In this issue, we offer a symposium-in-print that is focused on several new advancements in fundamental research related to the family of GFP (green fluorescent protein)-like proteins. A few applied aspects are also included to illustrate the impact this amazing set of colored proteins has made on our understanding of cell biology at the molecular level. The six articles presented here cut across several disciplines ranging from biological function to protein structure to photophysical aspects. These highly original pieces of work include both experimental and computational approaches, and will provide the reader with significant insight into current, state-of-the-art research activities in this very dynamic and fast-paced field. In the first part of this perspective, I will give a brief overview of the history and salient features of GFPs, cite some examples that illustrate their impact on biotechnology, and provide a brief review of the structural and chemical features that lend these proteins their fascinating appearance. In the second part, I will introduce each of the peer-reviewed contributions of the participating authors.

Using in situ spectrometry data and visual system modeling, we investigate whether the colors conferred to the reef-building corals by GFP-like proteins would look colorful not only to humans, but also to fish occupying different ecological niches on the reef. Some GFP-like proteins, most notably fluorescent greens and nonfluorescent chromoproteins, indeed generate intense color signals. An unexpected finding was that fluorescent proteins might also make corals appear less colorful to fish, counterbalancing the effect of absorption by the photosynthetic pigments of the endosymbiotic algae, which might be a form of protection against herbivores. We conclude that GFP-determined coloration of corals may be an important factor in visual ecology of the reef fishes.

EosFP is a fluorescent protein from the coral Lobophyllia hemprichii that changes its fluorescence emission from green to red upon irradiation with near-UV light. Here we present the spectroscopic properties of wild-type EosFP and a variety of monomeric and dimeric mutants and provide a structural interpretation of its oligomerization and photoconversion, which is based on X-ray structure analysis of the green and red species that we reported recently. Because functional expression of the monomeric EosFP variant is limited to temperatures of 30°C, we have developed a tandem dimer. This construct, in which two EosFP subunits are connected by a flexible 12 amino acid linker, expresses well after fusion with the androgen and endothelin A receptors at 37°C. A variety of applications in cellular imaging, developmental biology and automated high-content screening applications are presented, which demonstrate that EosFP is a powerful tool for in vivo monitoring of cellular processes.

The pocilloporin Rtms5 and an engineered variant Rtms5^H146S undergo distinct color transitions (from blue to red to yellow to colorless) in a pH-dependent manner. pK_a values of 4.1 and 3.2 were determined for the blue (absorption λ_max, 590 nm) to yellow (absorption λ_max, ∼453 nm) transitions of Rtms5 and Rtms5^H146. The pK_a for the blue-yellow transition of Rtms5^H146S increased by 1.4 U in the presence of 0.1 M KI, whereas the pK_a for the same transition of Rtms5 was relatively insensitive to added halides. To understand the structural basis for these observations, we have determined to 2.0 Å resolution the crystal structure of a yellow form of Rtms5^H146S at pH 3.5 in the presence of iodide. Iodide was found occupying a pocket in the structure with a pH of 3.5, forming van der Waals contacts with the tyrosyl moiety of the chromophore. Elsewhere, it was determined that this pocket is occupied by a water molecule in the Rtms5^H146S structure (pH 8.0) and by the side chain of histidine 146 in the wild-type Rtms5 structure. Collectively, our data provide an explanation for the observed linkage between color transitions for Rtms5^H146S and binding to halides.

In the ground state of the highly conjugated green fluorescent protein (GFP), the chromophore should be planar. However, numerous crystal structures of GFP and GFP-like proteins have been reported with slightly twisted chromophores. We have previously shown that the protein cavity surrounding the chromophore in wild-type GFP is not complementary with a planar chromophore. This study shows that the crystal structure of wild-type GFP is not an anomaly: most of the GFP and GFP-like proteins in the protein databank have a protein matrix that is not complementary with a planar chromophore. When the π-conjugation across the ethylenic bridge of the chromophore is removed the protein matrix will significantly twist the freely rotating chromophore from the relatively planar structures found in the crystal structures. The possible consequences of this nonplanar deformation on the photophysics of GFP are discussed. A volume analysis of the cis-trans-isomerization of HBDI, a GFP chromophore model compound, reveals that its hula-twist motion is volume conserving. This means that, if the GFP chromophore or GFP chromophore model compounds undergo a cis-trans-isomerization in a volume-constricting medium, such as a protein matrix or viscous liquid, it will probably isomerize by means of a HT–type motion.

The time-resolved emission spectra of wild-type green fluorescent protein (wtGFP) and the T203V GFP mutant have been recorded with picosecond time resolution, allowing the separate characterization of the two spectral components associated with the neutral and anionic forms of the GFP chromophore. Significantly, neither component shifts as a function of time. It is suggested that the absence of spectral shift is a result of highly restricted movement of the protein residues in the vicinity of the chromophore. The shapes of the separated spectra are discussed and their relative ratio analyzed in a steady-state analysis.

The spectral evolution of three photoactive proteins has been investigated by measuring the fluorescence with good temporal and wavelength resolution and a high signal-to-noise ratio. Upon excitation at 400 nm wild-type (wt) PYP both at neutral pH and in the low-pH blueshifted pBdark state exhibited a strong quenching of the fluorescence, the major part of which could be described by lifetimes of about 1.7 and 7.7 ps. The remaining fluorescence decay occurred multiexponentially with lifetimes between 30 and 125 ps. Additionally, in wtPYP at neutral pH, a dynamic Stokes shift was found to occur with a time constant of about 0.25 ps. In a PYP preparation that was reconstituted with the chromophore 7-hydroxy-coumarin-3- carboxylic acid rather than the native coumaric acid, and which is therefore not capable of performing the cis-trans-isomerization that initiates the photocycle in wtPYP, the fluorescence was found to decay multiexponentially with lifetimes of 51 ps, 0.33 and 3.77 ns. Additionally, dynamic Stokes shifts were observed with time constants of about 0.1 and 3.5 ps. Upon comparison of the dynamics of this preparation with that of wtPYP the multiexponential decay with lifetimes of 1.7 and 7.7 ps found in wtPYP was attributed to photochemistry of the p-coumaric-acid chromophore. The emission from bacteriorhodopsin mutant D85S upon excitation at 635 nm decays biexponentially with estimated lifetimes of 5.2 and 19.1 ps. No dynamic Stokes shift was observed here. Four lifetimes were needed to describe the decay of the emission from the A* state in the green fluorescent protein. From a target analysis it was concluded that the longer lifetimes are accompanied by a decreasing probability of forming I*, which approaches zero with the longest A* lifetime of 1.5 ns. These observations may be explained by heterogeneity of A and by relaxation of A*. In all three systems studied, multiexponential decay of emission was present, suggesting that heterogeneity is a common feature of these chromophore protein complexes.

