In this study we sought the detection and characterization of bacterial membrane domains. Fluorescence generalized polarization (GP) spectra of laurdan-labeled Escherichia coli and temperature dependencies of both laurdan's GP and fluorescence anisotropy of 1,3-diphenyl-1,3,5-hexatriene (DPH) (r_DPH) affirmed that at physiological temperatures, the E. coli membrane is in a liquid-crystalline phase. However, the strong excitation wavelength dependence of r_laurdan at 37°C reflects membrane heterogeneity. Time-resolved fluorescence emission spectra, which display distinct biphasic redshift kinetics, verified the coexistence of two subpopulations of laurdan. In the initial phase, <50 ps, the redshift in the spectral mass center is much faster for laurdan excited at the blue edge (350 nm), whereas at longer time intervals, similar kinetics is observed upon excitation at either blue or red edge (400 nm). Excitation in the blue region selects laurdan molecules presumably located in a lipid domain in which fast intramolecular relaxation and low anisotropy characterize laurdan's emission. In the proteo-lipid domain, laurdan motion and conformation are restricted as exhibited by a slower relaxation rate, higher anisotropy and a lower GP value. Triple-Gaussian decomposition of laurdan emission spectra showed a sharp phase transition in the temperature dependence of individual components when excited in the blue but not in the red region. At least two kinds of domains of distinct polarity and order are suggested to coexist in the liquid-crystalline bacterial membrane: a lipid-enriched and a proteo-lipid domain. In bacteria with chloramphenicol (Cam)–inhibited protein synthesis, laurdan showed reduced polarity and restoration of an isoemissive point in the temperature-dependent spectra. These results suggest a decrease in membrane heterogeneity caused by Cam-induced domain dissipation.

Oxygen-dependent quenching of phosphorescence has been proven to be a valuable tool for the measurement of oxygen concentrations both in vitro and in vivo. For biological measurements the relatively long lifetimes of phosphorescence have promoted time-domain–based devices using xenon arc flashlamps as the most common excitation light source. The resulting complex form of the excitation pulse leads to complications in the analysis of phosphorescence lifetimes and ultimately to errors in the recovered pO₂ values. Although the problem has been recognized, the consequences on in vivo phosphorescence lifetime measurements have been neglected so far. In this study, the consequences of finite excitation flash duration are analyzed using computer simulations, and a method for the recovery of phosphorescence decay times from complex photometric signals is presented. The analysis provides an explanation as to why different calibration constants are reported in the literature and presents a unified explanation whereby calibration constants are not solely a property of the dye but also of the measuring device. It is concluded that complex excitation pulse patterns without appropriate analysis methods lead to device-specific calibration constants and nonlinearity and can be a potent source of errors when applied in vivo. The method of analysis presented in this article allows reliable phosphorescence lifetime measurements to be made for oxygen pressure measurements and can easily be applied to existing phosphorimeters.

The frequency-domain technique is applied to measure the photodegradation rate of fluorescein in aqueous solutions. The illuminating light is modulated, and the changes in fluorescence from the illuminated region are detected synchronously. A constant flow rate is imposed on the fluorescein solution to control the mass transport of fluorescein into the illuminated region. The fluorescence response is described by a model that assumes that photodegradation occurs from the triplet excited state. The predictions of the model are consistent with the observed variations in the fluorescence response with flow rate, modulation frequency and incident power. We discuss in this article how the dependence of the model parameters on experimental conditions can be used to infer the photodegradation rate as well as some of the details of the photodegradation mechanism. The results are consistent with the known mechanism of photodegradation of fluorescein. The frequency-domain technique gives a photodegradation rate of 53 s⁻¹ in an air-saturated solution and 37 s⁻¹ in solutions purged with argon gas.

