Laser excitation of aqueous solutions of fenofibric acid (FA) at pH 7.4 show the formation of two reaction intermediates, the triplet state and the hydrated electron. The former is longer lived in water than in acetonitrile; its anionic form decays irreversibly by decarboxylation to give a carbanion that protonates before or after rearrangement. Several spectroscopic and quenching studies suggest that in aqueous media the triplet state of FA has a π,π* character, in comparison with an n,π* character in organic media. Further, the known chemistry of the triplet, including decarboxylation and hydrogen abstraction, occurs predominantly from the n,π* state, and as a consequence, activation energies are higher when the lowest triplet has a π,π* character. Photoionization is more important in aqueous than in organic media and involves a biphotonic process. Hydrated electrons are trapped by FA, leading to the corresponding ketyl radical after protonation.

The fluorescence properties of thiazole orange, linked via a (1) hydrophobic alkyl or a (2) hydrophilic ethylene glycol chain to the central internucleotidic phosphate group of a pentadeca-2′-deoxyriboadenylate (dA₁₅), are evaluated. Linkage at the phosphate group yields two stereoisomers, S-isomer of the phosphorus chiral center (Sp) and R-isomer of the phosphorus chiral center (Rp); these are studied separately. The character of the linkage chain and the chirality of the internucleotidic phosphate linkage site influence the fluorescent properties of these thiazole orange–oligonucleotide conjugates (TO-probes). Quantum yields of fluorescence (Φ_fl) of between 0.04 and 0.07 were determined for the single-stranded conjugates. The fluorescence yield increased by up to five times upon hybridization with the complementary sequence (d^5′[CACT₁₅CAC^3′]); Φ_fl values of between 0.06–0.35 were determined for the double-stranded conjugates. The Φ_fl value (0.17) of thiazole orange, 1-(N,N′-trimethylaminopropyl)-4-[3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene]-quinolinium iodide (TO-Pro 1) in the presence of the oligonucleotide duplex (TO-Pro 1: dA₁₅·d^5′[CACT₁₅CAC^3′] (1:1)) is much less than that for some of the hybrids of the conjugates. Our studies, using steady-state and time-resolved fluorescence experiments, show that a number of discrete fluorescent association species between the thiazole orange and the helix are formed. Time-resolved studies on the four double-stranded TO-probes revealed that the fluorescent oligonucleotide–thiazole orange complexes are common, only the distribution of the species varies with the character of the chain and the chirality at the internucleotidic phosphate site. Those TO-probes in which the isomeric structure of the phosphate-chain linkage is Rp, and therefore such that the fluorophore is directed toward the minor groove, have higher Φ_fl values than the Sp isomer. Of the systems studied, thiazole orange linked by an alkyl chain to the internucleotidic phosphate (Rp isomer) has the highest Φ_fl and the greatest fraction of the longest-lived fluorescent thiazole orange species (in the hybrid form).

The reversible sequestration and release of metal ions is an important objective in biological and environmental research. Unfortunately, although there have been dramatic examples of metal ion activity control, there are very few quantitative investigations of stoichiometry, equilibria and kinetics. A significant contributor to this lack of quantitative work is the complexity of many photochromic systems. Therefore, we have attempted to create a simple, reversible photochromic metal-ion chelator that can be analyzed quantitatively. The chelator should have certain other attributes as well, namely, that it binds to divalent metal ions (because of their extreme biological importance) and that it binds metal ions in the dark so that light is used to release metal ions rather than sequester them. The photochromic chelator (1) binds to divalent metal ions [Zn(II), Cu(II), Pb(II), Hg(II), Fe(II), Co(II) and Cd(II); other metal ions have not yet been tested] in the dark with a significant binding strength. In both methanol (by spectrophotometry) and methanol–water (by voltammetry), the stoichiometry of the 1–Zn(II) complex is 2:1. The binding constant (K₁K₂) is on the order of 10¹²–10¹⁴ M⁻² in methanol and 5.0 × 10⁸ M⁻² in 50% aqueous methanol. The chelator 1 is photolabile, yielding 2 with a quantum efficiency of 0.91. In a solution containing excess Zn(II), so that over 99% of the ligand exists as the monodentate complex, photolysis produces 2 with a quantum efficiency of 0.15. A kinetic analysis leads to the conclusion that the complex itself is photolabile.