UV radiation (UVR) is a significant ecological factor in the marine environment that can have important effects on planktonic organisms and dissolved organic matter (DOM). The penetration of UVR into the water column is likely to change in the near future due to interactions between global warming and ozone depletion. In this study we report underwater instruments employed for the measurement of UVR and we review data dealing with the depth of UVR penetration in different oceanic areas including the open ocean, Antarctic waters and coastal waters. We provide the 10% irradiance depth (Z10%) for UV-A and UV-B as well as for DNA damage effective dose (DNA), which we calculated from the values of diffuse attenuation coefficients or vertical profiles reported in the literature. We observe a clear distinction between open ocean (high Z10%, no variation in the ratio UV-B/UV-A), Antarctic waters (increase in the ratio UV-B/UV-A during ozone hole conditions) and coastal waters (low Z10%, no variation in the ratio UV-B/UV-A). These variations in the penetration of UVR could lead to differences in the relative importance of photobiological/photochemical processes. We also compare in this study the penetration of UV-B (unweighted and weighted by the Setlow action spectrum) and DNA damage effective dose.

UVA is the major portion (90–99%) of solar radiation reaching the surface of the earth and has been described to lead to formation of benign and malignant tumors. UVA-mediated cellular damage occurs primarily through the release of reactive oxygen species and is responsible for immunosuppression, photodermatoses, photoaging and photocarcinogenesis. Pomegranate fruit extract (PFE) possesses strong antioxidant and anti-inflammatory properties. Our recent studies have shown that PFE treatment of normal human epidermal keratinocytes (NHEK) inhibits UVB-mediated activation of MAPK and NF-κB pathways. Signal transducers and activators of transcription 3 (STAT3), Protein Kinase B/AKT and Map Kinases (MAPKs), which are activated by a variety of factors, modulate cell proliferation, apoptosis and other biological activities. The goal of this study was to determine whether PFE affords protection against UVA-mediated activation of STAT3, AKT and extracellular signal–regulated kinase (ERK1/2). Immunoblot analysis demonstrated that 4 J/cm² of UVA exposure to NHEK led to an increase in phosphorylation of STAT3 at Tyr⁷⁰⁵, AKT at Ser⁴⁷³ and ERK1/2. Pretreatment of NHEK with PFE (60–100 μg/mL) for 24 h before exposure to UVA resulted in a dose-dependent inhibition of UVA-mediated phosphorylation of STAT3 at Tyr⁷⁰⁵, AKT at Ser⁴⁷³ and ERK1/2. mTOR, structurally related to PI3K, is involved in the regulation of p70S6K, which in turn phosphorylates the S6 protein of the 40S ribosomal subunit. We found that UVA radiation of NHEK resulted in the phosphorylation of mTOR at Thr²⁴⁴⁸ and p70S6K at Thr⁴²¹/Ser⁴²⁴. PFE pretreatment resulted in a dose-dependent inhibition in the phosphorylation of mTOR at Thr²⁴⁴⁸ and p70S6K at Thr⁴²¹/Ser⁴²⁴. Our data further demonstrate that PFE pretreatment of NHEK resulted in significant inhibition of UVA exposure–mediated increases in Ki-67 and PCNA. PFE pretreatment of NHEK was found to increase the cell-cycle arrest induced by UVA in the G1 phase of the cell cycle and the expression of Bax and Bad (proapoptotic proteins), with downregulation of Bcl-X_L expression (antiapoptotic protein). Our data suggest that PFE is an effective agent for ameliorating UVA-mediated damages by modulating cellular pathways and merits further evaluation as a photochemopreventive agent.

In studies involving mice in which doses of UVA (320–400 nm) and UVB (290–320 nm) radiation were administered alone or combined sequentially, we observed a protective effect of UVA against UVB-induced erythema/edema and systemic suppression of contact hypersensitivity. The UVA immunoprotection was mediated by the induction of the stress enzyme heme oxygenase-1 (HO-1) in the skin, protection of the cutaneous Th1 cytokines interferon-γ (IFN-γ) and IL-12 and inhibition of the UVB-induced expression of the Th2 cytokine IL-10. In this study, we seek evidence for an immunological waveband interaction when UVA and UVB are administered concurrently to hairless mice as occurs during sunlight exposure in humans. A series of spectra providing varying ratios of UVA/UVB were developed, with the UVA ratio increased to approximately 3.5 times the UVA component in solar simulated UV (SSUV). We report that progressively increasing the UVA component of the radiation while maintaining a constant UVB dose resulted in a reduction of both the erythema/edema reaction and the degree of systemic immunosuppression, as measured as contact hypersensitivity. The UVA-enhanced immunoprotection was abrogated in mice treated with a specific HO enzyme inhibitor. UVA-enhanced radiation also upregulated the expression of cutaneous IFN-γ and IL-12 and inhibited expression of both IL-6 and IL-10, compared with the activity of SSUV. The results were consistent with the previously characterized mechanisms of photoprotection by the UVA waveband alone and suggest that the UVA component of solar UV may have beneficial properties for humans.

Laser photothermal therapy of cancer with the use of gold nanoparticles immunotargeted to molecular markers on the cell surface has been shown to be an effective modality to selectively kill cancer cells at much lower laser powers than those needed for healthy cells. To elucidate the minimum light dosimetry required to induce cell death, photothermal destruction of two cancerous cell lines and a noncancerous cell line treated with antiepidermal growth factor receptor (anti-EGFR) antibody-conjugated gold nanoparticles is studied, and a numerical heat transport model is used to estimate the local temperature rise within the cells as a result of the laser heating of the gold nanoparticles. It is found that cell samples with higher nanoparticle loading require a lower incident laser power to achieve a certain temperature rise. Numerically estimated temperatures of 70–80°C achieved by heating the gold particles agree well with the measured threshold temperature for destruction of the cell lines by oven heating and those measured in an earlier nanoshell method. Specific binding of anti-EGFR antibody to cancerous cells overexpressing EGFR selectively increases the gold nanoparticle loading within cancerous cells, thus allowing the cancerous cells to be destroyed at lower laser power thresholds than needed for the noncancerous cells. In addition, photothermal therapy using gold nanoparticles requires lower laser power thresholds than therapies using conventional dyes due to the much higher absorption coefficient of the gold nanoparticles.

