Multilayer oriented Langmuir–Blodgett films of bacteriorhodopsin were prepared and their nonlinear optical properties, including second harmonic generation and photoresponse at a two phase-modulated beams mixing, were investigated. The nonlinear component of refractive index of the films was measured.

The reactions of several psoralen and coumarin radical cations with biological substrates such as nucleotides, amino acids and alkenes that serve as models for unsaturated fatty acids have been examined. The radical cations were generated by laser photoionization of the parent psoralen or coumarin in aqueous buffer in most cases. Easily oxidized substrates such as tyrosine, tryptophan and guanosine monophosphate react with the 8-methoxypsoralen and several methoxy-substituted coumarin radical cations with rate constants in excess of 2 × 10⁹ M⁻¹ s⁻¹. In each case reaction occurs via electron transfer, as demonstrated by the observation of quencher-derived radical cations or radicals by transient absorption spectroscopy. For other substrates such as histidine, methionine and adenosine monophosphate the measured rate constants are significantly slower and vary with the oxidation potential of both the parent psoralen or coumarin and the quencher, again indicative of electron transfer reactivity. Most of the alkenes studied also react with the psoralen or coumarin radical cations via electron transfer, although there is some evidence for addition for linoleic acid. Product studies carried out using both lamp and laser irradiation in the presence of deoxyguanosine as a radical cation trap lead to the formation of characteristic base-derived Type-I (electron transfer) products. This lends support to our previous hypothesis that photoionization occurs via a monophotonic process and is thus relevant to conditions used in clinical phototherapeutic applications of psoralens. The results demonstrate the relevance of electron transfer chemistry to the use of psoralens and related compounds as photoactivated drugs.

The photodecarboxylation reaction of 2-(3-benzoylphenyl)propionate (ketoprofen anion, KP⁻) was studied in water and in 0.1 M phosphate buffer solutions in the pH range 5.7–11.0 by laser-induced optoacoustic spectroscopy (LIOAS, T range 9.5–31.6°C). Upon exciting KP⁻ with 355 nm laser pulses under anaerobic conditions, two components in the LIOAS signals with well-separated lifetimes were found (τ₁ < 20 ns; 250 < τ₂ < 500 ns) in the whole pH range, whereas a long-lived third component (4 < τ₃ < 10 μs) was only detected at pH ≤ 6.1. The heat and structural volume changes accompanying the first step did not depend on pH or on the presence of buffer. The carbanion resulting from prompt decarboxylation within the nanosecond pulse (<10 ns) drastically reduces its molar volume ([−18.9 ± 2.0] cm³/mol) with respect to KP⁻and its enthalpy content is (256 ± 10) kJ/mol. At acid pH (ca 6), a species is formed with a lifetime in the hundreds of ns. The enthalpy and structural volume change for this species with respect to KP⁻ are (181 ± 15) kJ/mol and ( 0.6 ± 2.0) cm³/mol, respectively. This species is most likely a neutral biradical formed by protonation of the decarboxylated carbanion, and decays to the final product 3-ethylbenzophenone in several μs. At basic pH (ca 11), direct formation of 3-ethylbenzophenone occurs in hundreds of ns involving a reaction with the solvent. The global decarboxylation reaction is endothermic ([45 ± 15] kJ/mol) and shows an expansion of ( 14.5 ± 0.5) cm³/mol with respect to KP⁻. At low pH, the presence of buffer strongly affects the magnitude of the structural volume changes associated with the intermolecular proton-transfer processes of the long-lived species due to reactions of the buffer anion with the decarboxylated ketoprofen anion.

The osmotic fragility of the erythrocyte membrane to hypotonic solutions is investigated theoretically. The fragility curves exhibit a strong transmittance rise. This variation is assumed to result from changes in the scattering properties of erythrocytes under dialysis resulting from swelling and hemolysis. The refractive indices of erythrocytes are obtained through the Lorentz–Lorenz relation based on hemoglobin and water contents. The scattering cross sections (needed to calculate the collimated transmittance) and the forward scattered intensity (needed to calculate the incoherent transmittance) are expressed according to the simple algebraic relations of the anomalous diffraction approximation. It is shown that swelling (or shrinking) has no influence on the collimated transmittance. Hemolysis alone causes the abrupt sigmoidal increase of the collimated transmittance with time. The possible transmittance increase (decrease) observed during swelling (shrinking) is due to incoherent transmittance and depends on the detecting solid angle value of the experimental setup.

