A new method was developed to allow direct visualization of damaged sites on individual DNA molecules. Fluorescence in situ hybridization on extended DNA molecules was modified to detect a single abasic site. Abasic sites were specifically labeled with a biotinylated aldehyde-reactive probe and fluorochrome-conjugated streptavidin. The light emitted by a single fluorochrome–DNA complex was calibrated. The number of abasic sites on the DNA molecule was estimated by counting each fluorochrome–DNA complex. The present study directly visualized and characterized the abasic sites of single DNA molecules.

We tested correlations between lipophilicity parameters and the partitioning of sensitizers into membranes. For this purpose we investigated 17 porphyrins and two chlorins having various chemical structures. Some of these compounds possess an amphiphilic structure (including hematoporphyrin, deuteroporphyrin, mesoporphyrin, chlorin e₆ and more). The others are very symmetrical sensitizers [meso-tetra(N-methyl-4-pyridyl)porphyrin, tetrabenzoporphyrin, coproporphyrin I dihydrochloride (CP), meso-tetra(4-carboxyphenyl)porphyrin (TCP) and meso-tetra(m-hydroxyphenyl)chlorin]. Our investigation also included two series of hematoporphyrins and protoporphyrins with varying lengths of alkylcarboxylate side groups. The partitioning of these compounds between the bulk aqueous phase and liposomes was studied by fluorescence methods, and a liposome-binding constant, K_b, was obtained. It was found that CP and TCP do not incorporate into the lipid phase at pH 7.3. An n-octanol–water partition coefficient (log P) and a distribution coefficient (log D) were predicted with a modeling software. The values of log D were also obtained experimentally. We found that for the studied molecules, the predicted log D correlated well with the measured values. The values of log D as well as log P, in turn, did not correlate nicely, for the whole group of studied compounds, with the binding constants to liposomes. However, in the case of porphyrins that share a similar structure, the K_b showed good linear correlation with both log P and log D. For the series of hematoporphyrins and protoporphyrins with different lengths of alkylcarboxyl groups, it was shown that prolongation of this group caused an increase in the lipophilicity and the liposome-binding constant. This effect is more pronounced for the proto- than for the hematoporphyrin series. The results highlight the possible use, as well as limitations, of lipophilicity parameters for the prediction of membrane binding.

We report the synthesis and optical characterization of two new photoactivators and demonstrate their use for multiphoton excited three-dimensional free-form fabrication with proteins. These reagents were developed with the goal of cross-linking Type 1 collagen. This cross-linking process produces structures on the micron and submicron size scales. A rose bengal diisopropyl amine derivative combines the classic photoactivator and co-initiator system into one molecule, reducing the reaction kinetics and increasing cross-linking efficiency. This derivative was successful at producing stable structures from collagen, whereas rose bengal alone was not effective. A benzophenone dimer connected by a flexible diamine tether was also synthesized. This activator has two photochemically reactive groups and is highly efficient in cross-linking bovine serum albumin and Type 1 collagen to form stable, robust structures. This approach is more flexible in terms of cross-linking a variety of proteins than by traditional benzophenone photochemistry. The photophysical properties vary greatly from that of benzophenone, with the appearance of a new, lower energy absorption band (λ_max ∼370 nm in water) and broad, visible emission band (∼500 nm maximum). This absorption band is highly solvatochromic, suggesting it arises, at least in part, from a charge transfer interaction. Collagens are typically difficult to cross-link photochemically, and the results here suggest that these two new activators will be suitable for cross-linking other forms of collagen and additional proteins for biomedical applications such as the de novo assembly of biomimetic tissue scaffolds.