The importance of conidial pigmentation to solar UV radiation tolerance in the entomopathogenic fungus Metarhizium anisopliae var. anisopliae, was estimated by comparing the effects of exposure to simulated solar UV radiation on the wild-type parent strain U.S. Department of Agriculture (USDA)–Agricultural Research Service (ARS) Collection of Entomopathogenic Fungal Cultures (ARSEF) 23, which has dark green conidia, and three groups of color mutants with yellow, purple and white conidia. The comparisons included inactivation levels and the kinetics of germination of conidia exposed or not exposed to simulated solar UV radiation. In addition to significantly inactivating the conidia of different mutants, exposure to radiation delayed for several hours the germination of surviving conidia of the wild type and all mutants. In general, mutants with white conidia were more sensitive to simulated solar UV radiation than mutants with purple conidia, which were more sensitive than mutants with yellow conidia, which in turn were more sensitive than the green wild strain. A significant variation in tolerance to simulated solar radiation was observed among mutants within each color group, particularly among mutants with yellow conidia. Revertants with green conidia, DWR 179 and DWR 176, were obtained from the very sensitive UV mutants DWR 148 (yellow conidia) and DWR 149 (purple conidia), respectively. These revertants had levels of tolerance to simulated solar UV radiation similar to those of the wild-type ARSEF 23. This observation is strong evidence of the importance of green conidial pigmentation for tolerance to simulated solar UV radiation, a factor that could be manipulated to produce M. anisopliae strains with more tolerance to solar UV radiation.

Inhibition of c-Jun N-terminal kinase (JNK) with the pharmacologic inhibitor SP600125 in UVA-irradiated HaCaT cells and human primary keratinocytes resulted in dramatic phenotypic changes indicative of cell death. These phenotypic changes correlated with caspase 8, 9 and 3 activations as well as cleavage of the caspase substrate polyADP-ribose polymerase (PARP). Morphologic analysis and analysis of sub-G₀ DNA content confirmed apoptotic cell death in these keratinocytes after combination treatment. Addition of the general caspase inhibitor zVAD-fmk to combination-treated HaCaT cells was able to completely block caspase activation, PARP cleavage, the increase in sub-G₀ DNA content and the classic morphologic features of apoptosis, indicating that this combination treatment resulted in caspase-dependent apoptotic cell death. zVAD-fmk treatment of primary keratinocytes was able to completely inhibit caspase activation and PARP cleavage, reduce morphologic apoptosis at lower concentrations of SP600125 and decrease the sub-G₀ DNA content detected after UVA SP600125 treatment. However, cell death and a significant amount of debris was still detected after caspase inhibitor treatment, particularly with 125 nM SP600125. At subconfluent conditions and low passage, primary keratinocytes were more sensitive to UVA irradiation alone than HaCaT cells. In conclusion, we have observed that inhibition of UVA-induced JNK activity with the pharmacologic inhibitor SP600125 resulted in caspase-dependent apoptotic cell death in both the immortalized keratinocyte cell line HaCaT and primary keratinocytes. However, the increased sensitivity of primary keratinocytes to experimental stress may have also resulted in direct cellular injury and caspase-independent cell death.

In the present study we investigated the photosensitizing properties of two novel mono- and bis-cyclodextrin tetrakis (pentafluorophenyl) porphyrin derivatives in several tumor cell lines and in BALB/c mice bearing subcutaneously transplanted syngeneic mouse mammary carcinoma 4T1. Both studied sensitizers were localized mainly in lysosomes and were found to induce cell death by triggering apoptosis in human leukemic cells HL-60. In 4T1 and other cell lines both apoptotic and necrotic modes of cell death occurred depending on drug and light doses. Mono-cyclodextrin porphyrin derivative P(β-CD)1 exhibited stronger in vitro phototoxic effect than bis-cyclodextrin derivative P(β-CD)2. However, in vivo P(β-CD)2 displayed faster tumor uptake with maximal accumulation 6 h after application, leading to complete and prolonged elimination of subcutaneous tumors within 3 days after irradiation (100 J cm⁻²). In contrast, P(β-CD)1 uptake was slower (48 h) and the reduction of tumor mass was only transient, reaching the maximum at the 12 h interval when a favorable tumor-to-skin ratio appeared. Thus, P(β-CD)2 represents a new photosensitizing drug displaying fast and selective tumor uptake, strong antitumor activity and fast elimination from the body.

Exposure to UV radiation up-regulates the synthesis of matrix metalloproteinases (MMPs), a group of matrix-degrading enzymes. MMPs are regarded as promising therapeutic targets and the development of effective inhibitors is an important research focus. The plant Pothomorphe umbellata has been shown to exert a potent antioxidant activity on the skin and to delay the onset and reduce the incidence of UVB-induced chronic skin damage. The aim of the present study was to determine the effect of P. umbellata ethanolic root extract on MMP-2 and MMP-9. The in vitro inhibition of MMP-2 and MMP-9 was measured by gelatin zymography in the presence of different concentrations of P. umbellata extract, as well as in the presence of its isolated active principle 4-nerolidylcatechol (4-NC). The inhibitory effect of the P. umbellata extract was stronger than that of 4-NC. Gelatin zymography and histological analysis revealed that P. umbellata was able to inhibit constitutive MMP-9 activity in vivo in mice sacrificed 2 h after UVB irradiation. The intensity of the MMP-2 band was unchanged. Our data contribute to the elucidation of the mechanism of prevention of photoaging by P. umbellata and may provide a rational basis for the use of this plant in prophylaxis against and treatment of skin cancer.

