Atmospheric pressure chemical ionization mass spectroscopy (APCI-MS) was used to examine the light-induced oxidation products of retinoic acid under conditions that favor and preclude its aggregation. We observed that in conditions that favor aggregation, i.e. in aqueous solutions, retinoic acid undergoes superoxidation to yield highly oxidized species. Oxidation is limited, however, in the absence of such communication, i.e. when the polyene is fully solvated. From a comparison of the measured MS with that obtained from chemical oxidation of retinoic acid under conditions that promote radical oxidation and singlet oxygen–mediated oxidation, we conclude that superoxidation is mediated by reactive oxygen species other than singlet oxygen.

Recent evidence for involvement of internal water molecules in the mechanism of bacteriorhodopsin is reviewed. Water O–H stretching vibration bands in the Fourier transform IR difference spectra of the L, M and N intermediates of bacteriorhodopsin were analyzed by photoreactions at cryogenic temperatures. A broad vibrational band in L was shown to be due to formation of a structure of water molecules connecting the Schiff base to the Thr46-Asp96 region. This structure disappears in the M intermediate, suggesting that it is involved in transient stabilization of the L intermediate prior to proton transfer from the Schiff base to Asp85. The interaction of the Schiff base with a water molecule is restored in the N intermediate. We propose that water is a critical mobile component of bacteriorhodopsin, forming organized structures in the transient intermediates during the photocycle and, to a large extent, determining the chemical behavior of these transient states.

Xanthorhodopsin (XR), the light-driven proton pump of the halophilic eubacterium Salinibacter ruber, exhibits substantial homology to bacteriorhodopsin (BR) of archaea and proteorhodopsin (PR) of marine bacteria, but unlike them contains a light-harvesting carotenoid antenna, salinixanthin, as well as retinal. We report here the pH-dependent properties of XR. The pK_a of the retinal Schiff base is as high as in BR, i.e. ≥12.4. Deprotonation of the Schiff base and the ensuing alkaline denaturation cause large changes in the absorption bands of the carotenoid antenna, which lose intensity and become broader, making the spectrum similar to that of salinixanthin not bound to XR. A small redshift of the retinal chromophore band and increase of its extinction, as well as the pH-dependent amplitude of the M intermediate indicate that in detergent-solubilized XR the pK_a of the Schiff base counterion and proton acceptor is about 6 (compared to 2.6 in BR, and 7.5 in PR). The protonation of the counterion is accompanied by a small blueshift of the carotenoid absorption bands. The pigment is stable in the dark upon acidification to pH 2. At pH < 2 a transition to a blueshifted species absorbing around 440 nm occurs, accompanied by loss of resolution of the carotenoid absorption bands. At pH < 3 illumination of XR with continuous light causes accumulation of long-lived photoproduct(s) with an absorption maximum around 400 nm. The photocycle of XR was examined between pH 4 and 10 in solubilized samples. The pH dependence of recovery of the initial state slows at both acid and alkaline pH, with pK_as of 6.0 and 9.3. The decrease in the rates with pK_a 6.0 is apparently caused by protonation of the counterion and proton acceptor, and that at high pH reflects the pK_a of the internal proton donor, Glu94, at the times in the photocycle when this group equilibrates with the bulk.

Halorhodopsin from Natronobacterium pharaonis (pHR) is a light-driven chloride pump in which photoisomerzation of a retinal chromophore triggers a photocycle which leads to a chloride anion transport across the plasma membrane. Similarly to other retinal proteins the protonated Schiff base (PSB), which covalently links the retinal to the protein, does not experience hydrolysis reaction at room temperature even though several water molecules are located in the protonated Schiff base (PSB) vicinity. In the present studies we have revealed that in contrast to other studied archaeal rhodopsins, temperature increase to about 70°C hydrolyses the PSB linkage of pHR. The rate of the reaction is affected by Cl⁻ concentration and reveals an anion binding site (in addition to the Cl⁻ in the SB vicinity) with a binding constant of 100mM (measured at 70°C). We suggest that this binding site is located on the extracellular side and its possible role in the Cl⁻ pumping mechanism is discussed. The rate of the hydrolysis reaction is affected by the nature of the anion bound to pHR. Substitution of the Cl⁻ anion by Br⁻, I⁻ and SCN⁻ exhibits similar behavior to that of Cl⁻ in the region of 100mM but higher concentrations are needed for N₃⁻, HCOO⁻ and NO₂⁻ to achieve similar behavior. Steady state pigment illumination accelerates the reaction and reduces the energy of activation and the frequency factor. Adjusting the sample temperature to 25°C following the hydrolysis reaction led to about 80% pigment recovery. However, the newly reformed pigment is different from the mother pigment and has different characteristics. It is concluded that the apo-membrane adopts a modified conformation and/or aggregated state which rebinds the retinal to give a new conformation of the pHR pigment.

We report a unique λ_max shift of the absorption maximum of a photoactive yellow protein (PYP) analogue reconstituted with a fluorinated chromophore (F-PYP). The difference in λ_max between the free chromophore and the protein was significantly larger than that with the native chromophore. We concluded that the unusual λ_max shift is caused by the electronegative character of the fluorine atom and not by steric hindrance. This result suggests that formation of a hydrogen bond between the fluorine atom and one or more amino acid residues could neutralize its electron-withdrawing character. The properties of analogues of PYP with brominated and methylated chromophore could be explained as an effect of steric hindrance.

Genome sequencing projects resulted in the identification of a large number of new sequence homologs of archaeal rhodopsins in marine bacteria, fungi, and unicellular algae. It is an important task to unambiguously predict the functions of these new rhodopsins, as it is difficult to perform individual experiments on every newly discovered sequence. The transmembrane segments of rhodopsins have similar three-dimensional structures where the seven transmembrane helices form a tightly packed scaffold to accommodate a covalently bound retinal. We use geometric computations to accurately define the retinal-binding pockets in high-resolution structures of rhodopsins and to extract residues forming the wall of the retinal-binding pocket. We then obtain a tree defining the functional relationship of rhodopsins based on the short sequences of residues forming the wall of the retinal-binding pocket concatenated from the primary sequence, and show that these sequence fragments are often sufficient to discriminate phototactic vs transporting function of the bacterial and unicellular algal rhodopsins. We further study the evolutionary history of retinal-binding pockets by estimating the pocket residue substitution rates using a Bayesian Monte Carlo method. Our findings indicate that every functional class of rhodopsins has a specific allowed set of fast-rate amino acid substitutions in the retinal-binding pocket that may contribute to spectral tuning or photocycle modulation. The substitution rates of the amino acid residues in a putative retinal-binding pocket of marine proteorhodopsins together with the clustering of pocket sequences indicate that green-absorbing and blue-absorbing proteorhodopsins have similar function. Our results demonstrate that the evolutionary patterns of the retinal-binding pockets reflect the functional specificity of the rhodopsins. The approach we describe in this paper may be useful for large-scale functional prediction of rhodopsins.