We have synthesized several 3-arylallyl acetates 1, 2, 3, 5 and 6, and E-3-(1-naphthyl)-2-propene-1-ol 4 for studying ionic photodissociation. Compounds 1, 2 and 3 underwent an efficient ionic photodissociation in polar solvents like acetonitrile and methanol leading to the formation of rearranged acetate and methyl ether products, as well as undergoing an E–Z isomerization. The arylallyl alcohol 4 and the two arylallyl acetates 5 and 6 did not undergo ionic photodissociation. Quantum yields of product formation, quantum yields of fluorescence, solvent polarity effects and triplet-sensitization studies indicated that a highly polarized excited singlet state is responsible for the ionic photodissociation. Both the singlet- and triplet-excited states are effective in displaying E–Z isomerization in 1, 2, 3 and 4. Compounds 5 and 6 are highly fluorescent, and the fluorescence may be the excited state deactivation pathway along with internal conversion.

Singlet oxygen (¹O₂) is generated by a number of enzymes as well as by UV or visible light in the presence of a sensitizer and has been proposed as a damaging agent in a number of pathologies including cataract, sunburn, and skin cancers. Proteins, and Cys, Met, Trp, Tyr and His side chains in particular, are major targets for ¹O₂ as a result of their abundance and high rate constants for reaction. In this study it is shown that long-lived peroxides are formed on free Tyr, Tyr residues in peptides and proteins, and model compounds on exposure to ¹O₂ generated by both photochemical and chemical methods. The yield of these species is significantly enhanced in D₂O and decreased by azide. Nuclear magnetic resonance and mass spectroscopic analysis of reaction mixtures, or materials separated by high-performance liquid chromatography, are consistent with the initial formation of an (undetected) endoperoxide that undergoes rapid ring-opening to give a hydroperoxide situated at the C1 ring-position (i.e. para to the phenolic group). In the presence of a free α-amino group (e.g. with free Tyr), rapid ring-closure occurs to give an indolic hydroperoxide that decays into the corresponding alcohol, 3a-hydroxy-6-oxo-2,3,3a,6,7,7a-hexahydro-1H-indole-2-carboxylic acid. Hydroperoxides that lack a free α-amino group (e.g. those formed on 3-(4-hydroxyphenyl)propionic acid, N-Ac-Tyr and Tyr-containing peptides) are longer-lived, with half-lives of hours to days. These species undergo slow decay at low temperatures to give the corresponding alcohol. Their rate of decay is enhanced at 37°C, or on exposure to UV light or metal ions, and gives rise to reactive radicals, via cleavage of the peroxide bond. These radicals have been characterized by electron paramagnetic resonance spin trapping. These studies demonstrate that long-lived Tyr-derived peroxides are formed on proteins exposed to ¹O₂ and that these may promote damage to other targets via further radical generation.

Gold porphyrins are often used as electron-accepting chromophores in artificial photosynthetic constructs. Because of the heavy atom effect, the gold porphyrin first-excited singlet state undergoes rapid intersystem crossing to form the triplet state. The lowest triplet state can undergo a reduction by electron donation from a nearby porphyrin or another moiety. In addition, it can be involved in triplet–triplet energy transfer interactions with other chromophores. In contrast, little has been known about the short-lived singlet excited state. In this work, ultrafast time-resolved absorption spectroscopy has been used to investigate the singlet excited state of Au(III) 5,15-bis(3,5-di-t-butylphenyl)-2,8,12,18,-tetraethyl-3,7,13,17-tetramethylporphyrin in ethanol solution. The excited singlet state is found to form with the laser pulse and decay with a time constant of 240 fs to give the triplet state. The triplet returns to the ground state with a lifetime of 400 ps. The lifetime of the singlet state is comparable with the time constants for energy and photoinduced electron transfer in some model and natural photosynthetic systems. Thus, it is kinetically competent to take part in such processes in suitably designed supermolecular systems.