Photodynamic therapy (PDT), the use of light-activated drugs (photosensitizers), is an emerging treatment modality for tumors as well as various nononcologic conditions. Single-photon (1-γ) PDT is limited by low specificity of the photosensitizer, leading to damage to healthy tissue adjacent to the diseased target tissue. One solution is to use simultaneous two-photon (2-γ) excitation with ultrafast pulses of near-IR light. Due to the nonlinear interaction mechanism, 2-γ excitation with a focused beam is localized in three dimensions, allowing treatment volumes on the order of femtoliters. We propose that this will be valuable in PDT of age-related macular degeneration (AMD), which causes blindness due to abnormal choroidal neovasculature and which is currently treated by 1-γ PDT. Here, Photofrin has been used as the photosensitizer to demonstrate proof-of-principle of 2-γ killing of vascular endothelial cells in vitro. The 2-γ absorption properties of Photofrin were investigated in the 750–900 nm excitation wavelength range. It was shown that 2-γ excitation dominates over 1-γ excitation above 800 nm. The 2-γ absorption spectrum of Photofrin in the 800–900 nm excitation wavelength range was measured. The 2-γ cross section decreased from about 10 GM (1 GM = 10⁻⁵⁰ cm⁴ s/photon) at 800 nm to 5 GM at 900 nm. Adherent YPEN-1 endothelial cells were then incubated with Photofrin for 24 h and then treated by PDT at 850 nm where the 1-γ contribution was negligible. Cell death was monitored with the use of 2-γ scanning laser microscopy. The light doses required for killing were high (6300 J cm⁻² for ∼50% killing), but 2-γ cytotoxicity was unequivocally demonstrated. Although Photofrin is, per se, not a good choice for 2-γ PDT due to its low 2-γ cross section, this work provides baseline data to guide the development of novel photosensitizers with much higher 2-γ cross sections (>100 GM), which will be required for 2-γ PDT of AMD (and other conditions) to be clinically practical.

Photodynamic therapy using 5-aminolevulinic acid-induced protoporphyrin IX has been developed as a very useful therapeutic modality. Recently, several authors have reported on the efficacy of this procedure for acne. This approach is based on the fact that 5-aminolevulinic acid-induced protoporphyrin IX has strong selectivity for sebaceous glands. We used the immortalized human sebaceous gland cell line SZ95 to investigate cellular mechanisms of photodynamic therapy using 5-aminolevulinic acid-induced protoporphyrin IX. Quantification of induced protoporphyrin IX production showed dependence on the applied 5-aminolevulinic acid dose. When SZ95 sebocytes were differentiated by arachidonic acid treatment, there was no difference between them and the control cells with respect to both the amount of 5-aminolevulinic acid-induced protoporphyrin IX and the phototoxic effects. We altered protoporphyrin IX formation rates by growing cells scattered as single cells in the culture dishes. Single cells produced significantly lower protoporphyrin IX levels than those grown with intercellular contacts. Intracellular localization of protoporphyrin IX was imaged using confocal laser scanning microscopy. The differentiation-specific lipid droplets were virtually excluded from protoporphyrin IX fluorescence. In addition to weak mitochondrial and strong membrane fluorescence, distinctive spots with strong fluorescence were observed. These did not colocalize with fluorescent probes for mitochondria, lysosomes or the Golgi apparati.

Leukemia and lymphoma cells are much more sensitive to Merocyanine 540 (MC540)–mediated photodynamic therapy (PDT) than normal pluripotent hematopoietic stem cells and normal colony forming unit–granulocyte/macrophage progenitors (CFU-GM). By contrast, most solid tumor cells are only moderately sensitive to MC540-PDT. The limited activity against solid tumor cells has detracted from MC540's appeal as a broad-spectrum purging agent. We report here that noncytotoxic concentrations of amifostine (Ethyol, Ethiofos, WR-2721) and amphotericin B used either alone or in combination potentiate the MC540-sensitized photoinactivation of leukemia cells, wild-type small cell lung cancer cells and cisplatin-resistant small cell lung cancer cells. Amphotericin B also enhances the MC540-sensitized photoinactivation of normal CFU-GM, whereas amifostine protects CFU-GM against the cytotoxic action of MC540-PDT. The yield of CD34-positive normal hematopoietic stem and progenitor cells is only minimally diminished by pretreatment with amifostine, amphotericin B or combinations of amifostine plus amphotericin B. Purging protocols that combine MC540-PDT with amifostine or with amifostine plus amphotericin B could offer a simple and effective approach to the purging of autologous stem cell grafts that are contaminated with solid tumor cells or the purging of stem cell grafts from heavily pretreated leukemia patients that contain reduced numbers of normal stem and progenitor cells and, therefore, can ill afford additional losses caused by purging.

Pyocyanin (PyO⁻) (1-hydroxy-5-methylphenazine) is a cytotoxic compound secreted by Pseudomonas aeruginosa, an omnipresent bacterium and a human pathogen. We report that visible light illumination in the presence of rose bengal, or riboflavin, in aerated solutions (pH 7.0–7.2) induces irreversible loss of the pigment's characteristic absorption band at 690 nm, indicating its oxidation. This photobleaching was paralleled by generation of a multiline Electron Paramagnetic Resonance (EPR) spectrum attributed to a PyO⁻-derived radical. The reaction was dependent on the presence of air, sensitizers and light, was inhibited by sodium azide and was unaffected by ethanol. This suggests that PyO⁻ was oxidized largely via singlet oxygen and that hydroxyl radicals were not involved. The photochemically modified pigment was less efficient in oxidizing NAD(P)H and generated less superoxide (by ∼50%) than the intact PyO⁻, indicating its partial inactivation. 1-Methoxy-5-methylphenazine, a PyO⁻ analog in which the –O⁻ moiety was replaced by the methoxy group (–OMe), was resistant to oxidation, suggesting that oxidation of PyO⁻ involves its phenolate moiety. These results also suggest that photosensitization could be a potentially useful method for inactivation of PyO⁻ and, possibly, detoxification of superficial wounds (skin, eye) infected with P. aeruginosa.

The retinal pigment epithelium (RPE) is a monolayer of highly pigmented cells lining the inner aspect of Bruch's membrane. This pigmentation is due to eumelanin and a possible antioxidant role of melanin is reported here. The photo-oxidation of A2E, a constituent of RPE lipofuscin, leads to the sequential addition of up to nine oxygen atoms and/or the addition or loss of two hydrogen atoms. These photo-oxidations were investigated in the presence and absence of either calf or human RPE melanin in A2E-laden RPE cells. It was found that calf melanin was protective against the photo-oxidation of A2E, with an inhibition of oxidation of up to 50% in the case of the addition of two oxygen atoms. Calf melanin was also protective against blue light–induced damage to RPE cells. In addition this ability appears to decrease in humans as they grow older. With aging, a melanin-lipofuscin complex called melanolipofuscin forms. It is suggested that the oxidation or photo-oxidation of A2E in vivo may contribute to the age-related deterioration of the anti-oxidant role of RPE melanin and lead to various retinal disorders, such as age-related macular degeneration.