Time-resolved absorbance difference spectra were collected at delays from 1 to 128 μs after photolysis of membrane and detergent suspensions of rhodopsin at 20°C. Fitting both sets of data with two exponential decays plus a constant showed a similar fast process (lifetime 11 μs in membrane, 12 μs in 5% dodecyl maltoside) with a small but similar spectral change. This demonstrates that the Lumi I → Lumi II process, previously characterized in detergent suspensions, has similar properties in membrane without significant effect of detergent. The slower exponential process detected in the data is quite different in membrane compared to detergent solubilized samples, showing that the pronounced effect of detergent on the later rhodopsin photointermediates begins fairly abruptly near 20 μs. Besides affecting the late processes, the data collected here shows that detergent induces a small blue shift in the 1 μs difference spectrum (the Lumi I minus rhodopsin difference spectrum). The blue shift is similar to one induced by chloride ion in the E181Q rhodopsin mutant and may indicate that the ionization state of Glu181 in rhodopsin is affected by detergent.

The 9-methyl group of 11-cis retinal is important in the efficient formation of the active conformation of rhodopsin, Meta II. Here, Tyr191 rhodopsin mutants were generated because of its proximity to that methyl group in the dark structure. If photoactivation results in movement of the 9-methyl group toward Tyr191, the steric interactions involved with activation and/or deactivation might not be as tightly coupled in mutant proteins with smaller amino acids at that position. Tyr191 mutations have no effect on the dark pigment. However, after photobleaching, the lifetime of Meta II is shorter; Meta II decays quickly into two inactive species: (1) a Meta III or Meta III-like species and (2) opsin and free retinal. The Meta III-like fraction maintains the covalent Schiff base linkage of the chromophore much longer than the wild type. On the other hand, the fast chromophore release is similar to cone pigments. Taken together, the data suggest that the role of the 9-methyl group after photoisomerization is not only to form Meta II efficiently, but also to maintain its active conformation and allow for the timely hydrolysis of the Schiff base. Perturbation of this interaction effects changes in the hydrolysis of the Schiff base and for the case of the Y191A mutation the folded structure of the protein after photobleaching.

Vertebrate rod photoreceptors are the ultimate light sensors, as they can detect a single photon. In darkness, rods maintain a high concentration of the intracellular messenger cyclic guanosine monophosphate (cGMP), which binds to and keeps open cationic channels on the plasma membrane of the outer segment. Absorption of a photon by the visual pigment of the rod, rhodopsin, initiates a biochemical amplification cascade that leads to a reduction in the concentration of cGMP and closure of the channels, thereby converting the incoming light to an electrical signal. Because the absorption of a photon and the ensuing reactions are localized events, the magnitude of the response of the rod to a single photon depends on the spread of the decrease in the cGMP concentration along the length of the outer segment. The longitudinal diffusion of cGMP depends on the structural parameters of the rod outer segment, specifically the area and the volume available for diffusion. To characterize the effect of rod outer segment cytoarchitecture on diffusion, we have used fluorescence recovery after photobleaching (FRAP) and examined the mobility of a fluorescent polar tracer, calcein, in the rod outer segments from three species with different outer segment structures: frog (Rana pipiens), mouse (Mus musculus domesticus) and gecko (Gekko gekko). We found that the diffusion coefficient is similar for all three species, in the order of 8–17 μm² s⁻¹, in broad agreement with the predictions by Holcman and Korenbrot (Biophys. J. 2004:86;2566–2582) based on the known cytoarchitecture of rod outer segments. Consequently, the results also support their prediction that the longitudinal spread of light excitation in rods is similar across species.

The retinal photoreceptors of the nocturnal Tokay gecko (Gekko gekko) consist exclusively of rods by the criteria of morphology and key features of their light responses. Unlike cones, they display robust photoresponses and have relatively slow recovery times. Nonetheless, the major and minor visual pigments identified in gecko rods are of the cone type by sequence and spectroscopic behavior. In the ongoing search for the molecular bases for the physiological differences between cones and rods, we have characterized the molecular biology and biochemistry of the gecko rod phototransduction cascade. We have cloned cDNAs encoding all or part of major protein components of the phototransduction cascade by RT-PCR with degenerate oligonucleotides designed to amplify cone- or rod-like sequences. For all proteins examined we obtained only cone-like and never rod-like sequences. The proteins identified include transducin α (Gα_t), phosphodiesterase (PDE6) catalytic and inhibitory subunits, cyclic nucleotide-gated channel (CNGα) and arrestin. We also cloned cDNA encoding gecko RGS9-1 (Regulator of G Protein Signaling 9, splice variant 1), which is expressed in both rods and cones of all species studied but is typically found at 10-fold higher concentrations in cones, and found that gecko rods contain slightly lower RGS9-1 levels than mammalian rods. Furthermore, we found that the levels of GTPase accelerating protein (GAP) activity and cyclic GMP (cGMP) phosphodiesterase activity were similar in gecko and mammalian rods. These results place substantial constraints on the critical changes needed to convert a cone into a rod in the course of evolution: The many features of phototransduction molecules conserved between those expressed in gecko rods and those expressed in cones cannot explain the physiological differences, whereas the higher levels of RGS9-1 and GAP activity in cones are likely among the essential requirements for the rapid photoresponses of cones.

The protein RPE65 is essential for the generation of the native chromophore, 11-cis retinal, of visual pigments. However, the Rpe65 knockout (Rpe65^−/−) mouse shows a minimal visual response due to the presence of a pigment, isorhodopsin, formed with 9-cis retinal. Isorhodopsin accumulates linearly with prolonged dark-rearing of the animals. The majority of Rpe65^−/− mice have an agouti coat color. A tan coat color subset of Rpe65^−/− mice was found to have an enhanced visual response as measured by electroretinograms. The enhanced response was found to be due to increased levels of 9-cis retinal and isorhodopsin pigment levels. Animals of both coat colors reared in cyclic light have minimal levels of regenerated pigment and show photoreceptor degeneration. On dark-rearing, pigment accumulates and photoreceptor degeneration is decreased. In the tan Rpe65^−/− mice, the level of photoreceptor degeneration is less than in the agouti animals, which have an increased pigment and decreased free opsin level. Therefore, photoreceptor damage correlates with the amount of the apoprotein present, supporting findings that the activity from unregenerated opsin can lead to photoreceptor degeneration.