A spectral resolution procedure was used to resolve the absorption, excitation and emission spectra of the fluorescein monoanion in a number of solvent–water mixtures. This permitted an analysis of the effect of the solvent environment on the spectral properties of the monoanion and on the lactone/monoanion/dianion transitions of fluorescein. The monoanion excitation and emission spectra show relatively small changes with changing environment, a behavior that is related to the hydrogen-bonding environment of the solvent–water mixtures. There is also a general increase in the quantum yield of the monoanion from 0.36 in water to values up to 0.49 in the solvent–water mixtures. The presence of solvent also results in a general increase in the lactone content and in the monoanion:dianion and lactone:monoanion ratios. General polarity effects alone cannot account for the observed effects on the prototropic transitions indicating that specific solute–solvent effects involving hydrogen bonding perturb the prototropic equilibria of fluorescein.

Human adenocarcinoma cells of the line WiDr were incubated with 5-aminolevulinic acid to induce protoporphyrin IX (PpIX) and then exposed to laser light of wavelength 635 nm. The PpIX fluorescence decreased with increasing exposure. The decay rate was slightly dependent on the initial PpIX concentration. The PpIX fluorescence was halved by a fluence of about 40 J/cm². Several fluorescing photoproducts were formed. The main one, supposedly the chlorin-type photoprotoporphyrin (Ppp), had a fluorescence excitation spectrum stretching out to about 680 nm with a maximum at around 668 nm. The formation kinetics of this product was dependent on the initial PpIX concentration. Moreover, it was selectively bleached by exposure to light at 670 nm. A photoproduct with an emission maximum at 652 nm, different from Ppp, remained after this exposure. Traces of a photoproduct(s) with fluorescence emission slightly blueshifted compared with that of PpIX, supposedly water-soluble porphyrins, were also detected after light exposure.

The study identifies the relative contribution of various bio-optical factors to the total attenuation of ultraviolet radiation (UVR) wavelengths and photosynthetically active radiation (PAR) in temperate coastal waters of Japan by surveying the physical properties of the water column, UVR and PAR penetration, and the absorption characteristics of dissolved and particulate material. Spectral absorbance properties of pigment (a_ph), detritus (a_d) and chromophoric dissolved organic material (a_CDOM) displayed both seasonal and wavelength specific variability. On an annual basis, absorbance by a_CDOM was the highest absorbing fraction (47–59%) for the UVR wavelengths measured (305, 320, 340 and 380 nm) but decreased (32%) at 450 nm. Contribution of pigments to total absorbance was highest (40–60%) during a spring bloom for both UVR and PAR. A large variability (C.V. > 42%) for annual average attenuation coefficients (K_d[λ]) at respective wavelengths observed suggests that the spectral composition of the water column changes throughout the year in this region. A significant relationship was observed between K_d(λ) and a_CDOM at 305, 320, 340 and 380 nm only (P < 0.01) but not for 450 nm (PAR) indicating the role of CDOM in regulating variations in K_d(λ), particularly in the UVR range. The slope S, obtained from a natural-log plot of the absorption coefficient of CDOM against wavelength, ranged between 0.014 and 0.036 nm⁻¹ annually (average = 0.020±0.007, C.V. = 35%) and suggests seasonal changes in the origin of CDOM between terrestrial (low S) and biogenous (high S) CDOM.