The cysteine residues of yeast alcohol dehydrogenase (YADH) were covalently modified by N-(1-pyrenyl) maleimide (PM). A maximum of 3.4 cysteines per YADH monomer could be modified by PM. The secondary structure of PM-YADH was found to be similar to that of the native YADH using far-UV circular dichroism. The covalent modification of YADH by PM inhibited the enzymatic activity indicating that the active site of the enzyme was altered. PM-YADH displayed maximum excimer fluorescence at an incorporation ratio of 2.6 mol of PM per monomeric subunit of YADH. Nucleotide adenine dinucleotide (NAD) divalent zinc and ethanol reduced the excimer fluorescence of PM-YADH indicating that these agents induce conformational changes in the enzyme. Guanidinium hydrochloride (GdnHCl)-induced unfolding of YADH was analyzed using tryptophan fluorescence, pyrene excimer fluorescence and enzymatic activity. The unfolding of YADH was found to occur in a stepwise manner. The loss of enzymatic activity preceded the global unfolding of the protein. Further, changes in tryptophan fluorescence with increasing GdnHCl suggested that YADH was completely unfolded by 2.5 M GdnHCl. Interestingly, residual structures of YADH were detected even in the presence of 5 M GdnHCl using the excimer fluorescence of PM-YADH.

The effect of cloud cover on the amount of solar UV radiation that reaches pedestrians under tree cover was evaluated with a three-dimensional canopy radiation transport model. The spatial distribution of UVB irradiance at the base of a regular array of spherical tree crowns was modeled under the full range of sky conditions. The spatial mean relative irradiance (I_r) and erythemal irradiance of the entire below-canopy domain and the spatial mean relative irradiance and erythemal irradiance in the shaded regions of the domain were determined for solar zenith angles from 15° to 60°. The erythemal UV irradiance under skies with 50% or less cloud cover was not remarkably different from that under clear skies. In the shade, the actual irradiance was greater under partly cloudy than under clear skies. The mean ultraviolet protection factor for tree canopies under skies with 50% or less cloud cover was nearly equivalent to that for clear sky days. Regression equations of spatially averaged I_r as a function of cloud cover fraction, solar zenith angle and canopy cover were used to predict the variation in erythemal irradiance in different land uses across Baltimore, MD.

RH421 is a widely used voltage-sensitive fluorescent membrane probe. Its exposure to continuous illumination with 577 nm light from an Hg lamp leads, however, to an increase in its steady-state fluorescence level when bound to lipid membranes. The increase occurs on the second time scale at typical light intensities and was found to be due to a single-photon excited-state isomerization. Modifications to the dye structure are, therefore, necessary to increase photochemical stability and allow wider application of such dyes in kinetic studies of ion-transporting membrane proteins. The related probe ANNINE 5, which has a rigid polycyclic structure, shows no observable photochemical reaction when bound to DMPC vesicles on irradiation with 436 nm light. The voltage sensitivity of ANNINE 5 was tested with the use of Na ,K -ATPase membrane fragments. As long as ANNINE 5 is excited on the far red edge of its visible absorption band, it shows a similar sensitivity to RH421 in detecting charge-translocating reactions triggered by ATP phosphorylation. Unfortunately the wavelengths necessary for ANNINE 5 excitation are in a region where the Hg lamps routinely used in stopped-flow apparatus have no significant lines available for excitation.

To determine the role of microwaves in the stress resistance of plants to enhanced ultraviolet-B (UV-B) radiation, Isatis indigotica Fort. seeds were subjected to microwave radiation for 8 s (wavelength 125 mm, power density 1.26 mW mm⁻², 2450 MHz). Afterwards they were cultivated in plastic pots in an artificial-glass greenhouse maintained at 25°C, 70% relative humidity, and 400 μmol mol⁻¹ CO₂, under visible-light conditions of 1500 μmol m⁻² s⁻¹ for 8 h day⁻¹. When the seedlings were 10 days old, they were subjected to 10.08 kJ m⁻² UV-B (PAR: 220 μmol m⁻² s⁻¹) radiation for 8 days. Changes in a number of physiological and biochemical characteristics and in the thermal decomposition enthalpy of biomass were measured and used as indicators of the protective capacity of microwave radiation in this experiment. Our results revealed that microwave pretreatment of seeds enhanced UV-B stress resistance in the seedlings by decreasing the concentration of malondialdehyde (MDA) and increasing the concentration of ascorbic acid (AsA) and UV-B–absorbing compounds, increasing the activities of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD), and increasing the energy accumulation of photosynthesis. All these results suggest that microwave radiation enhances plant metabolism and results in increased UV-B stress resistance. This is the first investigation reporting the use of microwave pretreatment to protect the cells of Isatis indigotica from UV-B-induced lesions.

The calibration of the erythemal irradiance measured by a Yankee Environmental System (YES) UVB-1 biometer is presented using two methods of calibration with a wide range of experimental solar zenith angles (SZAs) and ozone values. The calibration is performed through simultaneous spectral measurements by a calibrated double-monochromator Brewer MK-III spectrophotometer at “El Arenosillo” station, located in southwestern Spain. Because the range of spectral measurements of the Brewer spectrophotometer is 290–363 nm, a previously validated radiative transfer model was used to account for the erythemal contribution between 363 and 400 nm. Both methods are recommended by the World Meteorological Organization and we present and discuss here a wide range of results and features given by modified procedures applied to these two general methods. As is well established, the calibration factor for this type of radiometric system is dependent on atmospheric conditions, the most important of which are the ozone content and the SZA. Although the first method is insensitive to these two factors, we analyze this behavior in terms of the range used for the SZA and the use of two different mathematical approaches for its determination. The second method shows the dependence on SZA and ozone content and, thus, a polynomial as a function of SZA or a matrix including SZA and ozone content were determined as general calibration factors for the UV radiometric system. We must note that the angular responses of the YES radiometer and Brewer spectroradiometer have not been considered, because of the difficulty in correcting them. The results show in detail the advantages and drawbacks (and the corresponding associated error) given by the different approaches used for the determination of these calibration coefficients.