We previously identified three genes that encode putative visual cycle proteins that are homologues of retinal G-protein coupled receptor (Ci-opsin3), cellular retinaldehyde-binding protein (Ci-CRALBP) and β-carotene 15,15′-monooxygenase (Ci-BCO) in the ascidian Ciona intestinalis. Ci-opsin3 and Ci-CRALBP are localized in both ocellus photoreceptor cells and surrounding non-photoreceptor cells in the brain vesicle of the larva. In the present study, we investigated the possible role and evolutionary origin of the BCO/RPE65 family in the visual cycle by analyzing Ci-BCO localization by immunohistochemistry and by identifying a novel gene that encodes a homologue of retinal pigment epithelium–specific 65 kDa protein (Ci-RPE65) in C. intestinalis. In situ hybridization and expressed sequence tag (EST) profiles consistently suggest that Ci-RPE65 is not significantly expressed in the ocellus and brain vesicle of the larva. Ci-RPE65 is expressed in the neural complex, a photoreceptor organ of the adult ascidian, at a level comparable to that of Ci-opsin3 and Ci-CRALBP. Ci-RPE65 is also expressed in various adult tissues, including the gill, body wall and intestine, suggesting that Ci-RPE65 plays a role in addition to that in the visual cycle. In contrast, Ci-BCO is predominantly localized in ocellus photoreceptor cells of the larva. The larval visual cycle seems to use Ci-opsin3 as a photoisomerase. Our results also suggest that the RPE65-dependent visual cycle is used in the adult photoreceptors of a primitive chordate.

Melanosomes and lipofuscin were isolated from 14-, 59-, and 76-year-old, human retinal pigment epithelium specimens and examined. The morphological features of these samples were studied by scanning electron microscopy and atomic force microscopy, and the photoionization properties were examined by photoelectron emission microscopy. Ovoid- and rod-shaped melanosomes were observed. The size of the granules and the distribution between the two shapes show no significant age-dependent change. However, there is a higher occurrence of irregularly shaped aggregates of small round granules in older samples which suggests degradation or damage to melanosomes occurs with age. The melanosomes from the 14-year-old donor eye are well characterized by a single photoionization threshold, 4.1 eV, while the two older melanosomes exhibit two thresholds around 4.4 and 3.6 eV. Lipofuscin from both young and old cells show two thresholds, 4.4 and 3.4 eV. The similarity of the potentials observed for aged melanosomes and lipofuscin suggest that the lower threshold in the melanosome sample reflects lipofuscin deposited the surface of the melanosome. The amount, however, is not sufficient to alter the density of the melanosome, and therefore these granules do not separate in a sucrose gradient at densities characteristic of the typical melanolipofuscin granule. These data suggest that thin deposits of lipofuscin on the surface of retinal pigment epithelium melanosomes are common in the aged eye and that this renders the melanosomes more pro-oxidant.

CRALBP, cellular retinaldehyde-binding protein, is a retinoid-binding protein necessary for efficient regeneration of rod and cone visual pigments. The C terminus of CRALBP binds to the PDZ domains of EBP50/NHERF-1, which in turn bind to ezrin and actin, proteins localized to the apical processes of the retinal pigment epithelium. In this study, we examined structural features associated with the interaction of the two proteins. The C-terminal amino-acid sequence of 11 orthologous CRALBPs is either ENTAL, ENTAF or EDTAL. Peptides ending in each of these sequences inhibited the interaction of CRALBP and EBP50/NHERF-1 with the use of an overlay assay. Molecular modeling showed that both NTAL and NTAF formed similar networks of H bonds with PDZ1 of EBP50/NHERF-1, and the side chains of both C-terminal Leu and Phe fit into the peptide-binding groove of PDZ1. CRALBP·11-cis-retinal and EBP50/NHERF-1 migrated as single components when analyzed individually by gel filtration and as a complex when mixed together before gel filtration. Complex formation was abolished by preincubation of EBP50/NHERF-1 with peptide EVENTAL. The ligand absorption spectrum of the complex was identical with that of CRALBP·11-cis-retinal, demonstrating that complex formation did not perturb the ligand-binding domain of CRALBP.

Methods that allow specific manipulations of the mouse genome have made it possible to alter specific aspects of photoreceptor function within the mouse retina. Mice with photoreceptors that have altered photosensitivities and altered photoresponse kinetics are now available. Methods are needed that can show how those perturbations in photoreceptor response characteristics translate into perturbations in visual sensitivity and perception. We have adapted a previously described method to evaluate visual threshold, spatial discrimination and temporal discrimination in mice swimming in a water maze. In this report we describe the sensitivities of rod-mediated and cone-mediated vision using GNAT1−/− and GNAT2−/− mice. Cone-mediated vision is ∼10 000 times less sensitive than rod-mediated vision in mice. We also demonstrate that mice can distinguish striped from solid objects in the water maze and that they can distinguish flickering from continuous illumination.

Exposure to UV radiation is recognized to suppress cell-mediated immunity and therefore could adversely affect the course of a viral infection. Rodent models of viral infection confirm this possibility but the situation in human subjects is not so clear, apart from two exceptions. These are herpes simplex, in which sunlight exposure can cause reactivation, and certain papillomavirus types in which sunlight exposure can lead to the development of nonmelanoma skin cancer. In both cases, there are UV response elements in the viral genomes that alter the normal interactions between the viruses and the host following exposure, and UV-induced effects on the immune response occur in addition. These complex mechanisms are discussed, and the situation regarding UV radiation and viral exanthems plus other viruses, including the retroviruses, summarized. Finally viral vaccination is considered in the context of UV exposure and the importance of the host's genetic background emphasized. Further research is required to evaluate whether sunlight can significantly affect the resistance to common viral infections and vaccines.

Iron is a limiting nutrient for primary production in marine systems, and photochemical processes play a significant role in the upper ocean biogeochemical cycling of this key element. In recent years, progress has been made toward understanding the role of biologically produced organic ligands in controlling the speciation and photochemical redox cycling of iron in ocean surface waters. Most (>99%) of the dissolved iron in seawater is now known to be associated with strong organic ligands. New data concerning the structure and photochemical reactivity of strong Fe(III) binding ligands (siderophores) produced by pelagic marine bacteria suggest that direct photolysis via ligand-to-metal charge transfer reactions may be an important mechanism for the production of reduced, biologically available iron (Fe[II]) in surface waters. Questions remain, however, about the importance of these processes relative to secondary photochemical reactions with photochemically produced radical species, such as superoxide (O₂⁻). The mechanism of superoxide-mediated reduction of Fe(III) in the presence of strong Fe(III) organic ligands is also open to debate. This review highlights recent findings, including both model ligand studies and experimental/observational studies of the natural seawater ligand pool.

