We have developed a new model using liposome-encapsulated fluorescent probes, aiming at assessing both the physical and the biological protection provided by filter molecules such as those incorporated in sunscreens. The fluorescent indicator Indo-1 or 2′,7′-dichlorofluorescin (DCFH) was inside the liposomes, in the aqueous inner compartment, whereas the filter molecules octyl methoxycinnamate (OMC), benzophenone-3 (BP3) or avobenzone, widely used in sunscreens, were incorporated into liposome membranes. When liposome suspensions were placed in a fluorometer cuvette exposed to an incident UV beam, the decrease of Indo-1 fluorescence as a function of filter concentration was related to the extinction coefficient of the filters. On the other hand, when liposome suspensions were exposed to moderate UVB doses allowing Indo-1 photobleaching, the remaining intact Indo-1 was linked to the protection provided by filter-containing liposome membranes. Finally, when liposome-encapsulated DCFH was exposed to UVB, the degree of photo-oxidation of the fluorescent probe into 2′,7′-dichlorofluorescein accounted for the photoprotection provided by the filter contained in liposome membranes. BP3 was more potent and slightly less efficient than the other two filters in preventing Indo-1 fluorescence; all three filters provided a similar concentration-dependent protection of Indo-1 photobleaching, whereas only OMC was able to prevent the photooxidation of DCFH. The liposome model presented here has the advantage of combining both physical and biological parameters to assess the photoprotection provided by filter molecules, and the lack of photoprotection by two sunscreen molecules having a good filter capacity highlights the need for such a biological parameter when talking about the safety of sunscreens.

The influence of the surfactants sodium dodecyl sulphate, cetyltrimethyl-ammonium bromide and triton X-100 on the photochemically induced dynamic nuclear polarization (CIDNP) of N-acetyl tyrosine has been investigated. Three photosensitizers were used to generate polarization: thionin, eosin Y and flavin mononucleotide. 600 MHz ¹H photo-CIDNP experiments, supported by laser flash photolysis transient absorption measurements, indicate that the neutral triton surfactant has no influence on the nuclear polarization, but that the other two, charged, amphiphiles affect the photochemistry in a variety of ways, depending on the surfactant concentration and the identity of the sensitizer.

This work concerns a combined photophysical, photochemical and photobiological study of three drugs (psychotherapeutic agents) of the phenothiazine series: perphenazine, fluphenazine hydrochloride and thioridazine hydrochloride. The excited-state properties were first investigated by stationary and time-resolved fluorimetry and by laser flash photolysis. The spectral description was assisted by quantum-mechanical calculations with the INDO/1-CI method. In organic media the lowest excited singlet state was found to decay by fluorescence (small quantum yield) and mainly by intersystem crossing to the lowest triplet state, which is responsible for oxygen photosensitization (high yields of singlet oxygen production) and photodegradation. A further decay pathway in aqueous solutions was the photoionization process, which led to the formation of the phenothiazine radical cations and the solvated electron. After the preliminary study of the photobehavior in organic solvents and in water, the phototoxicity of the three drugs was investigated on various biological substrates through a series of in vitro assays under UVA irradiation. Photohemolysis of mouse erythrocytes and phototoxicity on cultured murine fibroblasts were observed for all three compounds. Lipid photoperoxidation was then investigated using linoleic acid as the unsaturated lipid model and isolated red blood cell membranes. The drug-induced photodamage was also evaluated on proteins by measuring the photosensitizing cross-linking in erythrocyte ghosts. The combined approach proved to be useful in understanding the mechanism by which these phenothiazine derivatives induce skin photosensitization. In particular, the photophysical properties of the compounds under investigation and the results of the study on their phototoxicity are in agreement with a mechanism that involves the radical cation of the drugs as a main intermediate.

