stress

Melanosomes of the retinal pigment epithelium (RPE) are long lived organelles that may undergo photobleaching with aging, which can diminish the antioxidant efficiency of melanin. Here, isolated porcine RPE melanosomes were experimentally photobleached with visible light to simulate aging and compared with untreated granules or control particles (black latex beads) for their effects on the survival of photically stressed ARPE-19 cultures. Particles were delivered to cultures for uptake by phagocytosis then cells were exposed to violet light and analyzed by a new live cell imaging method to identify the time of apoptotic blebbing as a dynamic measure of reduced cell survival. Results indicated that untreated melanosomes did not decrease photic injury to ARPE-19 cells when compared with cells lacking particles or with cells containing control particles, as might be expected if melanin performed an antioxidant function. Instead cells with untreated melanosomes showed reduced survival indicated by an earlier onset of blebbing and a lower fraction of surviving cells after photic stress. Cell survival was reduced even further in stressed cells containing melanosomes that were photobleached, and survival decreased with increasing photobleaching time. Photobleaching of RPE melanosomes therefore makes cells containing them more sensitive to light-induced cytotoxicity. This observation raises the possibility that aged melanosomes increase RPE cell photic stress in situ, perhaps contributing to reduced tissue function and to degeneration of the adjacent retina that the RPE supports. How melanosomes (photobleached or not) interact with their local subcellular environment to modify RPE cell survival is poorly understood and is likely determined by the physicochemical state of the granule and its constituent melanin. The live cell imaging method introduced here, which permitted detection of a graded effect of photobleaching, provides a sensitive bioassay for probing the effects of melanosome modifications.     

Effects of photodegradation on the physical and antioxidant properties of melanosomes isolated from retinal pigment epithelium

Melanosomes of the retinal pigment epithelium (RPE) are relatively long-lived organelles that are theoretically susceptible to changes induced by exposure to visible light. Here melanosomes were isolated from porcine RPE cells and subjected to high intensity visible light to determine the effects of illumination on melanosome structure and on the content and antioxidant properties of melanin. As compared to untreated melanosomes, illuminated granules showed morphologic changes consistent with photodegradation, which included variable reductions in electron density demonstrated by transmission electron microscopy (TEM), and particle fragmentation and surface disruption revealed by scanning electron microscopy (SEM) and atomic force microscopy. Illuminated melanosomes had lower melanin content, indicated by measures of absorbance and electron spin resonance (ESR) signal intensity, and reduced ability to bind iron, shown by chemical and ESR analyses. Compared to untreated melanosomes, ESR-spin trapping analyses further indicated that illuminated melanosomes show increased photogeneration of superoxide anion and reduced ability to inhibit the iron ion-catalyzed free radical decomposition of hydrogen peroxide. It appears therefore that visible light irradiation can disrupt the structure of RPE melanosomes and reduce the amount and antioxidant properties of melanin. Some of these changes occur in human RPE melanosomes with aging and the results obtained here suggest that visible light irradiation is at least partly responsible. The consequence of light-induced changes in RPE melanosomes may be a diminished capacity of melanin to help protect aged cells from oxidative damage, perhaps increasing the risk of diseases with an oxidative stress component such as age-related macular degeneration.     

In vitro assays for evaluating the ultraviolet B-induced damage in cultured human retinal pigment epithelial cells

The present study demonstrates broadband UV-B-induced damage of cultured human retinal pigment epithelial cells as an effort to develop an in vitro model that can be used, along with in vivo research and other in vitro efforts, to evaluate the need for retinal UV protection in humans after cataract removal. The human retinal pigment epithelial cell line, ARPE-19, was cultured in two groups: control and treated. Treated cells were irradiated with three broadband UVB radiations at energy levels of 0.05, 0.1 and 0.2J/cm(2). After irradiation, cells were incubated for 48h while cellular viability, morphology, and phagocytotic activity were analyzed using the Alamar blue assay, confocal microscopy, and fluorescent microspheres. Confocal analysis concentrated on the study of the cell nuclei and mitochondria. The Alamar blue assay of UV-B-exposed cells showed dose and time-dependent decreases in cellular viability in comparison to control cells. Loss of cell viability was measured at the two higher energy levels (0.2 and 0.1J/cm(2)), but the cell group exposed to 0.05J/cm(2) showed no significant viability change at 1-h time point. Morphological evaluation also showed dose and time-dependent degradation of mitochondria and nucleic acids. Cells exposed with 0.05J/cm(2) UVB did not show significant degradation of mitochondria and nucleic acids during the entire culture period. Phagocytotic activity assay data for UVB-exposed cells showed dose-dependent decreases in phagocytotic activity in comparison with the control cells. The control cells have significantly greater capacities for uptake than the 0.1 and 0.2J/cm(2) UV-B-exposed cells, while the 0.05J/cm(2) UV-B-exposed cell group showed no significant difference from the control cell group. The findings suggest that UVB radiation-induced cultured RPE cell damage can be evaluated by assays that probe cellular viability, morphological change, and phagocytotic activity, and that these assay methods together provide a valuable in vitro model for ultraviolet radiation-induced retinal toxicology research.     