This study reports 5 years of (1998–2003) data on continuous solar-irradiation measurements from a scanning spectroradiometer (SUV-100) in Valdivia, Chile (39° S), accompanied by evaluation of the impact of ultraviolet radiation (UVR) on marine macroalgae of this site. UVR conditions showed a strong seasonal variation, which was less pronounced toward longer wavelengths. Daily maximum dose rates (clear days) averaged in winter–summer: UV-B_{(290–315 nm)} 0.30–2.1, UV-B_{(290–320 nm)} 0.70–3.7, UV-A_{(315–400 nm)} 20.6–62.1, UV-A_{(320–400 nm)} 20.2–60.5 W m⁻², and photosynthetically active radiation (PAR) 969–2423 μmol m⁻² s⁻¹. The corresponding daily doses (all the days) ranged: UV-B_{(290–315 nm)} 2.6–40.7, UV-B_{(290–320 nm)} 6.7–78.5, UV-A_{(315–400 nm)} 228–1539, UV-A_{(320–400 nm)} 224–1501, and PAR 2008–13308 kJ m⁻² d⁻¹. Taking into consideration action spectra of a biological interest, the risk of UV exposure could be up to 37 times higher in summer than in winter. The photosynthetic activity (as maximum quantum yield of chlorophyll fluorescence, F_v/F_m) of the brown alga Lessonia nigrescens from the infralittoral zone was markedly more sensitive to UVR than of the green alga Enteromorpha intestinalis from the upper midlittoral, and the UV-B wave band increased markedly photoinhibition. In L. nigrescens, maximal photoinhibition (40%) took place at weighted (the action spectrum for photoinhibition of photosynthesis) UVR doses of 800 kJ m⁻², irrespective of the season (corresponding midsummer daily dose in Valdivia is 480 kJ m⁻²). In winter, when this alga was at its most sensitive, the weighted UV dose causing 35–40% photoinhibition was around 200 kJ m⁻². In E. intestinalis, weighted doses of 800 kJ m⁻² resulted in low photoinhibition (<10 %) and no clear seasonal patterns could be inferred. These results confirm that midday summer levels of UV-B and their daily doses in southern Chile are high enough to produce stress to intertidal macroalgae.

Thioridazine is a phenothiazine derivative that has been used as an antipsychotic; it rarely causes photosensitization. However, we noticed that this drug induced an erythematous reaction in a photopatch test. Six volunteers were patch tested with various concentrations of thioridazine and irradiated with a range of UVA doses, and the time courses of the color of and blood flow to the test sites were monitored. The free-radical metabolites of thioridazine generated under UVA irradiation and its effects on ascorbate radical formation were examined with an electron paramagnetic resonance (EPR) spectrometer in vitro. As a result, immediate erythema developed during UVA irradiation in most subjects when 1% thioridazine was applied for 48 h and irradiation doses were higher than 4 J cm⁻². Another peak of erythematous reaction was observed 8–12 h after irradiation. The in vitro examination detected an apparent EPR signal, which appeared when 2 mM thioridazine in air-saturated phosphate buffer was irradiated with UVA, whereas this reaction was attenuated under anaerobic conditions. The EPR signal of the ascorbate radical was augmented under both aerobic and anaerobic conditions. Thioridazine-derived oxidants and/or thioridazine radicals generated during UVA irradiation seem to play an important role in this unique phototoxic reaction.

We used Shiga-like toxin B subunit (SLTB) to deliver the photosensitizer, chlorin e6 (Ce6), to Vero cells expressing the Gb₃ receptor. Our aim was to provide an example of carrier-enhanced photodynamic cell killing with which to start a systematic consideration of photosensitizer delivery at the subcellular level. SLTB, in contrast to many other potential protein carriers, is delivered intracellularly to the Golgi apparatus and endoplasmic reticulum (ER). Ce6 was chosen both for its phototoxic properties and its potential for covalent conjugation with SLTB. Ce6-SLTB after cleanup contained ≤10% noncovalently bound Ce6. The noncovalent binding of porphyrins and chlorins to protein conjugates has been well documented, and hence the effective cleanup procedure is a significant accomplishment. We demonstrate that Ce6-SLTB enhances delivery of Ce6 to target cells as compared to free Ce6. In Vero cells, Ce6-SLTB was over an order of magnitude more photodynamically toxic than free Ce6. Moreover, we show that in the case of Ce6-SLTB, photosensitizer accumulation is in a combination of subcellular sites including mitochondria, Golgi apparatus, ER and plasma membrane. The occurrence in nature of diverse B subunit binding sites and the possibilities of varied intracellular delivery make optimized use of B subunit carriers attractive.

The C-40 xanthophylls zeaxanthin and astaxanthin were confirmed to form radical cations, Car^· , in the electron-accepting solvent chloroform by direct excitation using subpicosecond time-resolved absorption spectroscopy in combination with spectroelectrochemical determination of the near-infrared absorption of Car^· . For the singlets, the S₂(1B_u ) state and most likely the S_x(3A_g⁻) state directly eject electrons to chloroform leading to the rapid formation of Car^· on a timescale of ∼100 fs; the lowest-lying S₁(2A_g⁻) state, however, remains inactive. Standard reduction potential for Car^· was determined by cyclic voltametry to have the value 0.63 V for zeaxanthin and 0.75 V for astaxanthin from which excited state potentials were calculated, which confirmed the reactivity toward radical cation formation. On the other hand, Car^· formation from the lowest triplet excited state T₁ populated through anthracene sensitization is mediated by a precursor suggested to be a solute–solvent complex detected with broad near-infrared absorption to the shorter wavelength side of the characteristic Car^· absorption. However, ground state carotenoids are able to react with a secondary solvent radical to yield Car^· , a process occurring within 16 μs for zeaxanthin and within 21 μs for astaxanthin. Among the two xanthophylls together with lycopene and β-carotene, all having 11 conjugated double bonds, zeaxanthin ranks with the highest reactivity in forming Car^· from either the S₂(1B_u ) or the ground state. The effects of substituent groups on the reactivity are discussed.

