The fluorescence properties of 3-methylindole (MI), 3-indoleacetic acid (IAA), 3-indoleethyltrimethylammonium bromide (IETA), l-tryptophan (Trp) and tryptamine hydrochloride (TA) were studied in reverse micelles solutions made with the cationic surfactant benzylhexadecyldimethylammonium chloride (BHDC) in benzene as a function of the molar ratio water/surfactant R (= [H₂O]/[BHDC]). The fluorescence quenching of the model compound MI by benzene in cyclohexane solutions and by BHDC in benzene solutions were also studied in detail. The fluorescence of MI in benzene is characteristic of a charge-transfer exciplex. The exciplex is quenched by the presence of BHDC, due to the interactions of the surfactant ion pairs with the polar exciplex. In reverse micelle solutions at low R values, all the indoles show exciplex-type fluorescence. As R increases, the fluorescence behavior strongly depends on the nature of the indole derivative. The anionic IAA remains anchored to the cationic interface and its fluorescence is quenched upon water addition due to the increases of interface's micropolarity. For IETA, TA and Trp an initial fluorescence quenching is observed at increasing R, but a fluorescence recovery is observed at R > 5, indicating a probe partition between the micellar interface and the water pool. For the neutral MI, the fluorescence changes with R indicate the partition of the probe between the micellar interface and the bulk benzene pseudophase. A simple two-site model is proposed for the calculation of the partition constants K as a function of R. In all cases, the calculation showed that even at the highest R value, about 90% of the indole molecules remain associated at the micellar interface.

The excited-state intramolecular H-atom transfer of hypericin (Hyp) was investigated as a function of pH in monodispersed reverse micelles formed by sodium bis(2-ethylhexyl)sulfosuccinate/heptane/water and in complexes with Tb³ under conditions in which one of the two carbonyl groups of Hyp is incapable of accepting a hydrogen atom. The results of pump-probe transient absorption experiments provide no evidence for a concerted H-atom transfer mechanism.

The natural product hypericin was tested in recent years as a biological photosensitizer with a potential for viral and cellular photodamage. We thus studied extensively its spectroscopy and membrane partitioning. Absorption, fluorescence excitation and emission spectra of the sodium salt (HyNa) were measured in 36 protic and aprotic, polar and apolar, solvents. Electronic transition bands as well as vibrational progressions were identified. Aggregation in some nonpolar solvents and protonation in organic acids were demonstrated. Modeling solvatochromism was done by Lippert equation, by the E_T(30) parameter and by the Taft multiparameter approach. In all cases, separation into protic and aprotic solvents gave much better fits to the models. ¹³C chemical shift data could also be correlated with solvent polarity. They correlated best with Lippert's Δf polarity measure, but tended to fall into two distinct solvent groups—each along different lines—corresponding to protic and aprotic media, respectively. This interesting phenomenon suggests that in the case of the charged and slightly water soluble HyNa, two mechanisms of solvation are involved, each resulting in its own line equation. In aprotic media, dipole–dipole interaction is the predominant solvation mechanism. In protic solvents, the most effective means of solvation is likely to be hydrogen bonding. When intercalated into the liposomal phospholipid bilayer, HyNa is oriented at an angle to the interface, thus experiencing a gradient of solvent polarities: a highly polar environment (similar to methanol) for C-2/5, suggesting that they lie not far from the interface; a moderately polar environment (similar to that of n-propanol) for C-6a/14a, which are somewhat deeper within the bilayer; and a more lipophilic environment (akin to n-hexanol) for C-10/11. The fluorescence excitation peak in liposomes also correlates with an aprotic medium of relatively high polarity, as might be excepted from a molecule in a shallow position in the bilayer.

Ketoprofen (KP) is a potent nonsteroidal anti-inflammatory drug. However, application to the skin is problematic because the photosensitizing properties of the benzophenone moiety may cause phototoxic effects when the treated skin region is exposed to UVA light. Using capillary electrophoresis with electrochemical detection we are able to differentiate the peroxides formed during illumination of KP-containing solutions of linoleic acid. Contrary to other profens a high amount of hydrogen peroxide was found among the reaction products. For investigation of the skin damaging effect human keratinocytes were used as models. Cell viability, DNA synthesis efficiency and intracellular concentration of peroxides were determined. Viability and proliferation behavior was not altered under the influence of KP. While lower concentrations of KP (10–100 nM) led to a protection against the UVA-induced (8 J/cm²) cell proliferation damage, higher concentrations (10–100 μM) led to an amplification of the proliferation decrease. With UVB irradiation at relevant doses the effects were lower than using UVA. Furthermore, intracellular peroxide content was increased after UV irradiation and KP addition. In conclusion some efforts have to be done to avoid these side effects in the use of KP for topical or transdermal application.