Anthocyanins protect against A2E photooxidation and membrane permeabilization in retinal pigment epithelial cells

The pyridinium bisretinoid A2E, an autofluorescent pigment that accumulates in retinal pigment epithelial cells with age and in some retinal disorders, can mediate a detergent-like perturbation of cell membranes and light-induced damage to the cell. The photodynamic events initiated by the sensitization of A2E include the generation of singlet oxygen and the oxidation of A2E at carbon-carbon double bonds. To assess the ability of plant-derived anthocyanins to modulate adverse effects of A2E accumulation on retinal pigment epithelium (RPE) cells, these flavylium salts were isolated from extracts of bilberry. Nine anthocyanin fractions reflecting monoglycosides of delphinidin, cyanidin, petunidin and malvidin were obtained and all were shown to suppress the photooxidation of A2E at least in part by quenching singlet oxygen. The anthocyanins tested exhibited antioxidant activity of variable efficiency. The structural characteristics relevant to this variability likely included the ability to form a stable quinonoidal anhydro base at neutral pH, a conjugated diene structure in the C (pyrane) ring, the presence of hydroxyl groups on the B (benzene) ring and the relative hydrophobicity conferred by the arrangement of substituents on the B ring. Cells that had taken up anthocyanins also exhibited a resistance to the membrane permeabilization that occurs as a result of the detergent-like action of A2E.     

Study of the morphological and adhesion properties of collagen fibers in the Bruch's membrane

The Bruch's membrane is located beneath the retina in vertebrate eyes. We have used atomic force microscopy to examine the morphological and adhesion properties of collagen fibers located in different portions of the membrane. The D-periodicity of the fibers was 62.54 +/- 4.25 nm and 63.78 +/- 4.14 nm for regions away from the optic nerve and close to it, respectively. The adhesion properties of the collagen fibers were evaluated using force volume imaging on a number of different eye samples. The adhesion force we recorded in regions away from the optic nerve was different compared to regions close to the optic nerve. The reported results allow us to understand the nanoscopic properties of connective tissues in the eye and are important for the design of new and improved biomaterials.     

Nitric oxide-dependent pigment migration induced by ultraviolet radiation in retinal pigment cells of the crab Neohelice granulata

The purpose of this study was to verify the occurrence of pigment dispersion in retinal pigment cells exposed to UVA and UVB radiation, and to investigate the possible participation of a nitric oxide (NO) pathway. Retinal pigment cells from Neohelice granulata were obtained by cellular dissociation. Cells were analyzed for 30 min in the dark (control) and then exposed to 1.1 and 3.3 J cm⁻² UVA, 0.07 and 0.9 J cm⁻² UVB, 20 n m β-PDH (pigment dispersing hormone) or 10 μ m SIN-1 (NO donor). Histological analyses were performed to verify the UV effect in vivo . Cultured cells were exposed to 250 μ m L-NAME (NO synthase blocker) and afterwards were treated with UVA, UVB or β-PDH. The retinal cells in culture displayed significant pigment dispersion in response to UVA, UVB and β-PDH. The same responses to UVA and UVB were observed in vivo . SIN-1 did not induce pigment dispersion in the cell cultures. l-NAME significantly decreased the pigment dispersion induced by UVA and UVB but not by β-PDH. All retinal cells showed an immunopositive reaction against neuronal nitric oxide synthases. Therefore, UVA and UVB radiation are capable of inducing pigment dispersion in retinal pigment cells of Neohelice granulata and this dispersion may be nitric oxide synthase dependent.     