We examined the effects of UV radiation (UVR) on metabolic rates of the freshwater cladoceran Daphnia catawba. We exposed D. catawba to UVB for 12 h in a lamp phototron at levels of 2.08 and 4.16 kJ m⁻² both with and without concomitant exposure to UVA and visible photorepair radiation (PRR). We also included a group that received PRR only and a dark control group. Respiration rates were measured for 6 h following exposure. Respiration rates increased by 31.8% relative to the dark control at the lowest level of UVB stress (2.08 kJ m⁻² UVB with PRR), whereas respiration was inhibited by 70.3% at the highest stress level (4.16 kJ m⁻² UVB without PRR). Survival rates in the group that received PRR only and the group exposed to 2.08 kJ m⁻² and PRR were not significantly different from that in the control group; however, the survival rate was reduced for all other UVR exposures. We hypothesize that enhanced respiration rates reflect energetic costs related to repair of cellular components damaged by sublethal levels of UVR. Increases in respiration rate of the magnitude we found in our experiment could significantly reduce energetic reserves available for growth and reproduction, especially in cases where these costs are incurred repeatedly during a series of days with high levels of UVR.

Photo-oxidative processes occurring in wool can lead to significant photoyellowing of the fiber. In particular, wool that has been chemically bleached photoyellows more rapidly and to a greater degree than untreated wool. Direct identification of the chromophores responsible for such yellow discoloration in irradiated wool has proven to be elusive for many years. This article describes the characterization and location of yellow photo-oxidation products within the proteins of photoyellowed bleached wool fabric, using advanced protein chemistry techniques. The discolored fabric was enzymatically digested and chromatographed by high-pressure liquid chromatography, with monitoring at 400 nm, to select out fractions containing yellow chromophoric species. Thorough tandem mass spectrometric analysis was then used to sequence peptides and, in turn, to characterize modifications to key amino acid residues that had resulted in yellow chromophore formation. In total, 11 separate yellow chromophoric species were identified, ten derived from tryptophan residues and one from tyrosine. The tryptophan-derived modifications characterized included hydroxytryptophan, N-formylkynurenine, hydroxyformylkynurenine, kynurenine, hydroxykynurenine, carbolines, tryptophandiones and nitrotryptophan. The tyrosine-derived modification of tyrosine to dopa was also identified. The range of photomodifications we observed provides insight into the photo-oxidation pathways occurring within irradiated fibrous proteins leading to the formation of yellow chromophores.

Tumor treatment by photodynamic therapy (PDT) provokes a host-protective inflammatory and acute-phase response and an immune reaction. Neutrophilia manifested in this context is driven by multiple mediators of neutrophil chemotaxis orchestrated by an activated complement system. Mouse FsaR fibrosarcoma was used in this study to further investigate neutrophilia induced by Photofrin-based PDT. The complement anaphylatoxin C3a was identified as a major chemoattractant in the advanced phase of PDT-induced neutrophilia, because injecting mice with antibodies blocking its receptor C3aR significantly inhibited the increase in neutrophil levels 8 h after PDT. At the same time point, an increased C3aR expression was detected in neutrophils, monocytes and B lymphocytes in the blood of host mice. Peritoneal macrophages and mast cells harvested from treatment-naive mice exhibited elevated C3aR expression after coincubation in vitro for 8 h with PDT-treated FsaR cells. Thus, C3a emerges as one of the key effector molecules engaged in PDT-induced host response.

The formation of inclusion complexes of hydroxypropyl-β-cyclodextrin, heptakis(2,6-di-O-methyl)-β-cyclodextrin and heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin with 5,10,15,20-tetrakis(4-pyridyl)porphyrin (TpyP) has been studied in aqueous buffer solution (phosphate buffer pH = 7 and I = 0.01 M) to give a structural and spectroscopic characterization of a new class of potential sensitizers for photodynamic therapy. The interaction was investigated by a combination of UV/Vis absorption, fluorescence anisotropy, time-resolved fluorescence and circular dichroism. The experimental results point to the presence of the pigment in water in a monomeric complexed form. The fluorescence anisotropy measurements suggest that TpyP forms 1:1 complexes with heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin and hydroxypropyl-β-cyclodextrin, while 1:2 complexes are obtained with heptakis(2,6-di-O-methyl)-β-cyclodextrin.

Immunocompetent cells of earthworms (coelomocytes) contain adherent amoebocytes and large eleocytes (chloragocytes); the latter are filled with numerous granules. We have previously shown that eleocytes of several (but not all) earthworm species exhibit strong autofluorescence detectable by fluorescent microscopy and flow cytometry. In the present article, the molecular origin of eleocytes autofluorescence was elucidated in coelomocytes expelled via dorsal pores in the integument of Eisenia fetida subjected to electric shock (1 min at 4.5 V). Spectrofluorometry (excitation and emission spectra and fluorescence lifetime), together with HPLC analysis of coelomocyte suspensions and supernatants, indicated that riboflavin but not FMN (flavin mononucleotide) or FAD (flavin-adenine dinucleotide) is the main fluorophore responsible for eleocyte fluorescence in this species. Additionally, lipofuscins are suspected to participate in this phenomenon.

Repetitive exposure of the skin to UV radiation induces various harmful changes, such as thickening, wrinkle formation, inflammation and carcinogenesis. A variety of natural compounds and synthetic compounds have been studied to determine whether they can prevent UV-induced harmful effects. In this study, we investigated the effect of a novel compound, Melanocin A, which was isolated from Eupenicillium shearii F80695, on UV-induced premature skin aging. First, we studied the effect of Melanocin A on UV-induced matrix metalloproteinase (MMP)–9 expression in an immortalized human keratinocyte cell line, HaCaT, in vitro. Acute UV irradiation induced MMP-9 expression at both the mRNA and protein levels and Melanocin A suppressed this expression in a dose-dependent manner. We then investigated the effect of Melanocin A on UV-induced skin changes in hairless mice in vivo. Chronic exposure of hairless mouse dorsal skin to UV increased skin thickness and induced wrinkle formation and the gelatinase activities of MMP-2 and MMP-9. Moreover, Melanocin A significantly suppressed UV-induced morphologic skin changes and MMP-2 and MMP-9 expression. Taken together, these results show that Melanocin A can prevent the harmful effects of UV that lead to skin aging. Therefore, we suggest that Melanocin A should be viewed as a potential therapeutic agent for preventing and/or treating premature skin aging.