Hypericin is the active ingredient in the over the counter antidepressant medication St. John's Wort. Hypericin produces singlet oxygen and other excited state intermediates that indicate it should be a very efficient phototoxic agent in the eye. Furthermore it absorbs in the UV and visible range, which means it can potentially damage both the lens and the retina. Lens α-crystallin, isolated from calf lenses, was irradiated in the presence of hypericin (5 × 10⁻⁵ M, 10 mM ammonium bicarbonate, pH 7.0) and in the presence and absence of light (>300 nm, 24 mW/cm²). Hypericin-induced photosensitized photopolymerization as assessed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Further analysis of the oxidative changes occurring in α-crystallin using mass spectrometry showed specific oxidation of methionine, tryptophan and histidine residues, which increased with irradiation time. Hypericin did not damage the lens protein in the dark. Damage to α-crystallin could undermine the integrity of the lens directly by protein denaturation and indirectly by disturbing chaperone function. Therefore, in the presence of light, hypericin can induce changes in lens protein that could lead to the formation of cataracts. Appropriate precautions should be taken to protect the eye from intense sunlight while on this antidepressant medication.

We investigated the dependence on solvents of optical absorption and emission of the bacteriochlorophyll a-serine (BChl-ser), a water soluble bacteriochlorophyll (BChl) derivative. Comparison between the experimental data and those collected for BChl in nonaqueous solvents shows that only a minor interaction takes place between serine and the macrocycle's π-electron system. Nevertheless, the coupling with serine results in a small enhancement of the nonradiative relaxation rate from the first excited singlet state S₁. In buffered aqueous solution (pH = 7.4), the Stokes shift of the BChl-ser fluorescence and its nonradiative relaxation rate are enhanced compared with those in nonaqueous solutions (Scherz, A., S. Katz, Y. Vakrat, V. Brumfeld, E. Gabelmann, D. Leupold, J. R. Norris, H. Scheer and Y. Salomon (1998) Photosynthesis: Mechanisms and Effects, Vol. V (Edited by G. Garab), pp. 4207–4212. Kluwer Academic, Dordrecht.), probably as a result of a hydrogen bonding between the BChl macrocycle and the water molecules. In aprotic solvents, without hydrogen bonds, the permanent dipole moment of the first excited singlet state in both BChl and BChl-ser is increased compared with the ground state by at least 2.5 Debye.

Oxidative stress induced by light activation of photosensitizers is regarded to have a role in triggering cell death pathways during photodynamic therapy (PDT). Reactive oxygen species have been proposed to act as signal transduction molecules activating downstream reactions that lead to apoptosis. Mainly debated is the cooperating role of other signaling systems like calcium or pH. The present work contributes to this discussion by studying PDT effects in cell cultures of rat bladder epithelial cells for the hydrophilic tetrasulfonated aluminum phthalocyanine (AlPcS₄). Cells were coincubated with the photosensitizer and the calcium-sensitive probe Fluo-3. The light-induced reactions were analyzed with a confocal laser scanning microscope. The dynamics of the process during light activation was observed with subcellular resolution. A transient calcium elevation during the irradiation process was detected, especially in the cell's nuclei, followed by a more sustained increase. The evaluation of the energy-dose–dependent phototoxicity after an incubation time with the photosensitizer of 1 and 24 h, showed enhanced phototoxicity when the drug was present for 24 h. Surprisingly, stimulation of cell proliferation was observed at very low light doses (at 0.2 J/cm²) when the drug was incubated for 24 h (cell viability 160%). Induction of apoptosis could be observed after irradiation with fluences between 1 and 3 J/cm². Apoptotic cells were identified with fluorescein isothiocyanate–labeled Annexin V, which binds to phosphatidylserine after its translocation to the outer plasma membrane. In the presence of the antioxidant pyrrolidinedithiocarbamate the transient calcium elevation was totally inhibited, as was the subsequent translocation of PS. In contrast, N-acetyl-l-cysteine did not suppress the transient calcium increase. Our data might be consistent with calcium regulated processes during AlPcS₄-PDT and the involvement of oxygen radicals.

Studies on the synthesis, singlet oxygen and fluorescence yields and pharmacokinetic properties of three different dimeric porphyrins with an amide linkage (D2–D4) are described and compared with the results recently reported for a dimeric porphyrin (D1). The pharmacokinetic behavior of all dimers were examined in Balb/c mice bearing MS-2 fibrosarcomas. The maximal efficiency and selectivity of photosensitizer accumulation in each tumor tissue takes place at 24 h after drug administration of 1.0 mg kg⁻¹ into dl-α-dipalmitoylphosphatidylcholine liposomes by intravenous injection. Since the dimeric porphyrins exhibit high quantum yields of singlet oxygen generation, long triplet lifetimes and high photostability, the results obtained suggest that the evaluated dimeric structures may be promising candidates for further use in PDT experiments. The results also allow the possibility to establish a correlation between the chemical structure of the dyes and the efficiency/selectivity of the tumor accumulation and can be used for building up optimal photosensitizing agents for tumors.