At the air–water interface, interfacial molecular structure, intermolecular interactions, film relaxation and film respreading of model lung surfactant monolayers were studied using vibrational sum frequency generation (VSFG) spectroscopy combined with a Langmuir film balance. Chain-perdeuterated dipalmitoylphosphatidylcholine (DPPC-d62), palmitoyloleoylphosphatidylglycerol (POPG), palmitic acid (PA) and tripalmitin were investigated. In the DPPC-d62–PA binary monolayer, PA showed a condensing effect on the DPPC chains. On the contrary, in the DPPC-d62–POPG binary monolayer, POPG showed a fluidizing effect on the DPPC chains. In the ternary monolayer system of DPPC-d62–POPG–PA, the balance between the fluidizing and the condensing effect was also observed. In addition, the film relaxation behavior of DPPC-d62 and the enhanced film stability of DPPC-d62 caused by the addition of tripalmitin were observed. Real-time VSFG was also employed to study the respreading properties of a complex lung surfactant mixture containing DPPC-d62, POPG, PA and KL₄ (a mimic of SP-B) peptide, which revealed DPPC enrichment after film compression.

An overview of the evolution of artificial photosynthetic charge transfer systems containing porphyrin donors and pyromellitic or naphthalene diimide acceptor units is presented. Progression in this area of research is highlighted by the complexity of the systems, the nature of the medium separating donor and acceptor as well as the progression in the lifetime of the charge-separated state upon photoexcitation. A number of supramolecular systems that utilize hydrogen bonding or axial ligation of zinc porphyrins as a means for spatial orientation are highlighted.

Piperine, the major alkaloid of black pepper (Piper nigrum L.; Piperaceae), stimulates melanocyte proliferation and dendrite formation in vitro. This property renders it a potential treatment for the skin depigmentation disorder vitiligo. However, piperine does not stimulate melanin synthesis in vitro, and treatments based on this compound may therefore be more effective with concomitant exposure of the skin to ultraviolet (UV) radiation or sunlight. The present study investigated the effect of UVA and simulated solar radiation (SSR) on the chemical stability of piperine, its melanocyte stimulatory effects and its ability to bind protein and DNA. Chromatographic and spectroscopic analysis confirmed the anticipated photoisomerization of irradiated piperine and showed the absence of any hydrolysis to piperinic acid. Isomerization resulted in the loss of ability to stimulate proliferation of a mouse melanocyte cell line, and to bind to human serum albumin. There was no evidence of DNA binding by piperine either before or after irradiation, showing the absence of photoadduct formation by either piperine or its geometric isomers. This is unlike the situation with psoralens, which form DNA adducts when administered with UVA in treating skin diseases. The present study suggests that exposure to bright sunlight should be avoided both during active application of piperine to the skin and in the storage of piperine products. If UVA radiation is used with piperine in the treatment of vitiligo, application of the compound and irradiation should be staggered to minimize photoisomerization. This approach is shown to effectively induce pigmentation in a sparsely pigmented mouse strain.

Photophysical properties of sunscreens are commonly studied in solvent media, which do not mimic the skin, or in complex artificial skin systems, which are difficult to handle. In an earlier study, we showed that polystyrene nanosphere suspensions mimic the mixed polarity environment of skin cell systems. This paper presents a new method to quantify the effectiveness of sunscreens in the polystyrene nanosphere environment. This method utilizes the intrinsic UV-B fluorescence of polystyrene nanospheres. We studied three UV-B sunscreens by this new method and compared their extinction coefficients with observed values in solvent. The values follow the trend observed in solvents, but the ratio of their extinction coefficient in solvent to the value obtained by this new method is 1.3–1.8 instead of 1. This difference might be caused by the mixed polarity or the microgeometry of the nanosphere system. Regardless of the difference in the extinction coefficients, this new system can be used to test hundreds of chemicals for their sunscreening potential in a cost-effective way. One marked advantage of this new method is its ability to test both hydrophobic and hydrophilic sunscreening chemicals in the same environment. This is virtually impossible for current solvent-based models, which require different solvents for hydrophobic and hydrophilic chemicals. The new method also allows the simultaneous evaluation of a host of photophysical properties of sunscreening chemicals.

Sunscreens applied to the skin are retained primarily in the stratum corneum, where they adsorb and act as a barrier preventing UV penetration to deeper layers. Photophysical properties of sunscreens have traditionally been studied either in solvents, which are very different from skin, or in skin or complex artificial skin systems, which are difficult to handle. The purpose of this study was to determine whether polystyrene nanospheres could serve as an improvement over solvents for evaluation of the photophysical properties of sunscreens without the presence of autofluorescence from and interactions with specific skin biomolecules. We used HaCat cells and excised skin for this comparative study with nanospheres. Fluorescence spectral properties of common hydrophobic sunscreens octyl salicylate, padimate O (2-ethylhexyl-4-dimethylaminobenzoate) and octyl methoxycinnamate adsorbed to 220 nm polystyrene spheres are similar to those of sunscreens adsorbed to HaCat cells and excised skin. Specifically, similarity in the emission peaks and their approximate positions, excitation peak positions and a measurable reduction in scattering upon sunscreen addition suggest that polystyrene nanospheres constitute a useful system to evaluate the photophysical properties of topical sunscreens and may serve as a model system for high-throughput evaluation of potential sunscreens. An unexpected result of this comparative study was the observation of an increase in a specific skin component emission caused by addition of padimate O.

The calcium release channel (CRC) of the skeletal sarcoplasmic reticulum is rich in thiol groups and is strongly regulated by covalent modification of these thiols. Oxidizing reagents activate the release channel, whereas reducing reagents inhibit the channel. However, most CRC regulators are not thiol reagents. Here, we propose that reversible redox interactions are involved in regulation of the CRC by nonthiol reagents. This hypothesis was tested with several CRC regulators. The local anesthetics tetracaine, procaine and QX-314, which block the CRC, behaved as electron donors in reactions with dye free radicals. In contrast, ryanodine, caffeine, doxorubicin and daunorubicin, compounds known to activate the release channel, all accepted electrons from dye anion radicals. Moreover, release of Ca² from SR initiated by doxorubicin (acceptor) was antagonized by local anesthetics (donors). Based on the redox characterization of CRC modulators, we propose a functional model in which channel inhibitors and activators act as weak electron donors and acceptors, respectively, and shift the thiol–disulfide balance within the release protein. The consequence of this model is that, in spite of the chemical diversity of CRC modulators, a common mechanism of channel regulation involves the transient exchange of electrons between the activator/inhibitor and the CRC.