There are a range of UV models available, but one needs significant pre-existing knowledge and experience in order to be able to use them. In this article a comparatively simple Web-based model developed for the SoDa (Integration and Exploitation of Networked Solar Radiation Databases for Environment Monitoring) project is presented. This is a clear-sky model with modifications for cloud effects. To determine if the model produces realistic UV data the output is compared with 1 year sets of hourly measurements at sites in the United Kingdom and Thailand. The accuracy of the output depends on the input, but reasonable results were obtained with the use of the default database inputs and improved when pyranometer instead of modeled data provided the global radiation input needed to estimate the UV. The average modeled values of UV for the UK site were found to be within 10% of measurements. For the tropical sites in Thailand the average modeled values were within 1120% of measurements for the four sites with the use of the default SoDa database values. These results improved when pyranometer data and TOMS ozone data from 2002 replaced the standard SoDa database values, reducing the error range for all four sites to less than 15%.

Bioluminescence (BL) (λ_max ≈ 535 nm) of Vibrio fischeri strain Y1 has been previously characterized in terms of the fluctuation in intracellular levels of yellow fluorescent protein (YFP). In this study fluorescence microscopic analysis has revealed that yellow fluorescence, as well as blue fluorescence attributable to a luciferase intermediate, is localized to the periphery of V. fischeri Y1 cells. This finding indicates that both YFP and the luciferase are present in the vicinity of the cell membrane. By using cyanide to enhance yellow BL, it has been shown that BL modulation is coupled with the fluctuations in the intracellular levels of YFP and the primary emitter. On the basis of the BL characterization, combined with results of a sedimentation experiment, it has been shown that larger cells produce a relatively stronger yellow BL. Two-dimensional gel electrophoresis of cell-protein extracts has shown that the YFP level is more alterable than the luciferase level. It is postulated that the yellow BL modulation takes place in connection with cell growth.

The acid-base, spectroscopic, photophysical and liposome-binding properties of the recently synthesized free base, 29H,31H,1,4,8,11,15,18,22,25-octafluoro-2,3,9,10,16,17,23, 24-octakisperfluoro(isopropyl) phthalocyanine, F₆₄PcH₂, are reported. The perfluoroalkylation of the phthalocyanine core renders the hydrogen atoms acidic, with a pK_a = 6. The F₆₄Pc⁻² dianion is detected already at pH 3, by singular-value decomposition analysis of electronic spectra. F₆₄Pc⁻² generates ¹O₂ with quantum yields Φ_Δ = 0.252 (in MeOH) and 0.019 in liposomes. Metallation of the Pc macrocycle to yield F₆₄PcZn increases Φ_Δ to 0.606 and 0.126 in MeOH and liposomes, respectively. Surprisingly, F₆₄Pc⁻² (but not F₆₄PcH₂ or F₆₄PcZn) binds strongly to liposomes, with a binding constant K_b = 25 (mg/mL)⁻¹. The fully protonated F₆₄PcH₂, but not the zwitterionic F₆₄Pc⁻², might favor hydrogen bonding, thus reducing its lipophilicity. Similarly, the Lewis acidity of Zn in F₆₄PcZn, and thus its ability to bind water within a hydrophobic perfluoroalkyl pocket, is significantly enhanced by the fluorinated substituents.

An important goal of photodynamic therapy (PDT) for treatment of various cancers is to shorten PDT-performing time and simultaneously enhance PDT efficacy. Here, we investigated the nontumor tissue distribution of and the tumor vascular damage caused by a new photosensitizer, DH-I-180-3, in mice with implanted EMT6 mammary tumor cells. In addition, we performed cell-based assays to evaluate the basic antitumor effect of DH-I-180-3/PDT in EMT6 cells. After administration of PDT, the type of cell death was characterized to be apoptosis, and a change in the mitochondrial membrane potential was also observed within minutes. On the other hand, tumor growth was remarkably retarded in vivo in mice that received DH-I-180-3/PDT, compared with mice in the control group, which were exposed to light irradiation alone. Finally, tumors in some mice nearly healed. The antitumor drug reached a maximum concentration approximately 3 h after administration. However, PDT was most effective when there was substantial accumulation of DH-I-180-3 in the tumor vasculature and in healthy tissue. The histological demonstration provided further evidence of tumor vascular damage. On the basis of these findings, we suggest that PDT with the photosensitizer DH-I-180-3 induces vascular damage with blood vessel shutdown, in addition to direct killing of tumor cells, in mice.

An important step in predicting the effects of future increases in UV radiation (UVR) is to evaluate the mechanisms that organisms use to prevent and repair DNA damage and determine how those mechanisms influence UVR sensitivity. Damage is prevented to varying degrees through photoprotection and repaired via two main pathways: nucleotide excision repair and photoenzymatic repair. At present, little is known about the generality or similarity of these defenses among temperate freshwater fishes. We used laboratory experiments to compare UVR defense mechanisms among five freshwater fish species representing four families and three orders. Purified DNA, freeze-killed larvae and live larvae were exposed to UVB radiation for 12 h in the presence or absence of photorepair radiation. After exposure, we quantified frequencies of cyclobutane pyrimidine dimers in each exposure treatment. All five species used photoprotection and proportional decreases in dimer frequency were similar for all species. Evidence of excision repair was also found for all species but proportional decreases in photoproduct frequencies varied among species. Finally, evidence of photoenzymatic repair was found for only two of the five species.

Thirty-seven Suprasil quartz spheres, each approximately 1 cm in diameter and containing an iodide-iodate actinometric solution, were attached to a metal rack and inserted into a bench-scale UV reactor filled with water. The spheres were located at various distances and heights around a 12.4 W low-pressure Hg lamp housed inside a 3.2 cm–radius quartz sleeve in the middle of an annular batch reactor. UV light exposure at 254 nm was performed with the percent transmittance of the water present in the reactor at either 73% or 100% defined over a 1 cm path length. The spheres were simultaneously exposed to the UV light for a given period of time, after which the solutions were removed from the spheres and the yield of triiodide determined from the increase in absorbance at 352 nm. The resulting fluence rate at each site was then calculated on basis of the yield of triiodide. These results were compared with the predictions of a mathematical model based on the multiple point source summation approximation, including reflection and refraction at the air-quartz-water interface. Initially, the agreement was not satisfactory, especially in regions at an oblique angle to the lamp. The model was modified from a multiple point source model to a multiple cylindrical segment model by incorporating a cosine factor. The agreement between the new model and the experimental data was excellent and these experiments provide a strong validation of the model, even under conditions in which the fluence rate varied by >1000-fold between extreme sites in the reactor.