Bacteriochlorophyll (BChl) derivatives (with central Mg replaced by metal “M”) ([M]-BChl with M = 2H, Mg, Zn, Pd, Cu) have been investigated for their photodynamic capacity and stability toward photodegradation in organic solvents and aqueous micellar solution. A protocol has been developed for screening new sensitizers. BChl and [Zn]-BChl are efficient sensitizers, but they are also quickly degraded by the reactive oxygen species (ROS) produced by autosensitization, as well as by hetero-sensitization with 17⁴-methyl-13²-demethoxycarbonyl-pheophorbide a (MPP). Photostable [Cu]-BChl is a poor sensitizer, whereas [Pd]-BChl and bacteriopheophytin a are not only very efficient sensitizers but are also very stable toward ROS. β-Carotene is no efficient physical quencher of ROS in the system; rather, it acts as a photochemical quencher that competes with [M]-BChl and undergoes photooxygenation at high rates. Photolability seems to depend on the pigment oxidation potential and, in parallel, on the presence of central metals preferring coordination numbers higher than 4, whereas photodynamic capacity depends on long excited state lifetimes of the pigment or efficient intersystem crossing (or both).

The synthesis of a molecule containing hypericin and luciferin moieties joined by a tether is reported. The light-induced (in vitro) antiviral activity as well as the photophysical properties of this new compound are measured and compared with those of the parent compounds, hypericin and pseudohypericin. This tethered molecule exhibits excited-state behavior that is very similar to that of its parent compounds and antiviral activity that is identical, within experimental error, to that of its more closely related parent compound, pseudohypericin. The implications for a photodynamic therapy that is independent of external light sources are discussed.

A polychromatic irradiation system for the outdoor exposure of plants or other samples to additional UV and longer-wavelength radiation has been constructed and tested. The system provides a range of wavebands in the ultraviolet-B radiation (280–315 nm) and ultraviolet-A radiation (315–400 nm) spectral regions, and the irradiance is fully adjustable. The performance of the lamp and filter system after over 4000 h of use is described; a brief description of filter degradation after over 30 000 h of use is also presented. Cellulose acetate filters were found to be adequate for some purposes in the UV waveband, but rigid plastic and glass-based filters were superior in terms of stability and the range of spectral combinations available. They also exhibited relatively small changes in transmittance with time. The system is also convenient for the measurement of differential polychromatic action spectra.

Predicting the effects of polychromatic light on biological systems is a central goal of environmental photobiology. If the dose–response function for a process is a linear function of the light incident on a system at each wavelength within the spectrum, the effect of a polychromatic spectrum is obtained by integrating the product of the cross section for the reaction at each wavelength and the spectral irradiance at that wavelength over both wavelength and time. This procedure cannot be used, however, if the dose–response functions for an effect are not linear functions of photon dose. Although many photochemical reactions are linear within the biologically relevant range of doses, many biological end points are not. I describe procedures for calculating the effects of polychromatic irradiations on systems that exhibit certain classes of dose–response functions, including power law responses typical of mutation induction and exponential dose–responses typical of cell survival. I also present an approach to predict the effects of polychromatic spectra on systems in which the ultraviolet components form pyrimidine dimers, and the longer-wavelength ultraviolet and visible components remove them by photoreactivation, thus generating complex dose–response functions for these coupled light–driven reactions.

A multiple linear correlation is done between atmospheric transmissivity for four biologically active radiation daily doses (UVB, erythemal, DNA and plant damage) T, and three parameters (daily sunshine fraction σ, cosine of the daily minimum solar zenith angle μ_min and daily total ozone column Ω). T is defined as the ratio of a daily dose to its extra-atmospheric value. The data used are spectral UV measurements (390–400 nm at 0.5 nm step) recorded along year 2000 and over 8 months of year 2001 at Briançon Station (Alps, 1300 m above sea level) that forms part of the French UV network. The coefficients obtained from year 2000 correlation permit to retrieve daily doses for year 2001 with an average error running from 3 to 9% for monthly mean values and from 2 to 4.5% for 3-monthly mean values, depending on daily dose type. The retrieval of yearly mean value gives an error between 4 and 7.5%. Retrieving the daily dose of a given day, where σ ≥ 0.2, introduces error running from 16 to 32% depending on daily dose. An attempt to retrieve the yearly mean UVB daily dose for a northern France site, from the previous coefficients, gives encouraging results.

Multireference configuration interaction calculations are carried out for ground and excited states of dichloromethanol, Cl₂CHOH, to investigate two important photofragmentation processes relevant to atmospheric chemistry. Five low-lying excited states (1¹A″, 2¹A′, 1³A″, 2³A″ and 1³A′) in the energy range between 6.4 and 7.5 eV are found to be highly repulsive for C–Cl elongation, leading to ClCHOH (X²A) and Cl (X²P). Photodissociation along the C–O bond resulting in CHCl₂ (X²A′) and OH (X²Π) has to overcome a barrier of about 0.5 eV because the low-lying excited states 1¹A″, 1³A′ and 1³A″ become repulsive only after the C–O bond is elongated by about 0.3 Å.