5-Aminolevulinic acid (ALA)–supported fluorescence endoscopy of the urinary bladder results in a detection rate of bladder cancer superior to that of white light endoscopy. The different accumulation of the metabolite protoporphyrin IX (PPIX) in tumor cells after ALA instillation is poorly understood; however, it is crucial to optimize diagnosis and potential phototherapy. For systematic analysis of cell-type specific PPIX accumulation and metabolism two human bladder carcinoma cell lines (RT4 and J82), a normal urothelial cell line (UROtsa), and a fibroblast cell line (N1) were chosen, and grown in two different growth states to model important tissue components of the urinary bladder, i.e. tumor, normal epithelium and stroma. To quantitate PPIX content, fluorescence intensities measured by flow cytometry were matched with cellular PPIX extraction values, and related to relative ferrochelatase activity, cellular iron content, number of transferrin receptors per cell and porphobilinogen deaminase (PBGD) activity. For in vitro experiments, the initial correlation of relative flow cytometric and spectrometric measurements of PPIX provides a calibration curve for consequent flow cytometric PPIX quantification. Lower fluorescence of normal cells could be explained by significant differences of ferrochelatase activity and iron content in comparison to tumor cells. However, the content of iron was not related to transferrin receptor content. PBGD activity seemed to play a minor role for the differential accumulation of PPIX in urothelial cells. In conclusion, the in vitro culture of urothelial cells and fibroblasts indicates that the most important metabolic step for PPIX accumulation in the urinary bladder is the transition from PPIX to heme. Further investigation of PPIX metabolism does support the validation of photodynamic diagnosis, and might also lead the way to a highly specific tumor related molecule.

Macromolecules accumulate in solid tumors and can thus be used as carriers for the delivery of attached contrast agents to tumors. We report the synthesis and use of serum protein–dye conjugates consisting of transferrin (Tf) or human serum albumin (HSA) and an indotricarbocyanine (ITCC) derivative as contrast agents for the optical imaging of tumors. The compounds were characterized with respect to their photophysical properties and tested in vitro for their ability to bind to tumor cells and in vivo for their potential to delineate experimental tumors. In contrast to HAS-ITTC, Tf-ITCC showed receptor-mediated uptake by HT29 human colon cancer cells in vitro. After intravenous injection into HT29 tumor-bearing nude mice both compounds induced increased fluorescence contrast of tumors in vivo. After 24 h the contrast between tumor and normal tissue was significantly higher for Tf-ITCC than for HAS-ITCC. Dye-induced fluorescence was found to be predominantly located in perinecrotic areas of the tumor. Furthermore, Tf-ITCC produced fluorescence of viable tumor cells, whereas HAS-ITCC fluorescence was recorded along connective tissue. We conclude that ITCC-labeled Tf and HSA can serve as macromolecular contrast agents for the optical imaging of tumors, with Tf-ITCC showing higher efficiency.

Fluorescence emission analysis (FEA) has proven to be very sensitive for the detection of elastin, collagen and lipids, which are recognized as the major sources of autofluorescence in vascular tissues. FEA has also been reported to detect venous thromboemboli. In this paper we have tested the hypothesis that FEA can reproducibly detect in vivo and in vitro triggered plaque disruption and thrombosis in a rabbit model. Fluorescence emission (FE) spectra, recorded in vivo, detected Russell's viper venom (RVV)–induced transformation of atherosclerotic plaque. FE intensity at 410–490 nm 4 weeks after angioplasty was significantly lower (P < 0.0033 by analysis of variance) in RVV-treated rabbits when compared to control animals with stable plaque. FE spectral profile analyses also demonstrated a significant change in curve shape as demonstrated by polynomial regression analysis (R² from 0.980 to 0.997). We have also demonstrated an excellent correlation between changes in FE intensity and the structural characteristics detected at different stages of “unstable atherosclerotic plaque” development using multiple regression analysis (R² = 0.989). Thus, FEA applied in vivo is a sensitive and highly informative diagnostic technique for detection of triggered atherosclerotic plaque disruption and related structural changes, associated with plaque transformation, in a rabbit model.