A fluence-dependent fluorescence technique was used to observe reverse intersystem crossing from a certain higher-lying triplet state of rose bengal populated by a single pulse of 532 nm light. The quantum yield of reverse intersystem crossing from this state was determined to be 0.12 ± 0.02 for rose bengal in phosphate-buffered saline. The importance of including molecular rotation effects in the analysis of fluorescence resulting from reverse intersystem crossing is discussed. Differences in the photochemical reactivity of upper triplet states in biological systems have been previously hypothesized to result from photophysical differences, particularly substantial differences in their reverse intersystem crossing yields. In this work this hypothesis is analyzed quantitatively, using numerical models of the population dynamics. These models suggest that reverse intersystem crossing alone cannot adequately explain the differences in biological response.

Dimerization of free acid and ester forms of disulfonated deuteroporphyrin is investigated in aqueous solution by absorbance and fluorescence spectroscopies. The dimerization equilibrium constant increases with the extent of esterification. In phosphate buffer saline (pH 7.4, 20°C), it ranges from 1.4 × 10⁶ M⁻¹ to 7.8 × 10⁷ M⁻¹ for the free acid and the diethyl ester forms, respectively. The dimer formation is favored by an increase of ionic strength, as predicted by the Debye–Hückel law. The dimers display a marked shift to the blue of their Soret band. In agreement with the exciton model, a cofacial stacking of the molecules with some offset is postulated. The sulfonate groups on each molecule are likely to stand on opposite directions to reduce repulsion. Both the analysis of porphyrin self-association and careful examination of the fluorescence excitation spectra show that the dimers of disulfonated deuteroporphyrins do not fluoresce at all. The quantum yield of formation of singlet oxygen by the disulfonated deuteroporphyrins in deuterated methanol is 0.71, a value typical of monomers. In deuterated water, the yield is 0.44 for all the compounds studied though they are dimerized. The fact that nonfluorescent dimers of porphyrins can be efficient photosensitizers is emphasized.

The fluorescence properties of ofloxacin (OFL), norfloxacin (NOR) and flumequine (FLU) were studied in H₂O–CH₃OH and H₂O–CH₃CN mixed solvents because these solvents were thought to behave as a biological mimetic system. The emission spectra of OFL and NOR were very sensitive to the composition of the solvents. In the Lippert–Mataga analysis of the steady-state fluorescence data of OFL and NOR, clear reverse solvatochromism was exhibited in both mixed solvents. This observation can be explained by the twisted excited-state intramolecular charge transfer, which is accelerated by water. Theoretical treatments further support these results. The radiative and nonradiative rate constants were analyzed as a function of solvent dipolarity–polarizability (π^*) and hydrogen-bond donor acidity (α). These results were well consistent with the suggested mechanism of the excited-state chemical process of OFL and NOR, which depended upon the solvent–solute interactions such as bulk dielectric effects and specific hydrogen-bonding interactions. However, the influence of dielectric effects was more significant. The solvent structures of H₂O–CH₃CN and the preferential solvation by water were also examined. The emission spectra of FLU do not exhibit any characteristic responses to the properties of the environment.