The photoinduced DNA damage by the berberine derivative coralyne is presented. The irradiation of coralyne in the presence of plasmid DNA namely, pBR322, leads to remarkably fast DNA damage by single-strand cleavage, as determined by agarose-gel electrophoresis. Even upon exposure to sunlight, almost all of the supercoiled plasmid is converted to the open circular form in less than a minute [c(pBR322) = 3.5 × 10⁻⁹ M; c(coralyne) = 4.3 × 10⁻⁵ M]. The efficiency of the DNA strand cleavage is not decreased in the presence of radical-trapping reagents such as tert-butanol or DMSO. Moreover, the extent of the DNA damage is the same under aerobic conditions and at reduced oxygen concentration. Thus, the formation of reactive intermediates such as hydroxyl radicals or singlet oxygen is excluded. These results show that the exposure of coralyne and derivatives thereof to light, even with moderate light intensity, needs to be avoided during experiments in which their biological activity is assessed by plasmid unwinding assays.

Increased ocean temperatures are thought to be triggering mass coral bleaching events around the world. The intracellular symbiotic zooxanthellae (genus Symbiodinium) are expelled from the coral host, which is believed to be a response to photosynthetic damage within these symbionts. Several sites of impact have been proposed, and here we probe the functional heterogeneity of Photosystem II (PSII) in three coral species exposed to bleaching conditions. As length of exposure to bleaching conditions (32°C and 350 μmol photons m⁻² s⁻¹) increased, the Q_A⁻ reoxidation kinetics showed a rise in the proportion of inactive PSII centers (PSII_X), where Q_B was unable to accept electrons. PSII_X contributed up to 20% of the total PSII centers in Pocillopora damicornis, 35% in Acropora nobilis and 14% in Cyphastrea serailia. Changes in F_v/F_m and amplitude of the J step along fast induction curves were found to be highly dependent upon the proportion of PSII_X centers within the total pool of PSII reaction centers. Determination of PSII antenna size revealed that under control conditions in the three coral species up to 60% of PSII centers were lacking peripheral light-harvesting complexes (PSIIβ). In P. damicornis, the proportion of PSIIβ increased under bleaching conditions and this could be a photoprotective mechanism in response to excess light. The rapid increases in PSII_X and PSIIβ observed in these corals under bleaching conditions indicates these physiological processes are involved in the initial photochemical damage to zooxanthellae.

In order to provide a thorough characterization of a system with which to study the dielectric response of a protein, a well-defined system complex of a fluorescent probe and protein is required. We have argued that such a system is provided by coumarin 153 and apomyoglobin (Photochem. Photobiol. 79, 440–446 [2004]). In order to demonstrate further that coumarin 153 exhibits negligible nonspecific binding to the surface of apomyoglobin, we study its interactions with both the apo and holo proteins. We further make a similar comparison with 8-anilino-1-naphthalenesulfonic acid, for which an NMR structure with apomyoglobin has been obtained. Our results confirm the appropriateness of the system of coumarin 153 and apomyoglobin for the investigation of solvation by the protein matrix.

The present study compares two methods for the determination of fetal lung maturity: the novel intrinsic fluorescence polarization ratio (IFPR) and the commercial TDx-FLMII. Amniotic fluid (AF) samples were collected from 69 women during the second and third trimesters of singleton pregnancies. Thirty-three samples were tested for IFPR only after centrifugation, and the rest were examined both before and after centrifugation. Of the latter 33 samples, 29 were assessed for lung maturity with the TDx-FLMII method as well. The results showed that IFPR values decreased with the advance in gestational age (r = 0.77, p < 0.05, n = 69). A significant correlation was found between IFPR of centrifuged and noncentrifuged samples (r = 0.94, p < 0.05, n = 36). A significant correlation was demonstrated between IFPR and TDx-FLMII values of centrifuged (r = 0.75, p < 0.05, n = 29) and noncentrifuged (r = 0.63, p < 0.05, n = 29) samples and moreover, samples considered mature by TDx-FLMII had low values of IFPR (n = 10). It can be concluded that the IFPR method can utilize noncentrifuged AF, thus suggested as a potential noninvasive method.

The efficacy of two chalcogenoxanthylium photosensitizers to reduce pathogens in red cell suspensions while maintaining red cell storage properties was investigated in the presence and absence of the red cell band-3 ligand and antioxidant, dipyridamole (DP). In the presence of DP, both sensitizers, TMR-S and DJD-42, displayed potent virucidal photoactivity (>6 log₁₀) and species-dependent bactericidal activities that ranged from 0.6 to 8.7 log₁₀. Addition of DP to red cell suspensions containing TMR-S or DJD-42 reduced Day 42 photoinduced hemolysis by approximately eight-fold and four-fold, respectively. Red cell binding studies revealed only a small degree of competition between DP and TMR-S, and no competition between DP with DJD-42 for binding to red cell membranes, suggesting that protection of red cells by DP in this system may primarily stem from its antioxidant properties.

The unicellular, green flagellate wild-type Euglena gracilis (strain Z) possesses two genes of the photoactivated adenylyl cyclase (PAC) family. The corresponding gene products were found to be responsible for step-up (but not step-down) photophobic responses as well as both positive and negative phototaxis. The proteins consist of two PACα (M_r 105 kDa) and two PACβ (90 kDa) subunits. In an effort to produce sufficient amounts of PAC proteins, several routes of overexpression have been tried including homologous expression in Euglena and heterologous expression in Escherichia coli. All these approaches were hampered by low yield or formation of inclusion bodies. Therefore we decided to attempt a heterologous expression in an insect cell line. PACα and PACβ were separately cloned in the transfer vector pBacPAK9 with a His tag attached. The transfer vector was subsequently cotransfected via baculovirus into the insect cells and amplified. For the expression both recombinant viruses (containing PACα and PACβ, respectively) were cotransfected simultaneously into insect cells. The expressed proteins were analyzed in Western blots using PACα and PACβ antibodies. Most of the proteins were found to be in soluble form in high yield. The recombinant PAC proteins were purified via their attached His tag on an anti-His resin. Adenylyl cyclase activity was quantified after blue-light excitation using a cAMP enzyme immunoassay kit.

The phototoxic and photoallergic effects of the once popular UV sunscreen p-aminobanzoic acid are related, in part, to its ability to sensitize the formation of singlet oxygen as well as other reactive oxygen species. In this work we demonstrate that the sunscreen-photoinduced inactivation of a model protein, horseradish peroxidase, is reduced by approximately a factor of three when the sunscreen is encaspsulated in zeolite sodium Y. These results provide evidence that using the technology of zeolite encapsulation to prepare a supramolecular sunscreen that minimizes the skin contact of active ingredients may reduce the adverse effects of “naked” sunscreens on biological systems. These radiation-induced effects, unfortunately, frequently accompany the desirable UV-screening role of these products. These results provide an important benchmark for the use of zeolite encapsulation as a means of improving the safety of UV sunscreens for topical application.