Orfelia fultoni is the only bioluminescent dipteran (Mycetophilidae) found in North America. Its larvae live on stream banks in the Appalachian Mountains. Like their Australasian relative Arachnocampa spp., they build sticky webs to which their bioluminescence attracts flying prey. They bear two translucent lanterns at the extremities of the body, histologically distinct from the single caudal lantern of Arachnocampa spp., and emit the bluest bioluminescence recorded for luminescent insects (λ_max = 460 nm versus 484 nm from Arachnocampa). A preliminary characterization of these two bioluminescent systems indicates that they are markedly different. In Orfelia a luciferin–luciferase reaction was demonstrated by mixing a hot extract prepared with dithiothreitol (DTT) under argon with a crude cold extract. Bioluminescence is not activated by adenosine triphosphate (ATP) but is strongly stimulated by DTT and ascorbic acid. Using gel filtration, we isolated a luciferase fraction of ∼140 kDa and an additional high molecular weight fraction (possibly a luciferin-binding protein) that activated bioluminescence in the presence of luciferase and DTT. The Arachnocampa luciferin–luciferase system involves a 36 kDa luciferase and a luciferin soluble in ethyl acetate under acidic conditions; the bioluminescence is activated by ATP but not by DTT. The present findings indicate that the bioluminescence of O. fultoni constitutes a novel bioluminescent system unrelated to that of Arachnocampa.

The understanding of photosensitizer photobleaching is important not only for mechanistic studies, but also for the development of monitoring techniques for clinical dosimetry in photodynamic therapy. In this study, we investigated the intracellular photobleaching of 5,10,15,20-tetrakis(m-hydroxyphenyl)chlorin (mTHPC, Foscan^®) in the murine macrophage cell line J774A.1, using quantitative fluorescence imaging microscopy, microspectrofluorometry and microspectrophotometry. Using 652 nm laser irradiation, it was found that mTHPC exhibits oxygen- and fluence rate–dependent intracellular photobleaching. The kinetics showed an inverse dose–rate behavior, i.e. a reduction of fluence rate resulted in more photobleaching at comparable fluences. The effect of deoxygenation was found to be more complex, with decreased bleaching at low fluence rates and increased bleaching at higher fluence rates. The intracellular formation of reactive oxygen species was measured using 2′,7′-dichlorodihydrofluorescein diacetate. The results are analyzed in terms of competitive Type-I and Type-II mechanisms.

In human endothelial cells ECV 304 and HMEC-1 photosensitized by pyropheophorbide-a methylester (PPME) in sublethal conditions transcription factor Nuclear Factor kappa B (NF-κB) activation takes place for several hours. Activated NF-κB was functional because it stimulated the transcriptional activation of either a transfected reporter gene or the endogenous gene encoding interleukin (IL)-8. Concomitant with NF-κB activation, inhibitor of NF-κBα (IκBα) was degraded during photosensitization and IκBβ, p100, p105 and IκBε were slightly modified. Reactive oxygen species (ROS) were shown to be crucial intermediates in the activation because antioxidants strongly decreased NF-κB activation. Using both a fluorescent probe and isotope substitution, it was shown that ROS, and especially singlet oxygen (¹O₂), were important in the activation process. Because NF-κB activation in the presence of ROS was suspected to proceed through a pathway independent of the IκB kinases (IKK), we demonstrated that the IKK were indeed not activated by photosensitization but required an intact tyrosine residue at position 42 on IκBα, suggesting the involvement of a tyrosine kinase in the activation process. This was further reinforced by the demonstration that herbimycin A, a tyrosine kinase inhibitor, prevented NF-κB activation by photosensitization but not by TNFα, a cytokine known to activate NF-κB through an IKK-dependent mechanism.

Restenosis results from intimal hyperplasia and constrictive remodeling following cardiovascular interventions. Photodynamic therapy (PDT) has been shown to inhibit intimal hyperplasia in vivo by preventing neointimal repopulation of the treated vessel. This study was undertaken in an attempt to further dissect the mechanisms by which PDT acts on secreted and extracellular matrix proteins to inhibit migration of cultured human vascular cells. PDT of three-dimensional collagen gels inhibited invasive human smooth muscle cell (SMC) migration, whereas cell-derived matrix metalloproteinase production remained unaltered. Additionally, PDT generated cross-links in the collagen gels, a result substantiated in an ex vivo model whereby PDT rendered the treated vessels resistant to pepsin digestion and inhibited invasive migration of SMC and fibroblasts. These data support the premise that by inducing matrix protein cross-links, rendering the vessel resistant to degradation, in vivo PDT inhibits repopulation of the vessel and therefore intimal hyperplasia.