The synthesis, characterization and photophysical properties of two perfluoroalkyl (5,10,15,20-tetrakis-[trifluoromethyl]- and [heptafluoropropyl]-porphyrin) and two perfluoroaryl (5,10,15,20-tetrakis-[2,6-difluorophenyl]- and [pentafluorophenyl]-porphyrin) are described, with reference to their potential in both photodynamic therapy (PDT) and in vivo imaging by fluorescence and ¹⁹F nuclear magnetic resonance spectroscopy. Absorption and fluorescence spectra, fluorescence lifetimes and triplet–singlet difference spectra are reported. Triplet yields have been obtained by flash photolysis and pulse radiolysis, whereas yields of sensitized singlet oxygen formation have been determined by time-resolved phosphorimetry. All four compounds show high yields of triplet formation and singlet oxygen sensitization. The spectral properties, stability and attractive solubility characteristics of the perfluoroalkyl derivatives make them particularly suitable candidates for future study for applications in PDT.

We developed a facile, cost-effective competitive binding assay for the analysis of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) in DNA, using a polyclonal rabbit antiserum raised against an 8-oxodGuo hapten coupled to bovine serum albumin and radiolabeled synthetic ligand containing multiple 8-oxodGuo residues. This radioimmunoassay (RIA) displays a high affinity for 8-oxodGuo in DNA, with a detection limit of ∼1 adduct in 10⁵ bases of DNA. 8-oxodGuo standards for RIA were quantified by high-performance liquid chromatography and electrochemical detection in DNA diluted in methylene blue and exposed to visible light. As an initial application we quantified 8-oxodGuo in dosimeters deployed at increasing depths in the Southern Ocean during the austral spring of the 1998 field season or at the surface at Palmer Station, Antarctica, throughout the 1999 field season. Cyclobutane pyrimidine dimers (CPD) were quantified using an established RIA. We found that the frequency of both photoproducts decreased with depth. However, CPD induction was attenuated at a faster rate than 8-oxodGuo, correlating with the differential attenuation of solar ultraviolet wavelengths in the water column. CPD induction was closely related with ultraviolet-B radiation (UVB) attenuation, whereas the lower attenuation of 8-oxodGuo suggests that oxidative damage is more closely related to ultraviolet-A radiation (UVA) irradiance. The ratio of 8-oxodGuo : CPD was also found to covary with changes in stratospheric ozone concentrations at Palmer Station. These data demonstrate the usefulness of these assays for environmental photobiology and the potential for their use in studying the relative impacts of UVB versus UVA, including ozone depletion events.

Ultraviolet radiation–induced DNA damage frequencies were measured in DNA dosimeters and natural plankton communities during the austral spring at Palmer Station, Antarctica, during the 1999–2000 field season. We found that the fluence of solar ultraviolet-B radiation (UV-B) at the earth's surface correlated with stratospheric ozone concentrations, with significant ozone depletion observed because of “ozone hole” conditions. To verify the interdependence of ozone depletion and DNA damage in natural microbial communities, seawater was collected daily or weekly from Arthur Harbor at Palmer Station, Antarctica, throughout “ozone season,” exposed to ambient sunlight between 0600 and 1800 h and fractionated using membrane filtration to separate phytoplankton and bacterioplankton populations. DNA from these fractions was isolated and DNA damage measured using radioimmunoassay. Under low-ozone conditions cyclobutane dimer concentrations in bacterioplankton and phytoplankton communities were maximal. DNA damage measured in dosimeters correlated closely with ozone concentrations and UV-B fluence. Our studies offer further support to the theory that stratospheric deozonation is detrimental to marine planktonic organisms in the Southern Ocean.

Spectral reflectance of maple, chestnut and beech leaves in a wide range of pigment content and composition was investigated to devise a nondestructive technique for total carotenoid (Car) content estimation in higher plant leaves. Reciprocal reflectance in the range 510 to 550 nm was found to be closely related to the total pigment content in leaves. The sensitivity of reciprocal reflectance to Car content was maximal in a spectral range around 510 nm; however, chlorophylls (Chl) also affect reflectance in this spectral range. To remove the Chl effect on the reciprocal reflectance at 510 nm, a reciprocal reflectance at either 550 or 700 nm was used, which was linearly proportional to the Chl content. Indices for nondestructive estimation of Car content in leaves were devised and validated. Reflectances in three spectral bands, 510 ± 5 nm, either 550 ± 15 nm or 700 ± 7.5 nm and the near infrared range above 750 nm are sufficient to estimate total Car content in plant leaves nondestructively with a root mean square error of less than 1.75 nmol/cm².