An analysis is made of experimental ultraviolet erythemal solar radiation data measured during the years 2000 and 2001 by the Spanish UV-B radiation evaluation and prediction network. This network consists of 16 Robertson–Berger type pyranometers for evaluating solar erythemal radiation and five Brewer spectroradiometers for evaluating the stratospheric ozone. On the basis of these data the Ultraviolet Index (UVI) was evaluated for the measuring stations that are located either in coastal regions or in the more densely populated regions inland on the Iberian Peninsula. It has been checked that in most cases the maximum irradiance values corresponded to solar noon, although there were exceptions that could be explained by cloudiness. The maximum experimental values of the UVI were around 9 during the summer, though frequently passing this value at the inland measurement stations. The annual accumulated dose of irradiation on a horizontal plane has also been studied, as well as the evolution through the year in units of energy, standard erythemal doses and minimum erythemal doses, according to different phototypes.

Cyanobacteria must cope with the negative effects of ultraviolet B (280–315 nm) (UV-B) stress caused by their obligatory light requirement for photosynthesis. The adaptation of the cyanobacterium Anabaena sp. to moderate UV-B radiation has been observed after 2 weeks of irradiation, as indicated by decreased oxidative stress, decreased damage, recovered photosynthetic efficiency and increased survival. Oxidative stress in the form of UV-B–induced production of reactive oxygen species was measured in vivo with the oxidative stress–sensitive probe 2′,7′-dichlorodihydrofluorescein diacetate. Photooxidative damage by UV-B radiation, including lipid peroxidation and DNA strand breakage, was determined by a modified method using thiobarbituric acid reactive substances and fluorometric analysis of DNA unwinding. Photosynthetic quantum yield was determined by pulse amplitude–modulated fluorometry. The results suggest that moderate UV-B radiation results in an evident oxidative stress, enhanced lipid peroxidation, increased DNA strand breaks, elevated chlorophyll bleaching as well as decreased photosynthetic efficiency and survival during the initial exposure. However, DNA strand breaks, photosynthetic parameters and chlorophyll bleaching returned to their unirradiated levels after 4–7 days of irradiation. Oxidative stress and lipid peroxidation appeared to respond later because decreases were observed after 7 days of radiation. The survival curve against irradiation time exhibited a close relationship with the changes in photosynthetic quantum yield and DNA damage, with little mortality after 4 days. Growth inhibition by UV-B radiation was observed during the first 7 days of radiation, whereas normal growth resumed even under UV-B stress thereafter. An efficient defense system was assumed to come into play to repair photosynthetic and DNA damage and induce the de novo synthesis of UV-sensitive proteins and lipids, allowing the organisms to adapt to UV-B stress successfully and survive as well as grow. No induction of mycosporine-like amino acids (MAA) was observed during the adaptation of Anabaena sp. to UV-B stress in our work. The adaptation of the cyanobacterium correlated with and could be caused by the oxidative stress and oxidative damage.

Photodynamic therapy (PDT) requires molecular oxygen during light irradiation to generate reactive oxygen species. Tumor hypoxia, either preexisting or induced by PDT, can severely hamper the effectiveness of PDT. Lowering the light irradiation dose rate or fractionating a light dose may improve cell kill of PDT-induced hypoxic cells but will have no effect on preexisting hypoxic cells. In this study hyperoxygenation technique was used during PDT to overcome hypoxia. C3H mice with transplanted mammary carcinoma tumors were injected with 12.5 mg/kg Photofrin^® and irradiated with 630 nm laser light 24 h later. Tumor oxygenation was manipulated by subjecting the animals to 3 atp (atmospheric pressure) hyperbaric oxygen or normobaric oxygen during PDT light irradiation. The results show a significant improvement in tumor response when PDT was delivered during hyperoxygenation. With hyperoxygenation up to 80% of treated tumors showed no regrowth after 60 days. In comparison, when animals breathed room air, only 20% of treated tumors did not regrow. To explore the effect of hyperoxygenation on tumor oxygenation, tumor partial oxygen pressure was measured with microelectrodes positioned in preexisting hypoxic regions before and during the PDT. The results show that hyperoxygenation may oxygenate preexisting hypoxic cells and compensate for oxygen depletion induced by PDT light irradiation. In conclusion, hyperoxygenation may provide effective ways to improve PDT efficiency by oxygenating both preexisting and treatment-induced cell hypoxia.