Our continuing survey of phototoxins from higher plants has led to the isolation and identification from the common rush, Juncus effusus L., of the phenanthrene, dehydroeffusol (1), and the dihydrophenanthrene, juncusol (2), compounds that display enhanced antimicrobial activities in light. The antimicrobial activities (minimum inhibitory concentrations) for these compounds against methicillin-resistant and -sensitive Staphylococcus aureus and Candida albicans were increased 16- and two-fold, respectively, by irradiation with ultraviolet A (UVA). Photosensitized DNA-binding activities (as possible covalent bond formation) of these compounds were determined by using restriction enzymes and a specially prepared 1.5 kb DNA fragment. Under UVA irradiation, dehydroeffusol strongly inhibited all the restriction enzymes (KpnI, XbaI, PmeI, DraI, PacI and BciVI) that have at least one 5′-TpA sequence in their recognition sites. Weak inhibitions were found for the restriction enzymes EcoRI, SacI, BamHI, SalI, PstI and HindIII, which do not possess a 5′-TpA sequence at their restriction sites and the restriction site sequences of which consist of all bases, A, T, G and C. Trace or no inhibition was found for AscI and SmaI, the restriction site sequences of which are composed of only C and G. The results indicate the necessity of thymine (adenine) for the photosensitized DNA-binding activity of dehydroeffusol. A strong inhibition against SphI, which does not have a 5′-TpA sequence in the restriction sequence, indicates that there are possibly other binding sequence(s) for dehydroeffusol. With juncusol and UVA, strong inhibitions for KpnI and BciVI and trace inhibitions for PacI, XbaI, PmeI and DraI were found. This result also showed a preference of juncusol for 5′-TpA, but the preference could be more selective than that of dehydroeffusol depending on the surrounding sequences of 5′-TpA in the respective restriction sites. A strong inhibition of SphI by juncusol with UVA also indicated the existence of an unknown binding sequence for this compound. Generally, the DNA-binding activity of this compound was weaker than that of dehydroeffusol.

Two-photon fluorescence imaging is used to detect UV-induced reactive oxygen species (ROS) in ex vivo human skin in this study. ROS (potentially H₂O₂, singlet oxygen or peroxynitrite [or all]) are detected after reaction with nonfluorescent dihydrorhodamine-123 (DHR) and the consequent formation of fluorescent rhodamine-123 (R123). The cellular regions at each epidermal stratum that generate ROS are identified. R-123 fluorescence is detected predominately in the lipid matrix of the stratum corneum. In contrast, the strongest R123 fluorescence signal is detected in the intracellular cytoplasm of the viable epidermal keratinocytes. A simple bimolecular one-step kinetic model is used for estimating the upper bound of the number of ROS that are generated in the skin and that react with DHR. After ultraviolet-B radiation (280–320 nm) (UVB) equivalent to 2 h of noonday summer North American solar exposure (1600 J m⁻² UVB), the model finds that 14.70 × 10⁻³ mol of ROS that react with DHR are generated in the stratum corneum of an average adult-size face (258 cm⁻²). Approximately 10⁻⁴ mol are potentially generated in the lower epidermal strata. The data show that two-photon fluorescence imaging can be used to detect ROS in UV-irradiated skin.

A system to determine the spectral responsivity of ultraviolet (UV) radiometers has been developed and is routinely operated at the Central Ultraviolet Calibration Facility, at the National Oceanic and Atmospheric Administration. The instrument and the measurement methodologies are described. Results of measurements from thermally controlled broadband UV radiometers of the Robertson-Berger (R-B)–type are described. Systematic differences in the spectral response curves for these instruments have been detected. The effect of these differences on the field operation of UV-B radiometers has been studied by calculating the instrumental response from modeled UV spectra. The differences of the weighted spectral UV irradiances, measured by two radiometers with different spectral response functions, caused by the daily variation in the position of the sun were estimated for fixed values of total ozone, altitude and albedo, and for cloud-free conditions. These differences increase with the solar zenith angle and are as large as 8%. Larger differences in the instrumental response may be produced by ozone variations. Thus, care must be taken when analyzing data from R-B radiometers and comparing results from different instruments. Routine cycling of UV-B radiometers in operative networks without a careful determination of the spectral responsivity, or small drifts of the spectral responsivity, may strongly affect the accuracy of UV radiation measurements and produce an erroneous trend. Because of the possible differences among radiometers, it would not be practical to derive the long-term behavior of UV radiation without routine and thorough characterization of the spectral responsivities of the instruments.