We report measurements performed on the normal skin of rats in vivo, which provide information on the photobleaching kinetics and mechanisms of the photosensitizer meso-tetrahydroxyphenyl chlorin (mTHPC). Loss of mTHPC fluorescence was monitored using in vivo fluorescence spectroscopy during photodynamic therapy (PDT) performed using 650 nm laser irradiation. The bleaching was evaluated for irradiances of 5, 20 and 50 mW cm⁻². Two distinct phases of mTHPC photobleaching were observed. In the first phase there was no obvious irradiance dependence in the loss of fluorescence vs fluence. The second phase was initiated by an irradiance-dependent discontinuity in the slope of the bleaching curve, after which the photobleaching rates showed an irradiance dependence consistent with an oxygen-dependent reaction process. To investigate the unusual shape of the in vivo bleaching curves, we measured the PDT-induced changes in O₂ concentrations in mTHPC-sensitized spheroids irradiated with 2, 5 and 20 mW cm⁻² of 650 nm light. The oxygen concentration data indicated no unusual features within the range of fluences where the discontinuities in fluorescence were observed during in vivo spectroscopy. The fluorescence from the in vivo bleaching experiments thus reports a phenomenon that is not reported by measurements of the photochemical oxygen consumption in the spheroids.

An implicit dosimetric model has been proposed in which biological damage caused by photodynamic therapy (PDT) is monitored through the decrease in sensitizer fluorescence during treatment. To investigate this, in vitro experiments were performed in which DP16 cells were incubated in meta-tetra(hydroxyphenyl)chlorin (mTHPC) and then irradiated with 514 nm light. Photosensitizer concentration, fluence rate and oxygenation were independently controlled and monitored during the treatment. Fluorescence of mTHPC was continuously monitored via a charge-coupled device–coupled spectrometer during treatment and, at selected fluence levels, cell viability was determined using a trypan blue exclusion assay. The relationship of cell viability to normalized fluorescence was obtained for the different treatment conditions. The relationship was independent of cell medium oxygenation, treatment fluence rate and sensitizer incubation concentration except at a high mTHPC concentration (4 μg/mL). This relationship suggests that fluorescence bleaching may be used to predict mTHPC PDT damage in vitro.

Treatment of cutaneous melanoma (M-3 and B16-F10 implanted in mice) with rapidly-scanned, tightly-focused near infrared light elicits selective destruction of tumor tissue. A single laser treatment yielded complete eradication in >90% of B16-F10 tumors with thicknesses of approximately 3 mm; amelanotic M-3 tumors proved less responsive (ca 25% clearance rate). In addition to local tumor destruction, laser treatment of B16-F10 tumors in immunocompetent mice stimulated enhanced cytokine levels (interleukin-2 and interleukin-10) within treated tumor tissues and rejection of tumor cells upon a subsequent challenge dose. Such an antitumor immune response may lead to improved outcomes at both the treatment site and at sites of distant metastasis.