Leaf expansion, comprising cell division and cell enlargement, is controlled by light quality and quantity. The role of UV-B irradiance on leaf cell enlargement has not been determined. We studied the effect of a wide range of UV-B irradiances on the cell-enlargement-driven expansion of Phaseolus vulgaris L., cv. Contender (bush bean) leaf discs. Our growth method allowed separation of the cell enlargement phase of leaf expansion from the cell division phase. In two series of experiments with different types of UV-B screening filters, the effect of increasing levels of UV-B on the area of excised P. vulgaris leaf discs was investigated. One set of experiments utilized polyester (UV-B-absorbing) and cellulose acetate (UV-B-transmitting) filters. The other set utilized UV-B-absorbing and UV-B-transmitting acrylic filters. Regardless of which type of filter was used for screening, high (above summer solstice) levels of supplemental UV-B inhibited cell enlargement in a linear, dose-dependent manner, resulting in smaller leaf discs than treatment with UV-B-absorbing filters. Conversely, low levels of supplemental UV-B enhanced cell enlargement in a linear, dose-dependent manner, resulting in larger leaf discs than did treatment with UV-B-absorbing filters. The results suggest a biphasic response to UV-B, and that there is an optimum UV-B level that results in maximum leaf expansion by cell enlargement.

We investigated the associations between latitude and the incidence of two different types of ocular melanoma, external ocular melanoma (exposed to sunlight) and internal melanoma (not exposed to sunlight), separately. Using 1992–2002 data from the Surveillance, Epidemiology, and End Results (SEER) Program of National Cancer Institute, we identified 2142 ocular melanoma cases in non-Hispanic whites, and then regressed the incidences of various types of ocular melanomas with latitude. Our analysis indicated that the higher the latitude (away from the equator, the less sun exposure), the lower the risk of external ocular melanoma (eyelid and conjunctival melanomas) among non-Hispanic whites (P for trend = 0.018). The incidence increased 2.48 fold from 47–48° to 20–22°. This trend is very similar to that of skin melanoma. The incidence of internal ocular melanoma (uveal melanoma) increased significantly with increasing latitudes (the less sun exposure, P for trend < 0.0001), it increased 4.91 fold from 20–22° to 47–48°. The latitudinal patterns of ocular melanomas may reflect the dual effects of sunlight exposure, i.e. a mutagenic effect of direct solar radiation on external ocular melanomas and a protective effect for internal uveal melanoma, which is similar to the sun radiation protective effects for various internal malignant tumors that are not exposed to the sunlight.

Light, oxygen or voltage (LOV) domains function as blue-light sensors in the phototropin family of photoreceptors found in plants, algae and bacteria. We detected putative LOV domains (Alr3170-LOV, All2875-LOV and Alr1229-LOV) in the genome of a filamentous cyanobacterium, Anabaena sp. PCC 7120. These cyanobacterial LOV domains are closely clustered with the known LOV domains. Alr3170-LOV and All2875-LOV carry the conserved cysteine residue unique to the photoactive LOV, whereas Alr1229-LOV does not. We expressed these three LOV domains in Escherichia coli and purified them. In fact, Alr3170-LOV and All2875-LOV that are conserved in Nostoc punctiforme, a related species, bound flavin mononucleotide and showed spectral changes unique to known LOV domains on illumination with blue light. Alr3170-LOV was completely photoreduced and dark reversion was slow, whereas All2875-LOV was slowly photoreduced and dark reversion was rapid. For comparison, AvAll2875-LOV in a closely related A. variabilis was also studied as a homolog of All2875-LOV. Finally, we observed that Alr1229-LOV that is not conserved in N. punctiforme showed no flavin binding.

Singlet oxygen is a highly reactive electrophilic species that reacts rapidly with electron-rich moieties, such as the double bonds of lipids, thiols, and ascorbate (AscH⁻). The reaction of ascorbate with singlet oxygen is rapid (k = 3 × 10⁸ M⁻¹ s⁻¹). Here we have investigated the stoichiometry of this reaction. Using electrodes to make simultaneous, real-time measurements of oxygen and hydrogen peroxide concentrations, we have investigated the products of this reaction. We have demonstrated that hydrogen peroxide is a product of this reaction. The stoichiometry for the reactants of the reaction (1¹O₂ 1AscH⁻ → 1H₂O₂ 1dehydroascorbic) is 1:1. The formation of H₂O₂ results in a very different oxidant that has a longer lifetime and much greater diffusion distance. Thus, locally produced singlet oxygen with a half-life of 1 ns to 1 μs in a biological setting is changed to an oxidant that has a much longer lifetime and thus can diffuse to distant targets to initiate biological oxidations.

Barrett's esophagus (BE) can experimentally be treated with 5-aminolevulinic acid–based photodynamic therapy (ALA-PDT), in which ALA, the precursor of the endogenous photosensitizer protoporphyrin IX (PpIX) and subsequent irradiation with laser light are applied to destroy the (pre)malignant tissue. Accurate dosimetry is critical for successful ALA-PDT. Here, in vivo dosimetry and kinetics of PpIX fluorescence photobleaching were studied in a rat model of BE. The fluence and fluence rate were standardized in vivo and PpIX fluorescence was measured simultaneously at the esophageal wall during ALA-PDT and plotted against the delivered fluence rather than time. Rats with BE were administered 200 mg kg⁻¹ ALA (n = 17) or served as control (n = 4). Animals were irradiated with 633 nm laser light at a measured fluence rate of 75 mW cm⁻² and a fluence of 54 J cm⁻². Large differences were observed in the kinetics of PpIX fluorescence photobleaching in different animals. High PpIX fluorescence photobleaching rates corresponded with tissue ablation, whereas low rates corresponded with no damage to the epithelium. Attempts to influence tissue oxygenation by varying balloon pressure and ventilation were shown not to be directly responsible for the differences in effect. In conclusion, in vivo dosimetry is feasible in heterogeneous conditions such as BE, and PpIX fluorescence photobleaching is useful to predict the tissue response to ALA-PDT.