A quantitative assessment of the light field produced by a Waldmann PDT 1200 lamp is presented. A photodiode detector array capable of measuring a beam diameter of 30 cm was used to map the light field. The irradiance was measured as a function of voltage. For lamp–detector distances of 10 cm (central axis irradiance = 250 mW/cm²), the spatial profile of irradiance was typically Gaussian. For lamp–detector distances of 30 cm (central axis irradiance = 79 mW/cm²), the spatial profile appeared more hemispherical in shape but with some asymmetry. The relative percentage variation between the maximum and minimum irradiance with respect to the central axis irradiance was approximately 13% and 3%, respectively, for a beam width of 12 cm. Beyond a lamp–detector distance of 50 cm (central axis irradiance = 32 mW/cm²), the spatial profile of irradiance was observed to become more crater-like in structure, with a minimum on the central axis and an approximately symmetric peak at a radial distance of 9 cm from the center. The relative percentage variation of this peak irradiance with respect to the central axis irradiance was approximately 17%. At lamp–detector distances of 70 and 90 cm (central axis irradiance = 19 and 13 mW/cm², respectively), the beam's profile was asymmetric, and the irradiance was observed to increase from the center to a radial distance of 15 cm (beam width 30 cm). For a lamp–detector distance of 70 and 90 cm, the relative percentage variation between the maximum irradiance and the central axis irradiance was approximately 25% and 35%, respectively.

Photodynamic therapy (PDT) is a promising treatment modality that has recently been accepted in clinics as a curative or palliative therapy for cancer and other nonmalignant conditions. Phthalocyanines (Pc) are attractive photosensitizers for PDT because of their enhanced photophysical and photochemical properties. The overall charge and solubility of Pc play a major role in their potential usefulness for PDT. A series of amphiphilic derivatives of tetrasulfonated aluminum Pc (AlPcS₄) was prepared by substituting one of the four sulfonate groups with aliphatic side chains of 4, 8, 12 and 16 carbon atoms. The photodynamic properties of the derivatives were compared with those of AlPcS₄ and the adjacent disulfonated aluminum Pc. Parameters studied included reversed-phase high-performance liquid chromatography (HPLC) retention times, capacity to generate singlet oxygen (¹O₂), in vitro cell uptake and phototoxicity, as well as PDT response of transplantable EMT-6 tumors in mice. The monomerized AlPcS₄ derivatives showed similar or higher capacities to generate ¹O₂ as compared with the parent AlPcS₄ as measured from relative l-tryptophan photooxidation yields. A549 cell uptake of the AlPcS₄ derivatives decreased in the following order: AlPcS₄(C16) > AlPcS₄(C12) > AlPcS₄(C8) > AlPcS₄(C4). Human low-density lipoprotein at high concentrations (40 μg/mL) completely prevented uptake, whereas at 4 μg/mL uptake was decreased for the more lipophilic compounds and yet remained unaffected for the more hydrophilic dyes. Using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, A549 cell survival was assessed; it showed that photocytotoxic activity varied directly with the HPLC retention times, i.e. more hydrophilic compounds were less phototoxic. As ¹O₂ yields were similar for the four substituted AlPcS₄ derivatives, it was postulated that the increased cytotoxic activity was caused by enhanced subcellular localization as a result of the long aliphatic side chains. These amphiphilic compounds proved to be photodynamically potent against the EMT-6 mouse mammary tumor model implanted in Balb/c mice. At dye doses of 0.2 μmol/kg and a fluence of 400 J/cm² complete tumor regression was observed with no morbidity. The substitution of AlPcS₄ with long aliphatic chains on the macrocycle greatly enhances its photodynamic efficacy both in vitro and in vivo.

