Photoreduction of methyl viologen (MV² ) by eosin-Y (EY²⁻) in the presence of triethanolamine (TEOA) has been investigated in water–methanol mixture by means of steady-state photolysis and laser-flash photolysis in the visible/near-infrared regions. The complete conversion to the persistent methyl viologen radical cation (MV^· ) was observed in the presence of lower concentrations of EY²⁻ and excess TEOA. By laser-flash photolysis measurements, electron transfer was confirmed to occur from the triplet state of EY²⁻ [³(EY²⁻)^*] to MV² in the rate constants of ca 2.0 × 10¹⁰ M⁻¹ s⁻¹. The rates and efficiencies of production of MV^· were found to be dependent on solvent compositions and concentrations of MV² ionic salt and TEOA. The back electron transfer reaction from MV^· to EY^·− was retarded in the presence of TEOA, which supports that EY²⁻ is reproduced by accepting an electron from TEOA. In the presence of excess TEOA, the indirect formation of MV^· from EY^·3− which was produced by accepting an electron from TEOA, was confirmed. The contributions of both the oxidative and reductive routes of ³(EY²⁻)^* for the MV^· formation have been confirmed.

Since 1992 solar ultraviolet (UV) spectral irradiance (290–325 nm) has been measured at two Italian stations of Rome (urban site) and Ispra (semirural site) using Brewer spectrophotometry. The data collected under all sky conditions, are compared with the output of a sophisticated radiative transfer model (System for Transfer of Atmospheric Radiation—STAR model). The STAR multiple scattering scheme is able to cope with all physical processes relevant to the UV transfer through the atmosphere. The experience so far acquired indicates that, in spite of the unavoidable uncertainties in the input parameters (ozone, aerosol, surface albedo, pressure, temperature, relative humidity, cloud cover), measured and computed clear sky irradiances are in reasonable agreement. The STAR model is applied to build up the solar UV geographic patterns in Italy: the daily dose in the range 290–325 nm is computed at about 70 sites where a thorough and homogeneous climatology is available. For each month the concept of an idealized “standard day” is introduced and the surface distribution of solar UV field determined. The map of solar UV patterns for Italy, available for the first time, meets the study requirements in the field of skin and eye epidemiology, as well as in other investigations dealing with the impact of UV on the biosphere. The results are interpreted in terms of atmospheric and meteorological parameters modulating UV radiation reaching the ground.

Hydrogen peroxide (H₂O₂) is widely distributed in surface waters where the primary photochemical formation pathway involves the interaction between dissolved organic carbon (DOC) and ultraviolet radiation (UVR). In laboratory studies using iron-rich water from Yellowstone's Chocolate Pots spring, H₂O₂ formation depended on sample treatment (unfiltered, <0.2 μm filtered, autoclaved) prior to irradiation, suggesting several formation pathways. Similar H₂O₂ formation in filtered and unfiltered water indicates that it is primarily soluble material that is responsible for H₂O₂ formation. H₂O₂ formation with soluble material probably includes only photochemical reactions with DOC and/or metals. Greater H₂O₂ formation in unfiltered and filtered water than in autoclaved water suggests that the agent(s) involved in H₂O₂ formation is (are) not stable at high temperatures and pressures and degrade to nonphotoreactive species. Such unstable agents may include DOC and/or dissolved complexes of iron or other metals. UVR absorbance occurs across the UV spectrum and, though slightly greater in the UVA range (320–400 nm), is similar to that of other surface waters. Increased UVR absorbance after autoclaving suggested degradation or alteration of some components, which in turn affected H₂O₂ formation. The spectral region used for irradiation affected net formation and yield. H₂O₂ formation in water irradiated with UVA radiation was 2.5–3 times that formed in water irradiated with UVB radiation (280–320 nm) in experiments using artificial light sources. Apparent quantum yields comparable to those reported by others could not be calculated because the instrumental designs are not the same. However, approximate quantum yields were calculated for these experiments but should be viewed with caution. Quantum yields were higher in these experiments (0.0040 mol H₂O₂ per mol photon at 310 nm and 0.0012 mol H₂O₂ per mol photon at 350 nm) than values reported by other researchers (<0.0007 mol H₂O₂ per mol photon at 300 nm and <0.0005 H₂O₂ per mol photon at 340 nm; [Scully, N. M., D. R. S. Lean, D. J. McQueen and W. J. Cooper (1996) Limnol. Oceanogr. 41, 540–548]). In natural solar source experiments, H₂O₂ formation was greater in experiments with UVA and photosynthetically active radiation (PAR; 400–700 nm) than with PAR alone or with UVB, UVA and PAR. However, H₂O₂ capacity (nM H₂O₂ W⁻¹ h⁻¹ m²) was greatest with UVB radiation and lowest with PAR radiation. Source regions could not be studied separately. Dark decay of H₂O₂ occurred via two mechanisms. The main mechanism responsible for H₂O₂ decay involved particulate matter (probably microorganisms), whereas a secondary mechanism involved soluble matter (i.e. DOC, metal ions and other dissolved species involved in Fenton reactions).