Formamidopyrimidine-DNA glycosylase (FPG) catalyzes the initial steps in the repair of DNA containing oxidized purines. Two complementary DNA clones encoding homologs of bacterial FPG, designated Atfpg-1 and Atfpg-2, have been isolated from Arabidopsis thaliana. They are products of alternative splicing of the transcript of a single gene. Proteins encoded by both clones, AtFPG-1 and AtFPG-2, engineered to contain oligohistidine sequences on their C-terminal ends, were expressed in Escherichia coli and purified, and their activities were assayed. Both proteins cleaved DNA that contained apurinic sites, indicating that they have abasic lyase activity. AtFPG-1, but not AtFPG-2, showed significant cleavage of a double-stranded oligonucleotide that contained 8-oxo-guanine, indicating that the structural differences between the two proteins influence their enzymatic activities. However, both proteins were able to cleave the same sites in DNA that was treated with visible light in the presence of methylene blue.

Ocular UV exposure is a function of both the direct and diffuse components of solar radiation. Broadband global and diffuse UV measurements were made in the morning, noon and afternoon. Thirty sets of measurements were made in summer and 50 in each of the other seasons at each of the periods in full sun. Corresponding sets were made in the shade of Australian evergreen trees: 42 trees in summer and 50 in each of the other seasons. The percentage diffuse UV was higher for the shorter 320–400 nm range (UVB) than for 280–320 nm (UVA). The percentage diffuse UVB ranged from 23 to 59%, whereas the percentage diffuse UVA ranged from 17 to 31%. The percentage diffuse UV was lower at noon than in the morning and afternoon with the difference more pronounced for the UVB. The average percentage diffuse UVB over all the measurements in the tree shade for the morning, noon and afternoon was 62, 58 and 71%, respectively, and the average percentage diffuse UVA was 52, 51 and 59%, respectively.

We tested the effects of irradiances of 920 and 1200 mW m⁻² (weighted irradiance) on the conidia and germinants of the entomopathogenic Hyphomycete Metarhizium anisopliae. The conidia were exposed to the two irradiances for 1, 2, 4, 6, 7 or 8 h. Increased exposure decreased relative percent culturability. The inactivation provoked by the irradiance of 1200 mW m⁻² was higher than for the 920 mW m⁻², with a reduction in the 50% lethal time (LT₅₀) from 6 h 40 min to 4 h 26 min. Reciprocity was not observed when conidia in water suspension and germinants in different stages of the germinative process were exposed to a 17.3 kJ m⁻² total dose at both irradiance levels. Although nonreciprocity was observed in all situations, its magnitude varied as a function of metabolic state and/or cell-cycle phase in which the conidia were at the exposure time. The least difference between the effects of the two irradiance levels was observed when nongerminating conidia in suspension were exposed, and the greatest was observed when conidia were exposed during an advanced germination phase. Doses of 6.6 and 17.3 kJ m⁻² supplied through the two irradiance levels delayed the germination of the surviving conidia. At both doses, delay was greater during exposure to the higher irradiance. Nonreciprocity was higher for the 17.3 kJ m⁻² dose. Nonreciprocity magnitude, in addition to depending on the conidial physiological state, also depended on dose. The results demonstrate the importance of evaluating the impact of the increase in irradiance during the different stages of the fungal life cycle, especially during the stages which are more sensitive to UV, and not simply in dormant conidia.

The depletion of stratospheric ozone causes related increase in UV light below about 310 nm, which significantly affects biological and ecological systems. To understand the wavelength-specific effects of UV light, Molt4 cells (human T lymphoma cells) were irradiated with a series of monochromatic UV lights and the activities of three members of the mitogen-activated protein (MAP) kinase group were examined. Extracellular signal–regulated kinase was specifically activated within 1 min after UV irradiation in the range 320–360 nm. In contrast, P38 kinase was activated by 270–280 nm light with a peak at 1 min after irradiation. c-Jun N-terminal kinase activation was observed in a narrow range of UV light with a sharp peak at 280 nm occurring in 10 min. JNK translocated from the cytosol to the nucleus upon irradiation, while P38 remained in the cytosol even after UV irradiation. The activation of three MAP kinases was prevented by antioxidant reagents, suggesting that an oxidative signal initiates these responses. These results confirm that UV light affects various cellular functions through the activation of intracellular signaling systems including MAP kinase family proteins. However, the UV-induced activities of the separate MAP kinases show distinctly different dose, time and wavelength dependencies.