Comparative study of the dark and light-induced toxicity of lipofuscin granules from human retinal pigment epithelium and their chromophore A2E on the cardiolipin liposome model

The damaging effect of lipofuscin granules from the human retinal pigment epithelium and fluorophore A2E was studied on models of calcein- and ascorbate-loaded cardiolipin liposomes and outer segments of the bovine eye photoreceptor cells in dark and under visible light irradiation. In dark fluorophore A2E induces the release of calcein from calcein-loaded liposomes and reduces the lifetime of the artificial bilayer lipid membrane prepared from dioleyl phosphatidilcholine. A similar detergent-like action A2E exhibits towards ascorbate-loaded liposomes, significantly accelerating the release of ascorbate in dark. In the presence of A2E, irradiation with the full visible light (390?C700 nm) stimulates both the release of ascorbate from liposomes and accelerates the destruction of the bilayer lipid membrane. Retinal pigment epithelium lipofuscin granules also accelerate the release of ascorbate from ascorbate-loaded liposomes under visible light irradiation; the blue light (457.9 nm) was twice as more efficient as the green light (514.5 nm). The preliminary irradiation of A2E with the visible light decreases its detergent-like action on the cardiolipin liposomal membranes under the dark conditions and the photosensitizing effect on the lipid peroxidation of the outer segments of photoreceptor cells. Unlike A2E, the visible light irradiation of a suspension of lipofuscin granules under similar conditions does not noticeably decrease their sensitizing activity towards lipid peroxidation. It is assumed that the phototoxicity of retinal pigment epithelium lipofuscin granules is related not only to A2E in their composition, but depends mainly on the content of other photosensitizers (chromophores) in the granules.     

Hormonal and dim light effects in retinal photodamage.

Abstract—Exposure of albino rats to continuous visible dim light results in the rapid. but graded. loss of photoreceptor cells. The age at which albino rats are exposed to light influences the degree of photoreceptor damage. The severity of cell destruction per unit exposure time gradually increases in rats exposed at different ages from postweaning to adulthood, and by 8 weeks of age, reduction in the thickness of the outer nuclear layer (photoreceptor cell nuclei) is highly significant. The period of increasing susceptibility to light damage is coincident with the occurrence of sexual maturation, which suggests a relationship between photoreceptor destruction and the maturation of the pituitary gland-gonadal axis. To determine whether the absence of the pituitary gland and ovarian hormones influences photoreceptor damage, animals were either hypophysectomized or ovariectomized and exposed to continuous illumination for up to 45 days. The degree of photoreceptor destruction is significantly reduced in operated, as compared with sham-operated control, rats exposed to an identical light regimen. Small, physiologic doses of estradiol reverse the protective effects of ovariectomy, but larger, pharmacologic doses do not. Retinal damage is significantly enhanced by injection of pituitary homogenates during the light-exposure period and by transplantation of pituitary glands under the kidney capsule of rats in which the pituitary has been removed. Pituitary glands transplanted in this location produce high levels of prolactin, while other pituitary hormones are found at unusually low, and sometimes nonfunctional, levels. Prolactin administration to rats during the light exposure period effectively reverses the protection afforded by removal of the pituitary gland. Pituitary gland hormones as well as hormones from some of the pituitary target organs, appear to have a regulatory influence on the severity of light-induced photoreceptor damage in this species.     

Mice spermatogonial stem cells transplantation induces macrophage migration into the seminiferous epithelium and lipid body formation: high-resolution light microscopy and ultrastructural studies