Inflammatory stimuli result in the production of cutaneous eicosanoids, which are known to contribute to the process of tumor promotion. Cyclooxygenase (COX), the rate-limiting enzyme for the production of prostaglandins (PG) from arachidonic acid, exists in at least two isoforms, COX-1 and COX-2. COX-1 is constitutively expressed in most tissues and plays various physiological roles, whereas increased COX-2 expression is known to occur in several types of epithelial neoplasms. Enhanced PG synthesis is a potential contributing factor in UVB-induced nonmelanoma skin cancers (NMSC). Increased COX-2 staining occurs in murine skin neoplasms after chronic exposure to carcinogenic doses of UVB. In this study, immunohistochemical and Western blot analyses were employed to assess longitudinally COX-2 expression in a standard mouse UVB complete carcinogenesis protocol and in human basal cell carcinomas (BCC) and squamous cell carcinomas (SCC). During UVB irradiation of mice, COX-2 expression consistently increased in the hyperplastic skin, the benign papillomas and the SCC. COX-2 expression was also increased in human actinic keratoses, SCC and BCC as well as in murine SCC and BCC. The pattern of COX-2 expression was quite variable, occurring in a patchy distribution in some lesions with staining confined mainly to suprabasal cell layers. In general, COX-2 expression progressively became more extensive in benign papillomas and well-differentiated murine SCC. The staining was predominantly cytoplasmic and perinuclear in some focal areas in tissue stroma around both murine and human tumors. Western blot analysis confirmed negative COX-2 expression in normal skin, whereas acute UVB exposure resulted in increased enzyme expression, which continued to increase in developing papillomas and SCC. Because of the evidence indicating a pathogenic role for eicosanoids in murine and human skin neoplasms, we performed studies to assess the anti-inflammatory and anticarcinogenic effects of green tea extracts, which are potent antioxidants. Acute exposure of the human skin to UVB (minimum erythema dose × 4) caused a transient enhancement of the COX-2 expression, which reverted to baseline within hours; however, in murine skin the expression persisted for several days. Pretreatment with the topically applied green tea extract (1 mg/cm²) largely abrogated the acute COX-2 response to UVB in mice or humans. In summary, enhanced COX-2 expression serves as a marker of epidermal UVB exposure for murine and human NMSC. These results suggest that COX-2 inhibitors could have potent anticarcinogenic effects in UVB-induced skin cancer.

δ-Aminolevulinic acid–photodynamic therapy (ALA-PDT) has emerged as a useful technique in the treatment of superficial basal cell carcinoma, actinic keratosis, squamous cell carcinoma and tumors of other organs. Earlier reports mention that there is reappearance of protoporphyrin IX (PpIX) after photoirradiation of tumors. This property of reappearance of PpIX is being utilized to treat nodular tumors by fractionated light dose delivery. However, there is still no unanimously accepted reason for this reappearance phenomenon and the rate of resynthesis after PDT. On account of this, studies are carried out on the estimation of the pharmacokinetics of the ALA-induced PpIX in mice tumor models and the surrounding normal tissues before and after PDT. Further, a mathematical model based on a multiple compartment system is proposed to estimate the rate parameter for the diffusion of PpIX from the surrounding normal tissues into the tumor tissue (k_m) caused by photobleaching during PDT with irradiating fluences of 36.0 and 57.6 J/cm². The k_m value at two different fluences, 36.0 and 57.6 J/cm², are estimated as 3.0636 ± 0.7083 h⁻¹ and 6.9231 ± 2.17651 h⁻¹, respectively. Further, the rate parameter for the cleavage and efflux of ALA (k₁) and the rate parameter for the evasion of PpIX from the tumor tissues after PDT (k_t) were also estimated by fitting the experimental data to the developed mathematical model. The statistical significance of the estimated parameters was determined using Student's t-test. The experimental results and the rate parameters obtained using the proposed compartment model suggest that in addition to the earlier reported reasons, the invasion or diffusion of PpIX from the surrounding tissues to the tumor tissues after photoirradiation might also contribute to the reappearance of PpIX after PDT.