The spectral properties and Z,E-photoisomerizations of three 4-styrylstilbenes, a 4,4′-bis(β-methylstyryl)benzene and a 4,4′-distyrylstilbene were investigated in solution and in the solid state. Some notable features of the absorption and fluorescence spectra due to the structures and the phases (solution or solid) were observed, especially the Stokes shifts. Interesting photochemical behaviors in solution and in the solid state were also found. While the (E,Z)-4-styrylstilbenes undergo a one-way photoisomerization to their E,E-isomers with almost equal rates in solution, their quantum yields in the solid state decrease with increasing substituent size at the terminal aromatic rings. On the other hand, the 4,4′-bis(β-methylstyryl)benzene undergoes a mutual photoisomerization in solution, but its E,Z-isomer isomerizes in a one-way manner to the E,E-isomer in the solid state. Additionally, the (Z,E,Z)-4,4′-styrylstilbene was found to undergo a one-way photoisomerization to the E,E,E-isomer via the E,E,Z-isomer in solution and the crystalline E,E,Z-isomer to the E,E,E-isomer in the solid state.

5-methyltetrahydrofolate (5MTHF) absorbs UV radiation and has an absorption coefficient of 24 250 ± 1170 M⁻¹ cm⁻¹ at 290 nm. It has a weak fluorescence emission in the wavelength region around 360 nm. Our data demonstrated induction of 5-methyldihydrofolate by exposure to UVB and, after continues irradiation, p-aminobenzoyl-l-glutamic acid was found. The photodegradation of 5MTHF follows a first order kinetic with a degradation rate constant of 9.2 × 10⁻³ min⁻¹ under our conditions (fluence rate of 2.15 mW cm⁻², exposure wavelengths from 280 to 350 nm). Our results indicate that a direct degradation of 5MTHF by UV exposure in humans in vivo is rather unlikely. 5MTHF mainly absorbs, and is degraded by, UVB and UVC, radiation that does not penetrate the earth's atmosphere and the human skin well.

We examined age-related vulnerability of Daphnia magna to UV-B (ultraviolet-B radiation, 280–320 nm), the age-related relationships between UV-B dosage and reproduction, and the effects of duration of PRR (photorecovery radiation) on survival and fecundity. Animals were exposed to 0, 6, 9 or 12 h UV-B (peak emission 312 nm) at 1, 2, 3 or 4 days of age and followed to 12 days. Survival decreased with increasing exposure to UV-B, but increased with age at exposure. Duration of UV-B, but not age at exposure, decreased offspring production. Survival increased with an increase in duration of PRR for animals of all ages; however, the effect was not age related. Three-day-old animals were exposed to 6 h of UV-B followed by 0–600 min of PRR. Survival was greatest in controls, lowest in the group that received no PRR and similar for all other groups. Most mortality occurred within the first 72 h postirradiation. Offspring production was highest in light controls, lowest in the UV-B–exposed group that received no PRR and not significantly different among other groups. This study demonstrates the need to consider age when examining the effects of UV-B on zooplankton and the need to monitor responses over a sufficient length of time.

The present study reports the effect a cell permeabilizer, polyethylenimine (PEI) has on the photodynamic effect of methylene blue (MB) and nuclear fast red (NFR) in the presence of hydrogen peroxide (H₂O₂). The photosensitized destruction of the algae Chlorella vulgaris under irradiation with visible light is examined. The photodynamic effect was investigated under aerobic and anaerobic conditions. The presence of a permeabilizer during the photosensitized destruction of C. vulgaris does not enhance the activity of the MB, MB/H₂O₂ system or the NFR, NFR/H₂O₂ system under aerobic conditions. However under anaerobic conditions we have determined that when a cell permeabilizer was added to the MB/H₂O₂ system, the photosensitized destruction of C. vulgaris proceeded via a combination of Type I and Type II mechanisms. The presence of PEI enforces MB/H₂O₂ to be active toward the destruction of C. vulgaris whether oxygen is present or absent. Under aerobic and anaerobic conditions the activity of NFR was suppressed in the presence of PEI as a result of electrostatic interactions between the photosensitizer and the cell permeabilizer. The decrease in fluorescence recorded is indicative of destruction of the chlorophyll a pigment.

Caffeic acid (CA) and its analogues such as rosmarinic acid are well known as antioxidative agents. Exposure to UVA is known to generate reactive oxygen species (ROS) such as singlet oxygen (¹O₂) and superoxide anion radical (^·O₂⁻) in the skin of animals, which in turn induces skin photodamage and photoaging. Because CA and its analogues quench ¹O₂, these compounds were topically applied to the abdominal skin of live hairless mice and were found to suppress ROS generation upon UVA exposure. Furthermore, the generation of UVA-induced ROS was also suppressed in the skin of mice that were orally given CA. In order to understand the mechanism by which CA blocks ROS production in UVA-exposed skin, the pharmacokinetics of CA upon oral administration to mice was followed and CA was found to efficiently distribute in the skin. These results suggest that skin damage by UVA-induced ROS generation is reduced by oral supplementation of CA, which has a scavenging and quenching activity against ROS.

The photosynthetic reaction center is an efficient molecular device for the conversion of light energy to chemical energy. In a previous study, we synthesized the hydrogenase/photosystem I (PSI) complex, in which Ralstonia hydrogenase was linked to the cytoplasmic side of Synechocystis PSI, to modify PSI so that it photoproduced molecular hydrogen (H₂). In that study, hydrogenase was fused with a PSI subunit, PsaE, and the resulting hydrogenase-PsaE fusion protein was self-assembled with PsaE-free PSI to give the hydrogenase/PSI complex. Although the hydrogenase/PSI complex served as a direct light-to-H₂ conversion system in vitro, the activity was totally suppressed by adding physiological PSI partners, ferredoxin (Fd) and ferredoxin-NADP -reductase (FNR). In the present study, to establish an H₂ photoproduction system in which the activity is not interrupted by Fd and FNR, position 40 of PsaE from Synechocystis sp. PCC6803, corresponding to the Fd-binding site on PSI, was selected and targeted for the cross-linking with cytochrome c₃ (cytc₃) from Desulfovibrio vulgaris. The covalent adduct of cytc₃ and PsaE was stoichiometrically assembled with PsaE-free PSI to form the cytc₃/PSI complex. The NADPH production by the cytc₃/PSI complex coupled with Fd and FNR decreased to approximately 20% of the original activity, whereas the H₂ production by the cytc₃/PSI complex coupled with hydrogenase from Desulfovibrio vulgaris was enhanced 7-fold. Consequently, in the simultaneous presence of hydrogenase, Fd, and FNR, the light-driven H₂ production by the hydrogenase/cytc₃/PSI complex was observed (0.30 μmol H₂/mg chlorophyll/h). These results suggest that the cytc₃/PSI complex may produce H₂ in vivo.