Photodynamic therapy (PDT) using the second-generation photosensitizer phthalocyanine (Pc) 4 causes mitochondrial damage and induces apoptosis through the release of cytochrome c to the cytosol. Another protein of the mitochondrial intermembrane space, Smac/DIABLO (second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI), is also released to the cytosol in response to apoptotic stimuli and promotes caspase activation by binding IAP. To investigate the possible role of Smac/DIABLO in apoptosis induced by Pc 4-PDT, we transfected Smac/DIABLO (tagged at its C-terminus with green fluorescent protein [GFP]) into MCF-7c3 cells (human breast cancer MCF-7 cells stably transfected with procaspase-3) and DU-145 cells (human prostate cancer cells that express no Bax because of a frameshift insertion mutation). Confocal microscopy showed that recombinant Smac/DIABLO, like cytochrome c, localized to mitochondria and colocalized with MitoTracker Red. Three hours after exposure of MCF-7c3 cells to PDT (200 nM Pc 4 and 150 mJ/cm² red light), Smac/DIABLO–GFP, as well as cytochrome c, was found largely in the cytosol. In contrast, for DU-145 cells, both Smac/DIABLO–GFP and cytochrome c remained in the mitochondria after PDT. By staining with Hoechst 33342, typical apoptotic nuclei were observed in MCF-7c3 cells, but not in DU-145 cells, after Pc 4-PDT. These results suggest that the release of Smac/DIABLO from mitochondria may be regulated by a Bax-mediated mechanism and that Smac/DIABLO may cooperate with the cytochrome c–dependent apoptosis pathway. In addition, in MCF-7c3 cells transfected by Smac/DIABLO–GFP, apoptosis induced by Pc 4-PDT was greater than in cells transfected with the GFP vector alone or in untransfected cells, as determined by flow cytometry. Thus, Smac/DIABLO promotes apoptosis after Pc 4-PDT in a Bax-dependent manner and may facilitate the passage of PDT-treated cells through the late steps of apoptosis.

A new photosensitizer, presently designated QLT0074, may have the potential for the treatment of immune and nonimmune conditions with photodynamic therapy (PDT). The activity of QLT0074 was tested against human peripheral blood T cells and Jurkat T lymphoma cells. At low nanomolar concentrations of QLT0074 in combination with blue light, apoptosis was rapidly induced in Jurkat and blood T cells in vitro as indicated by the expression of the apoptosis-associated mitochondrial 7A6 marker and Annexin-V labeling. Further studies performed with Jurkat T cells showed that PDT-induced apoptosis with QLT0074 was associated with caspase-3 activation and the cleavage of the caspase substrate poly(adenosine diphosphate–ribose)polymerase. Flow cytometry studies revealed that blood T cells with high expression of the interleukin-2 receptor (CD25) took up greater amounts of QLT0074 and were eliminated to a greater extent with PDT than T cells with low levels of this activation marker. This selective action of PDT was confirmed by similar reductions in the percentage of T cells that expressed other activation-related markers, including very late activation antigen-4 (CD49d), human leukocyte antigen DR (HLA-DR), intercellular adhesion molecule-1 (CD54) and Fas (CD95). For activated T cells treated with a specific dose of QLT0074 and light 24 h earlier, CD25 expression density was significantly less, whereas CD54, CD95 and HLA-DR levels were similar to those for control cells treated with light alone. This work shows that PDT with QLT0074 exerts selective, dose-related effects on T cells in vitro.

Expression of a carrot phenylalanine ammonia-lyase (PAL) gene (gDcPAL1) in suspension-cultured carrot cells is induced by dilution of the culture or by application of a fungal elicitor, as well as by ultraviolet B (UVB) irradiation. We demonstrated that among its upstream cis-elements (Takeda et al. [1997] Photochem. Photobiol. 66, 464–470), L₄ is UVB responsive, and L₁ is protoplastization- (dilution-) and elicitor responsive, from studies with transiently transformed mutated or truncated gDcPAL1 promoter–luc constructs. This conclusion is consistent with our observation that PAL activities induced by UVB and by protoplastization (dilution) or elicitor are additive.