Zinc-chlorin 3 (see Fig. 2 in text) possessing a tertiary 3¹-hydroxyl group and a 13-keto group was synthesized as a model for the antenna chlorophylls of green bacteria. Self-aggregation of 3 in nonpolar organic media was examined and compared to 1 and 2 possessing a primary and secondary 3¹-hydroxyl group, respectively. Zinc-chlorin 3 self-aggregated in 1 vol% CH₂Cl₂–hexane to form oligomers and showed a red-shifted Q_y maximum at 704 nm compared to the monomer (648 nm in CH₂Cl₂). This red-shift is larger than that of 2S (648 → 697 nm) and comparable to that of 2R (648 → 705 nm), but smaller than that of 1 (648 → 740 nm), indicating that while a single 3¹-methyl group (prim-OH → sec-OH) suppressed close and/or higher aggregation, the additional 3¹-methyl group (sec-OH → tert-OH) did not further suppress aggregation. The relative stability of the aggregates was in the order 1 > 2R ∼ 3 > 2S as determined by visible spectral analyses. Molecular modeling calculations on dodecamers of zinc-chlorins 1, 2R and 3 gave similar well-ordered energy-minimized structures, while 1 stacked more tightly than 2R and 3. In contrast, 2S gave a relatively disordered (twisted) structure. The calculated dodecameric structures could explain the visible spectral data of 1–3 in nonpolar organic media.

The transport mechanisms of 5-aminolevulinic acid methyl ester (5-ALA-ME) have been studied in a human adenocarcinoma cell line (WiDr) by means of ¹⁴[C]-labeled 5-ALA-ME. The transport was found to be partly Na dependent, while the extracellular Cl⁻ concentration did not affect the uptake. The transport of 5-ALA-ME into WiDr cells was dependent on the incubation temperature and was found to be completely blocked by the inhibitors of energy metabolism, 2-deoxyglucose and sodium azide. WiDr cells were treated with 10 mM of 14 different amino acids and the substrate specificity of the 5-ALA-ME transporter(s) was analyzed by treating the cells with 23 μM or 1 mM ¹⁴[C]-labeled 5-ALA-ME. The transport of 5-ALA-ME was found to be inhibited to the highest extent, i.e. about 60%, by the nonpolar amino acids l-alanine, l-methionine, l-tryptophan and glycine. The uptake of 5-ALA-ME followed an exponential decay with increasing concentration of glycine, reaching a maximum inhibition of uptake of 5-ALA-ME of 55%. Sarcosine, a specific inhibitor of system Gly, did not significantly inhibit 5-ALA-ME transport. In contrast to transport of 5-ALA, 5-ALA-ME does not seem to be taken up by system BETA transporters. In conclusion, the cellular uptake of 5-ALA-ME into WiDr cells seems to be due to active transport mechanisms, involving transporters of nonpolar amino acids.

Photodynamic therapy (PDT), an anticancer treatment modality, has recently been shown to be an effective treatment for several autoimmune disease models including antigen-induced arthritis. PDT was found to induce the expression of IL-10 messenger RNA (mRNA) and protein in the skin, and this expression has similar kinetics to the appearance of PDT-induced suppression of skin-mediated immune responses such as the contract hypersensitivity (CHS) response. Some aspects of the UVB-induced suppression of the immune response have been linked to the induction of IL-10. IL-10 has been shown to inhibit the development and activation of Th1 cells, which are critical for many cell-mediated immune responses, including CHS. We have examined the effect of PDT and UVB irradiation on the activity of the IL-10 gene promoter and on IL-10 mRNA stability using the murine keratinocyte line, PAM 212. In vitro PDT induces IL-10 mRNA and protein expression from PAM 212 cells, which can be correlated with an increase in AP-1 DNA binding activity and activation of the IL-10 gene promoter by PDT. Deletion of an AP-1 response element from the IL-10 gene promoter was shown to abrogate the PDT-induced promoter activity indicating that the AP-1 response element is critical to IL-10 induction by PDT. In addition, PDT results in an increase in IL-10 mRNA stability, which may also contribute to the increased IL-10 expression in PAM 212 cells following PDT. In vitro UVB irradiation also results in activation of the IL-10 promoter. However, in contrast to PDT, UVB-induced activation of the IL-10 promoter is not AP-1 dependent and did not increase IL-10 mRNA stability.

In vivo and ex vivo tissue autofluorescence (endogenous fluorescence) have been employed to investigate the presence of markers that could be used to detect tissue abnormalities and/or malignancies. We present a study of the autofluorescence of normal skin and tumor in vivo, conducted on 18 patients diagnosed with nonmelanoma skin cancers (NMSC). We observed that both in basal cell carcinomas (BCC) and squamous cell carcinomas (SCC) the endogenous fluorescence due to tryptophan residues was more intense in tumor than in normal tissue, probably due to epidermal thickening and/or hyperproliferation. Conversely, the fluorescence intensity associated with dermal collagen crosslinks was generally lower in tumors than in the surrounding normal tissue, probably because of degradation or erosion of the connective tissue due to enzymes released by the tumor. The decrease of collagen fluorescence in the connective tissue adjacent to the tumor loci was validated by fluorescence imaging on fresh-frozen tissue sections obtained from 33 NMSC excised specimens. Our results suggest that endogenous fluorescence of NMSC, excited in the UV region of the spectrum, has characteristic features that are different from normal tissue and may be exploited for noninvasive diagnostics and for the detection of tumor margins.