We have tested the hypothesis that exposure to ultraviolet light would inhibit T helper-1 (Th1) responses and stimulate T helper-2 (Th2) responses, and that thus in a mouse model of allergic (i.e. extrinsic) asthma (using ovalbumin [OVA] as the allergen) increased symptoms would be observed, while in a model of Th1-dependent occupational asthma (in which picryl chloride is the allergen) decreased symptoms would be observed. Whereas reduced interferon (IFN)-γ production, decreased inflammatory responses in the airways, and reduced airway reactivity to nonspecific stimuli were observed in UV-preexposed picryl chloride sensitized and challenged mice, the results in the OVA model were less clear. Increased interleukin (IL)-10 production as a result of UV exposure was observed, together with unchanged IL-4 and IFN-γ. In addition, decreased OVA-specific immunoglobin, IgG1 and IgE, titers were noted, as well as decreased nonspecific airway hyperreactivity. Eosinophilic inflammatory responses were not influenced. The results indicate that UV exposure can have systemic effects that influence ongoing immune responses in the respiratory tract. The effects are not only restricted to immune responses that are predominantly Th1 dependent (i.e. pulmonary delayed-type hypersensitivity and IFN-γ production in response to picryl chloride) but also to immune response that are predominantly Th2 dependent, i.e. decreased specific IgE titers.

The genus Xiphophorus is an important model for investigating the etiology and genetics of sunlight-induced melanoma as well as other cancers. We used immunological techniques to determine the induction, distribution and repair of cyclobutane pyrimidine dimers (CPD) and pyrimidine(6-4)pyrimidone dimers ([6-4]PD) in different tissues of Xiphophorus signum exposed to ultraviolet-B light. We found that the (6-4)PD was induced at 5 to 10-fold lower frequency than the CPD and that scalation provided considerable photoprotection against both photoproducts. Photoenzymatic repair (PER) was very efficient in X. signum with most of the lesions removed within 20 min; PER of CPD occurred at about twice the rate of (6-4)PD. Nucleotide excision repair (NER) was much less efficient than PER and the rates of CPD and (6-4)PD removal were comparable. PER was more efficient in the caudal fin compared to the lateral epidermis; the opposite was true for NER. Although the initial rate of CPD excision was five-fold faster in the lateral epidermis compared to the caudal fin a considerable amount of residual damage remained in both tissues. The diverse photochemical and photobiological responses observed in X. signum suggest that heritable traits governing deoxyribonucleic acid damage induction and repair may be involved in the susceptibility of other Xiphophorus species to melanomagenesis.

Chimeric proteins were produced using the green light–emitting luciferase of Phrixothrix vivianii (PxGr: λ_max = 548 nm) and the red light–emitting luciferase of Phrixothrix hirtus (PxRe: λ_max = 623 nm). Constructs containing residues 1–344 of the red light–emitting luciferase with residues 345–545 of the green light emitting one emitted red light (PxReGr; λ_max = 613 nm), while the reverse emitted green light (PxGrRe; λ_max = 552 nm). From these results we conclude that the region 1–344 determines the color of bioluminescence (BL) in railroad-worm luciferases, and that residues above 344 are not involved. The substitution R215S in the green light–emitting luciferase (PxGr) resulted in a ∼40 nm redshift on the BL spectrum (λ_max = 585 nm) and an associated decrease of activity, whereas the same mutation in PxRe luciferase had little effect. Guanidine was shown to cause blueshifts in the BL spectra and stimulate the activity of the red-emitting luciferases (from λ_max = 623 to λ_max = 600 nm) and in PxGr R215S (from λ_max = 585 to λ_max = 560 nm) mutant luciferase, but not in the green-emitting luciferases, suggesting that guanidine can simulate positively charged residues involved in BL color determination.