To determine whether the transcription factor activator protein-1 (AP-1) could be modulated by ultraviolet A (UVA) exposure, we examined AP-1 DNA-binding activity and transactivation after exposure to UVA in the human immortalized keratinocyte cell line HaCaT. Maximal AP-1 transactivation was observed with 250 kJ/m² UVA between 3 and 4 h after irradiation. DNA binding of AP-1 to the target 12-O-tetradecanoylphorbol-13-acetate response element sequence was maximally induced 1–3 h after irradiation. Both de novo transcription and translation contributed to the UVA-induced AP-1 DNA binding. c-Fos was implicated as a primary component of the AP-1 DNA-binding complex. Other components of the complex included Fra-2, c-Jun, JunB and JunD. UVA irradiation induced protein expression of c-Fos, c-Jun, Fra-1 and Fra-2. Phosphorylated forms of these induced proteins were determined at specific time points. A strong correlation existed between UVA-induced AP-1 activity and accumulation of c-Fos, c-Jun and Fra-1 proteins. UVA irradiation also induced c-fos and c-jun mRNA expression and transcriptional activation of the c-fos gene promoter. These results demonstrate that UVA irradiation activates AP-1 and that c-fos induction may play a critical role in the response of these human keratinocytes to UVA irradiation.

Two marine dinoflagellates, Lingulodinium polyedrum and Pyrocystis lunula, emit light in a reaction involving the enzymatic oxidation of its tetrapyrrole luciferin by molecular oxygen. The characteristic properties of P. lunula luciferase have not been clarified, whereas L. polyedrum luciferase, which has three active domains, has been characterized. A cloned partial cDNA of the P. lunula luciferase encodes an active fragment corresponding to part of domain 2 and all of domain 3 of L. polyedrum luciferase. The homology of the amino acid sequence between the two luciferases in domain 3 is about 84.3%. A recombinant His-tagged luciferase fragment containing domain 3 (M_r = 46 kDa) catalyzed the light-emitting oxidation of luciferin (λ_max = 474 nm). This protein was purified by a single affinity-chromatography procedure. The pH–activity profile and the bioluminescence spectrum of the recombinant enzyme having a third domain are almost identical to those of an extract from P. lunula cultured in vitro. The recombinant enzyme is active at pH 8.0, although the recombinant enzyme derived from the second domain of L. polyedrum luciferase is inactive at pH 8.0. Substitution of Glu-201 by histidine in the third domain of P. lunula luciferase showed a decrease of activity above pH 7.0, suggesting that histidine residues could be responsible for pH-sensitivity in dinoflagellate luciferase.

Rhodopsin is the dim light photosensitive pigment of animals. In this work, we undertook to study the structure of rhodopsin from swine and compare it with bovine and rat rhodopsin. Porcine rhodopsin was analyzed using methodology developed previously for mass spectrometric analysis of integral membrane proteins. Combining efficient protein cleavage and high performance liquid chromatography separation with the sensitivity of mass spectrometry (MS), this technique allows the observation of the full protein map and the posttranslational modifications of the protein in a single experiment. The rhodopsin protein from a single porcine eye was sequenced completely, with the exception of two single–amino acid fragments and one two–amino acid fragment, and the gene sequence reported previously was confirmed. The posttranslational modifications, similar to the ones reported previously for bovine and rat rhodopsin, were also identified. Although porcine rhodopsin has a high degree of homology to bovine and rat rhodopsins and most of their posttranslational modifications are identical, the glycosylation and phosphorylation patterns observed were different. These results show that rhodopsin from a single porcine eye can be characterized completely by MS. This technology opens the possibility of rhodopsin structural and functional studies aided by powerful mass spectrometric analysis, using the fellow eye as an internal control.

Consecutive spectral analyses of ultraweak photon emission from sweet potato showing a defense response were conducted to observe the process of physiological transition. The spectrum showed a drastic transition from 2 to 10 h after inoculation with Fusarium oxysporum, during which the emission intensity increased slowly. The spectrum was stable from 10 to 36 h after inoculation, whereas the emission intensity peaked approximately 20 h after inoculation. A change in the physiological state connected with the synthesis of defense-related substances is suggested as contributing to this phenomenon. The spectral transition was also detected in sweet potato treated with growth hormone or exposed to alternating temperature, although with an extremely weak emission intensity. This spectral analysis of ultraweak photon emission can be used as a new means for identifying the physiological state.