Peptides derived from the heavy chain of the HLA Class-I molecules have been shown to modulate immune responses both in vivo and in vitro. Using a computer-aided rational drug design approach, novel immunomodulatory peptides were designed based on peptide 2702.75–85, derived from HLA-B2702. Several peptides were identified which had increased immunomodulatory activity, including peptides RDP1258 and its d-isomer the peptide Allotrap 1258. The present study using Skh/hr hairless mouse skin model evaluated the in vivo effects of Allotrap 1258 on acute UVB-induced skin inflammation. Here we demonstrate that intraperitoneal administration of Allotrap 1258 1 h prior to UV exposure resulted in significantly diminished levels of UV-induced tumor necrosis factor (TNF)-α protein production in the epidermis but had no effect on other parameters of the acute UV-induced inflammatory response. By virtue of its ability to suppress TNF-α protein production, Allotrap 1258 could prove to be an effective modulator of inflammatory responses.

Lung cancer has long been considered a disease that might benefit from the dose escalation of radio/chemotherapy afforded by a stem cell transplant. However, the clinical experience with high-dose chemotherapy and autologous bone marrow transplantation in lung cancer has been disappointing, with most trials showing little or no improvement in long-term survival. Unfortunately, lung cancer has a tendency to metastasize to the bone marrow, and lung cancer cells are known to circulate in the peripheral blood. Therefore, there is concern that autologous stem cell grafts from lung cancer patients may reinoculate recipients with live tumor cells. Photochemical purging of stem cell grafts with Merocyanine 540 (MC540) is highly effective against a wide range of leukemia and lymphoma cells and is well tolerated by normal hematopoietic stem and progenitor cells. Most solid tumor cells (including lung cancer cells), however, are only moderately sensitive or refractory to MC540-mediated photodynamic therapy (PDT). We report here that postirradiation hyperthermia (≤42°C, 3 h) potentiates the MC540-mediated photoinactivation of both wild-type (H69) and cisplatin-resistant mutant (H69/CDDP) small cell lung cancer cells by several orders of magnitude, while only minimally enhancing the depletion of normal human granulocyte/macrophage progenitor cells. Our data suggest that postirradiation hyperthermia provides a simple and effective means of extending the utility of MC540-PDT to the purging of stem cell grafts contaminated with lung cancer and possibly other solid tumor cells.

Although many fundamental blue light responses have been identified, blue light dose-response curves are not well characterized. We studied the growth and development of soybean, wheat and lettuce plants under high-pressure sodium (HPS) and metal halide (MH) lamps with yellow filters creating five fractions of blue light. The blue light fractions obtained were <0.1, 2 and 6% under HPS lamps, and 6, 12 and 26% under MH lamps. Studies utilizing both lamp types were done at two photosynthetic photon flux levels, 200 and 500 μmol m⁻² s⁻¹ under a 16 h photoperiod. Phytochrome photoequilibria was nearly identical among treatments. The blue light effect on dry mass, stem length, leaf area, specific leaf area and tillering/branching was species dependent. For these parameters, wheat did not respond to blue light, but lettuce was highly sensitive to blue light fraction between 0 and 6% blue. Soybean stem length decreased and leaf area increased up to 6% blue, but total dry mass was unchanged. The blue light fraction determined the stem elongation response in soybean, whereas the absolute amount of blue light determined the stem elongation response in lettuce. The data indicate that lettuce growth and development requires blue light, but soybean and wheat may not.

Researchers studying plant growth under different lamp types often attribute differences in growth to a blue light response. Lettuce plants were grown in six blue light treatments comprising five blue light fractions (0, 2, 6% from high-pressure sodium [HPS] lamps and 6, 12, 26% from metal halide [MH] lamps). Lettuce chlorophyll concentration, dry mass, leaf area and specific leaf area under the HPS and MH 6% blue were significantly different, suggesting wavelengths other than blue and red affected plant growth. Results were reproducible in two replicate studies at each of two photosynthetic photon fluxes, 200 and 500 μmol m⁻² s⁻¹. We graphed the data against absolute blue light, phytochrome photoequilibrium, phototropic blue, UV, red:far red, blue:red, blue:far red and ‘yellow’ light fraction. Only the ‘yellow’ wavelength range (580–600 nm) explained the differences between the two lamp types.