Transplantation of spermatogonial stem cells (SSCs), the male germline stem cells, in experimental animal models has been successfully used to study mechanisms involved in SSC self-renewal and to restore fertility. However, there are still many challenges associated with understanding the recipient immune response for SSCs use in clinical therapies. Here, we have undertaken a detailed structural study of macrophages elicited by SSCs transplantation in mice using both high-resolution light microscopy (HRLM) and transmission electron microscopy (TEM). We demonstrate that SSCs transplantation elicits a rapid and potent recruitment of macrophages into the seminiferous epithelium (SE). Infiltrating macrophages were derived from differentiation of peritubular monocyte-like cells into typical activated macrophages, which actively migrate through the SE, accumulate in the tubule lumen, and direct phagocytosis of differentiating germ cells and spermatozoa. Quantitative TEM analyses revealed increased formation of lipid bodies (LBs), organelles recognized as intracellular platforms for synthesis of inflammatory mediators and key markers of macrophage activation, within both infiltrating macrophages and Sertoli cells. LBs significantly increased in number and size in parallel to the augmented macrophage migration during different times post-transplantation. Our findings suggest that LBs may be involved with immunomodulatory mechanisms regulating the seminiferous tubule niche after SSC transplantation.     

Evaluation of the photosensitizer Tookad for photodynamic therapy on the Syrian golden hamster cheek pouch model: light dose, drug dose and drug-light interval effects

We have evaluated the efficacy of the new photosensitizer (PS) Tookad in photodynamic therapy (PDT) in vivo. This PS is a palladium-bacteriopheophorbide presenting absorption peaks at 762 and 538 nm. The light dose, drug dose and drug injection-light irradiation interval (DLI), ranging between 100 and 300 J/cm2, 1 and 5 mg/kg and from 10 to 240 min, respectively, were varied, and the response to PDT was analyzed by staging the macroscopic response and by the histological examination of the sections of the irradiated cheek pouch. The level of PDT response, macroscopically and histologically, shows a strong dependence on the DLI, light dose and drug dose at the applied conditions in the normal hamster cheek pouch. A decay of the tissular response with increasing DLI is observed corresponding to a time of half-maximum response ranging from 10 to 120 min, depending on drug dose and light dose. The tissues affected at the lowest doses are predominantly the vascularized diffuse connective tissue situated between the inner and outer striated muscle (SM) layers as well as these muscle layers themselves. The highest response at the shortest DLI and the absence of a measurable response at DLI longer than 240 min at 300 J/cm2 and drug dose of 5 mg/kg are characteristics of a predominantly vascular effect of this PS. This observation suggests that Tookad could be effective in PDT of vascularized lesions or pathologies associated with the proliferation of neovessels.     

Functional expression, targeting and Ca2+ signaling of a mouse melanopsin-eYFP fusion protein in a retinal pigment epithelium cell line

Melanopsin, first discovered in Xenopus melanophores, is now established as a functional sensory photopigment of the intrinsically photosensitive retinal ganglion cells. These ganglion cells drive circadian rhythm and pupillary adjustments through projection to the brain. Melanopsin shares structural similarities with all known opsins. Comprehensive characterization of melanopsin with respect to its spectral properties, photochemical cascade and signaling partners requires a suitable recombinant system and high expression levels. This combination has not yet been described. To address this issue, we have expressed recombinant mouse melanopsin in several cell lines. Using enhanced yellow fluorescent protein (eYFP) as a visualization tag, expression was observed in all cell lines. Confocal microscopy revealed that melanopsin was properly routed to the plasma membrane only in retinal pigment epithelium (RPE)-derived D407 cells and in human embryonic kidney (HEK) cells. Further, we performed intracellular calcium measurements in order to probe the melanopsin signaling activity of this fusion protein. Transfected cells were loaded with the calcium indicator Fura2-AM. Upon illumination, an immediate but transient calcium response was observed in HEK as well as in D407 cells, while mock-transfected cells showed no calcium response under identical conditions. Supplementation with 11-cis retinal or all-trans retinal enhanced the response. After prolonged illumination the cells became desensitized. Thus, RPE-derived cells expressing recombinant melanopsin may constitute a suitable system for the study of the structural and functional characteristics of melanopsin.     