Meso-tetra-hydroxyphenyl-chlorin (mTHPC)-mediated photodynamic therapy (PDT) has shown to be effective in the treatment of patients with multiple basal cell carcinoma (BCC). In the present study we further optimized the drug-light interval and examined the correlation between plasma drug levels and treatment efficacy. Thirteen patients with multiple BCC (a total of 366 lesions) were included in the study. Following intravenous administration of 0.1 mg kg⁻¹ mTHPC, lesions were illuminated with 10 J cm⁻² light (652 nm, 100 mW cm⁻²) at 12, 24, 48, 72 or 96 h. Plasma samples were taken prior to each illumination for determination of mTHPC levels, and tumor response was evaluated at 6 months and 1 year. Both univariable and multivariable analyses showed that optimal treatment outcome was obtained for a drug-light interval of 24 h when plasma drug levels were high. Overall, good cosmetic results with little or no scarring were obtained in 87% of the treated lesions and no serious side effects were observed. We optimized mTHPC-mediated PDT for patients suffering from multiple BCC by determining the most effective drug-light interval and showed that this treatment offers significant advantages over surgical resection.

Radiotherapy is one of the most common therapies for treating human cancers. Several studies have indicated that irradiation induces reactive oxygen species (ROS), which play an important role in radiation damage of the cell. It has been shown that Nigella sativa L. (NS) and reduced glutathione (GSH) have both an antiperoxidative effect on different tissues and a scavenger effect on ROS. The purpose of this study was to determine the antioxidant and radioprotective roles of NS and GSH against irradiation-induced oxidative injury in an experimental model. The NS group was administrated NS (1 mL/kg body weight), the GSH group was injected GSH (150 mg/kg body weight) and the control group was given physiologic saline solution (1 mL/kg body weight) for 30 consecutive days before exposure to a single dose of 6 Gy of radiation. Animals were sacrificed after irradiation. Malondialdehyde, nitrate, nitrite (oxidative stress markers) and ascorbic acid, retinol, β-carotene, GSH and ceruloplasmin (nonenzymatic antioxidant markers) levels and peripheral blood lymphocytes were measured in all groups. There were statistically significant differences between the groups for all parameters (P < 0.05). Whole-body irradiation caused a significant increase in blood malondialdehyde, nitrate and nitrite levels. The blood oxidative stress marker levels in irradiated rats that were pretreated with NS and GSH were significantly decreased; however, nonenzymatic antioxidant levels were significantly increased. Also, our results suggest that NS and GSH administration prior to irradiation prevent the number of alpha-naphthyl acetate esterase peripheral blood T lymphocytes from declining. These results clearly show that NS and GSH treatment significantly antagonize the effects of radiation. Therefore, NS and GSH may be a beneficial agent in protection against ionizing radiation-related tissue injury.

The dangers of overexposure to sunlight have been well publicized, but less attention has been given to an acknowledged benefit of exposure to UV radiation; that being the cutaneous synthesis of vitamin D₃. Here we define a standard vitamin D dose on the basis of recently recommended requirements for vitamin D that take account of its risk reduction role in a variety of diseases, and present a web-based tool that enables the reader to calculate associated exposure times for any time and place using either default values or user-selected conditions. Either it is not possible to synthesize vitamin D₃ at high latitudes in winter, or the exposure time required to reach a standard dose is sometimes impractical. Where solar UV is sufficient, a risk-benefit analysis of sunburn vs. vitamin D₃ synthesis shows that the best time for brief sun exposure is in the middle of the day. For low solar elevation angles common at high latitudes, a fine line exists between adequate UV exposure for vitamin D₃ synthesis and a risk of sun burn.

The objective of the present study was to investigate the treatment of 9L gliosarcoma brain tumor in the rat with the combination of surgical resection and photodynamic therapy (PDT). Nude rats with intracranial 7-day-old 9L gliomas were randomly subjected to no treatment, PDT alone (Photofrin®: 2 mg kg⁻¹, optical: 80 J cm⁻²), surgical resection alone or resection combined with 2 mg kg⁻¹ Photofrin®-mediated PDT at an optical dose of 80 J cm⁻². All animals were sacrificed 14 days after tumor implantation. Hematoxylin-and-eosin and immunohistochemical stainings were performed to assess the tumor volume and the expression of vascular endothelial growth factor (VEGF) in the brain adjacent to the tumor (BAT) as well as the tumor cell apoptosis and proliferation. Our data show that both surgical resection alone and PDT alone significantly decreased tumor volume, but furthermore, surgical resection combined with PDT significantly reduced the tumor volume and reduced local tumor infiltration compared to either surgical resection or PDT treatment alone. PDT treatment with or without resection increased tumor apoptosis, but resection alone did not alter the tumor cell apoptosis compared with a nontreatment control group. Both surgical resection alone and PDT alone induced a significant increase in VEGF expression in the BAT; however intraoperative PDT did not further increase VEGF expression, compared with surgery alone or PDT alone. No significant differences were found in tumor cell proliferation as indicated by Ki67 immunoreactivity among the four groups. Our results suggest that PDT enhances the efficacy of surgical resection in the management of malignant gliomas without increasing VEGF expression in the BAT.

We recently reported that variations in cellular phototoxicity among a series of alkynyl-substituted zinc trisulfophthalocyanines (ZnPcS₃C_n) correlates with their hydrophobicity, with the most amphiphilic derivatives showing the highest cell uptake and phototoxicity. In this study we address the role of the plasma membrane in the photodynamic response as it relates to the overall hydrophobicity of the photosensitizer. The membrane tracker dye 1-[4(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), which is incorporated into plasma membranes by endocytosis, was used to establish plasma membrane uptake by EMT-6 cells of the ZnPcS₃C_n by colocalization, and TMA-DPH membrane uptake rates after photodynamic therapy were used to quantify membrane damage. TMA-DPH colocalization patterns show plasma membrane uptake of the photosensitizers after short 1 h incubation periods. TMA-DPH plasma membrane uptake rates after illumination of the photosensitizer-treated cells show a parabolic relationship with photosensitizer hydrophobicity that correlates well with the phototoxicity of the ZnPcS₃C_n. After a 1 h incubation period, overall phototoxicity correlates closely with the postillumination rate of TMA-DPH incorporation into the cell membrane, suggesting a major role of plasma membrane damage in the overall PDT effect. In contrast, after a 24 h incubation, phototoxicity shows a stronger but imperfect correlation with total cellular photosensitizer uptake rather than TMA-DPH membrane uptake, suggesting a partial shift in the cellular damage responsible for photosensitization from the plasma membrane to intracellular targets. We conclude that plasma membrane localization of the amphiphilic ZnPcS₃C₆–C₉ is a major factor in their overall photodynamic activity.