The worldwide rise in antibiotic resistance necessitates the development of novel antimicrobial strategies. In this study we report on the first use of a photochemical approach to destroy bacteria infecting a wound in an animal model. Following topical application, a targeted polycationic photosensitizer conjugate between poly-l-lysine and chlorin_e6 penetrated the Gram (−) outer bacterial membrane, and subsequent activation with 660 nm laser light rapidly killed Escherichia coli infecting excisional wounds in mice. To facilitate real-time monitoring of infection, we used bacteria that expressed the lux operon from Photorhabdus luminescens; these cells emitted a bioluminescent signal that allowed the infection to be rapidly quantified, using a low-light imaging system. There was a light-dose dependent loss of luminescence in the wound treated with conjugate and light, not seen in untreated wounds. Treated wounds healed as well as control wounds, showing that the photodynamic treatment did not damage the host tissue. Our study points to the possible use of this methodology in the rapid control of wounds and other localized infections.

We have evaluated the in vivo correlation between the expression of cell cycle markers and skin tumor development in SKH-1 hairless mice in a complete photocarcinogenesis protocol. Irradiated mice developed an average of 16 tumors per animal by week 23 with the average number of carcinomas per mouse being 2.1. The expression of p53 and cyclins A and D1 was confined initially to sporadic single cells and gradually developed into foci of patchy intense staining in the basal and granular layers of UVB-exposed epidermis. p53 was expressed in all the papilloma sections examined, whereas cyclins D1 and A were expressed in 68 and 71% of these lesions, respectively. In UVB-induced squamous cell carcinomas (SCC), p53 was expressed in >90% of the tumors, whereas cyclin D1 was detected in 55% of the lesions, and cyclin A staining was limited to 27%. These immunohistochemical observations were confirmed by Western blotting and protein kinase assays. We observed an early wave of cyclin A overexpression and cyclin A protein kinase activity preceding the appearance of detectable tumors. Cyclin D1 and p53 overexpression were coupled with the development of tumors, and these changes are likely to be relevant to the pathogenesis of these lesions.

In this study we have explored the potential of PUVB (8-MOP UVB) therapy for the reduction of luminal narrowing after arterial injury. In 15 rabbits, balloon dilation of iliac arteries was performed. In 20 arteries, dilation was combined with the delivery of pulsed ultraviolet light B (UVB) irradiation with 10 arteries being previously subjected to sensitizer infusion. Changes in vessel diameter, proliferation and extracellular matrix protein content at 6 weeks were evaluated by means of angiography and histomorphometry–immunohistochemistry. We found that PUVB, applied at the time of dilation, induced reduction in late loss (LL) at 6 weeks (percutaneous transluminal angioplasty vs UVB vs PUVB: 0.64 ± 0.15 mm vs 0.61 ± 0.05 mm vs 0.29 ± 0.05 mm; p = 0.018). The same holds true for constrictive remodeling (0.53 ± 0.15 mm vs 0.45 ± 0.06 mm vs 0.15 ± 0.05 mm; p = 0.016). In the irradiation groups, LL was independent of acute gain (AG), as opposed to the control. Collagen content increased significantly after PUVB in media and adventitia, without increased cellular proliferation in all vessel layers. Thus, PUVB at the time of dilation reduced luminal narrowing at follow-up without effecting proliferation. This effect was independent of AG and was associated with increased collagen content in media and adventitia.

Unidirectional blue light directs the rhizoid–thallus axis in the apolar zygote of the brown alga, Silvetia compressa. This effect is mediated by an increase in the intracellular concentration of guanosine 3′, 5′-cyclic monophosphate. In this study we show the identification of a rhodopsin-like protein, by means of antibody reaction, in the plasma membrane of Silvetia eggs. This new result suggests a role for opsins in Silvetia photopolarity.

Blue light inhibits the formation of asexual cycle spores (conidia) and stimulates the development of the sexual (female) reproductive structures (protoperithecia) in the nitrogen-starved mycelium of Neurospora crassa. The DNA methylation inhibitor, 5-azacytidine (3–300 μM), opposed the effect of light by suppressing the protoperithecia formation and stimulating a conidiation. The addition of 300 μM 5-azacytidine inhibited protoperithecia formation in the dark-cultivated mycelium by about two orders of magnitude and activated conidiation in the light-exposed mycelium by almost three orders of magnitude. Both in the dark-cultivated and the irradiated mycelium treated with various 5-azacytidine concentrations, the yield of conidia and protoperithecia demonstrated an inverse relationship. We suggest that DNA methylation and blue light are involved in the organism's selection of sexual or asexual reproductive cycle.