A study has been carried out to define the importance of the peripheral benzodiazepine receptor (PBR) as a binding site for a series of chlorin-type photosensitizers, pyropheophorbide-a ethers, the subject of a previous quantitative structure–activity relationship study by us. The effects of the PBR ligand PK11195 on the photodynamic activity have been determined in vivo for certain members of this series of alkyl-substituted ethers: two of the most active derivatives (hexyl and heptyl), the least active derivative (dodecyl [C12]) and one of intermediate activity (octyl [C8]). The photodynamic therapy (PDT) effect was inhibited by PK11195 for both of the most active derivatives, but no effect on PDT activity was found for the less active C12 or C8 ethers. The inhibitory effects of PK11195 were predicted by the binding of only the active derivatives to the benzodiazepine site on albumin, i.e. human serum albumin (HSA)-Site II. Thus, as with certain other types of photosensitizers, it has been demonstrated with this series of pyropheophorbide ethers that in vitro binding to HSA-Site II is a predictor of both optimal in vivo activity and binding to the PBR in vivo.

The Photofrin-resistant cell line (HT29-P14) was used in the present study to investigate the mechanism(s) involved in Photofrin-mediated photodynamic therapy (PDT). We compared gene expression profiles between the resistant cell line and its parental cell line (HT29) using DNA microarray analysis. A significant up-regulation of small heat shock protein 27 (Hsp27) was found in HT29-P14 cells. The elevated Hsp27 level may play an important role in the resistance of HT29-P14 to Photofrin-PDT. To test this hypothesis, we stably transfected HT29 cells with human Hsp27 complementary DNA. The potential role of Hsp27 in the resistance to PDT was examined in Hsp27-overexpressing cells. Stable trasnfected cells (H13) showed an increased survival after Photofrin-PDT, suggesting that the up-regulation of Hsp27 is related to the induced resistance to Photofrin-PDT. Phosphorylation of Hsp27 has been suggested to play an important role in cytoprotection. We have examined the phosphorylation activity of Hsp27 among the parental and resistant cells, as well as the overexpression cells. An elevated level of Hsp27 resulted in an increased ability of phosphorylation in both resistant and overexpressing cells after PDT. The activation of the phosphorylation of Hsp27 induced by PDT was not mediated by the p38 mitogen-activated protein kinase. These data suggest that Hsp27 may play an important role in mediating the adaptive response to Photofrin-PDT–induced oxidative stress and that the pathways leading to Hsp27 phosphorylation may contribute to the resistance of the cells to photooxidative damage.

A novel type of circadian and photoperiodic control of the cell division cycle was found in photoautotrophic Euglena gracilis. When algae entrained to 24 h light–dark (LD) cycles (14 h L) were transferred to continuous darkness (DD) at the eighth hour of the final LD photoperiod, cell-cycle transition was arrested in phase G1, S or G2. The subsequent exposure of these dark-arrested cells to a 6 h light-break allowed the dark-arrested cells to undergo cell-cycle progression in DD, in a manner dependent on the circadian phase; maximum photoinduction occurred around dusk. Inhibitor experiments suggested that the photoinduced commitment of G2 cells to cell division required light for a signal originating in noncyclic photosynthetic electron transport (PET), particularly cytochrome b₆–f but not for the metabolic energy required by the process. The fact that the circadian rhythm of photoinduction ran out-of-phase from that of noncyclic PET signaling suggests that the site of regulation by the former rhythm is downstream of noncyclic PET. The occurrence of maximum photoinduction around dusk suggests that the ‘external coincidence' model of photoperiodic induction describes the activation of the photoinductive phase. Further evidence supporting this hypothesis is the relationship between cell reproduction and day length; the resulting sigmoidal curve indicates a combined effect of photosynthesizing period and circadian stimulation around dusk. Circadian control is shown to be an integral part of the mechanism for 24 h LD cycle–induced synchronous cell division.

Two carotenoid pigments have been linked as axial ligands to the central silicon atom of a phthalocyanine derivative, forming molecular triad 1. Laser flash studies on the femtosecond and picosecond time scales show that both the carotenoid S₁ and S₂ excited states act as donor states in 1, resulting in highly efficient singlet energy transfer from the carotenoids to the phthalocyanine. Triplet energy transfer in the opposite direction was also observed. In polar solvents efficient electron transfer from a carotenoid to the phthalocyanine excited singlet state yields a charge-separated state that recombines to the ground state of 1.