Comparisons of the structural and chemical properties of melanosomes isolated from retinal pigment epithelium, iris and choroid of newborn and mature bovine eyes

Melanosomes were isolated from the retinal pigment epithelium (RPE), iris and choroid of mature (age >2 years) and newborn (age <1 week) bovine eyes. Scanning electron microscopy was utilized to analyze the morphology of the melanosomes, which were found to vary among different tissues and different ages. While the total content of amino acids differs slightly (ranging from 9% to 15% by mass), the distributions of the amino acids are similar. The pheomelanin content is low in the choroid and the RPE (0.1-0.5%), and moderate in the iris (<2%); therefore, the major melanin component of bovine eye melanosomes is eumelanin, independent of the shape of the melanosomes. The yields of pyrrole-2,3,5-tricarboxylic acid from melanosomes decrease in the following order: choroid > iris > RPE, and exhibit decreasing yields with age. 13C solid-state nuclear magnetic resonance (NMR) spectroscopic analysis of iris and choroid melanosomes indicates the same trends. These observations suggest that the 5,6-dihydroxyindole-2-carboxylic acid contents decrease in the following order: choroid > iris > RPE, and decrease with age. Moreover, the 13C solid-state NMR spectra show (1) for the same age samples, the CH:Cq ratio for choroid is larger than that for iris melanosomes; and (2) an increase in the concentration of carbonyl groups with age within each type of melanosome.     

Phototoxicity in human retinal pigment epithelial cells promoted by hypericin, a component of St. John's wort

St. John's wort (SJW), an over-the-counter antidepressant, contains hypericin, which absorbs light in the UV and visible ranges. In vivo studies have determined that hypericin is phototoxic to skin and our previous in vitro studies with lens tissues have determined that it is potentially phototoxic to the human lens. To determine if hypericin might also be phototoxic to the human retina, we exposed human retinal pigment epithelial (hRPE) cells to 10(-7) to 10(-5) M hypericin. Fluorescence emission detected from the cells (lambda(ex) = 488 nm; lambda(em) = 505 nm) confirmed hypericin uptake by human RPE. Neither hypericin exposure alone nor visible light exposure alone reduced cell viability. However when irradiated with 0.7 J cm(-2) of visible light (lambda > 400 nm) there was loss of cell viability as measured by MTS and lactate dehydrogenase assays. The presence of hypericin in irradiated hRPE cells significantly changed the redox equilibrium of glutathione and a decrease in the activity of glutathione reductase. Increased lipid peroxidation as measured by the thiobarbituric acid reactive substances assay correlated to hypericin concentration in hRPE cells and visible light radiation. Thus, ingested SJW is potentially phototoxic to the retina and could contribute to retinal or early macular degeneration.     

UV-induced changes in pigment content and light penetration in the fruticose lichen Cladonia arbuscula ssp. mitis

The response of the lichen, Cladonia arbuscula (Wallr.) Flot. ssp. mitis (Sandst.) Ruoss to enhanced UV-B (280-315 nm) radiation was investigated with respect to: (a) changes in phenolic content; (b) differential pigment accumulation under visible and UV radiation with increasing distance from thallus apices; and (c) the internal distribution of UV-B radiation within the thallus measured with quartz optical fibres. In a short-term experiment, lichens were exposed for 7 days in a growth chamber to visible light with or without additional UV-B radiation. For a longer term experiment, lichens were grown outdoors under both natural UV radiation, and supplemental UV-A (315-400 nm)+UV-B provided by lamps. Controls were placed under filters that removed the radiation below 290 nm from the natural sunlight. The concentration of total phenolic compounds was measured spectrophotometrically at the termination of the experiments, in different parts of the lichen podetia. UV-exposed lichens showed increased accumulation of phenolics compared to those not grown under UV. At the termination of the long-term experiment, fibre optic measurements of the penetration of radiation into lichen thallus reflected the influence of growth under UV radiation, whereby UV was more strongly attenuated as compared to that in lichens not exposed to enhanced levels of UV-B radiation. Results indicated that in Cladonia, UV-B radiation induces the accumulation of phenolic compounds that may have a protective role. In addition, the morphological distribution of phenolic compounds was different under visible and supplemental UV-B radiation. Internal radiation measurements served to visualise the attenuation of radiation with thallus depth for different wavelengths in the UV-B waveband.     

EPS biofouling in membrane filtration: an analytic modeling study

Biofouling is theoretically investigated by modeling solute transport within a biofilm, defined in this study as a swarm of solid biocolloids surrounded by liquid-like exopolymeric substances (EPS). A mathematical approach is employed to map the biofilm to an equivalent, simple spherical cell using a self-consistent method. It is found that the physical presence of EPS and their reaction with solute ions reduce the mass transfer coefficient, which significantly contributes to permeate flux decline in reverse osmosis and nanofiltration membrane processes.