Cutaneous and systemic immune function are believed to play an important role in cutaneous carcinogenesis. We therefore sought to determine whether the suntan parlor radiation sources commonly used in the United States cause measurable qualitative suppression of immune function and quantitative alterations in circulating T cell subpopulations. Subjects (n = 22) were recruited and randomly assigned to receive suntan parlor exposures (10 full-body UV exposures over a 2 week period, shielding only the right flexural arm) or no exposure. Baseline circulating T lymphocyte subpopulations (T helper lymphocyte, CD4; T suppressor/cytotoxic lymphocyte, CD8) were measured. Two weeks later (upon completion of UV exposures for those in this group), circulating T cell subpopulations were measured and dinitrochlorobenzene (DNCB) sensitization (in the UV group, on the UV-exposed buttock) was performed. Subsequent DNCB elicitation was performed in a bilateral fashion (in the UV group, on the right UV-shielded and the left UV-exposed upper arm). We found that subjects in the UV group demonstrated localized suppression of contact hypersensitivity sensitization and elicitation and also an increase in circulating CD8 cells when compared to the control group (P ≤ 0.05). We conclude that suntan parlor exposures, as typically received in this country, suppress contact hypersensitivity and increase the circulating T suppressor/cytotoxic cell number quantity.

In cyanobacterium Synechococcus sp. PCC 7942 the photosystem II reaction-center protein D1 is encoded by three psbA genes. The psbAI gene encodes D1:1 protein, the form used for acclimated growth, and psbAII and psbAIII genes encode the stress-induced form, D1:2 protein. Strong light and low temperature have been shown to induce the expression of psbAII/III genes and down-regulate the expression of psbAI gene. Recently, we reported the involvement of reduced thiols in the up-regulation of psbAII/III genes. In this study, we have analyzed the regulation of psbA gene expression in Synechococcus further, at both the transcriptional and post-transcriptional levels. We show that the inhibitors of the photosynthetic electron-transfer chain, which have different effects on the redox state of the plastoquinone (PQ) pool, have similar effect on the transcription of psbA genes. The inhibitors 3-(3,4 dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) do not cause any changes in psbA gene expression when added under low-light conditions, but dramatically reduce the high-light induction of psbAII/III genes when added upon a high-light shift. Moreover, when the thiol reductant, dithiothreitol, is added to Synechococcus cells together with DCMU concomitant with the high-light shift, no inhibition of psbAII/III gene up-regulation takes place, indicating that the thiol redox state rather than the redox state of the PQ pool regulates psbA gene transcription. We also provide evidence for post-transcriptional regulation of psbA gene expression, particularly, inhibition of translation of psbAI transcripts at high light, and demonstrate that the D1 protein synthesis and degradation processes are coregulated in Synechococcus.

We have studied the effect of the absence of carotenoids on the organization of bacteriochlorophylls (BChls) in chlorosomes of Chlorobium (Chl.) phaeobacteroides strain CL1401. Carotenoid-depleted chlorosomes were obtained by means of 2-hydroxybiphenyl–supplemented cultures. In the presence of the inhibitor, isorenieratene (Isr) and β-Isr biosynthesis were inhibited to more than 95%, leading to an accumulation of the colorless precursor phytoene inside the chlorosomes. In addition, there was a 30–40% decrease in the baseplate BChl a content. The absorption spectrum of the carotenoid-depleted chlorosomes showed a 10 nm blue shift in the BChl e Q_y absorption peak. Under reducing conditions, a decrease in the BChl a/BChl e fluorescence emission ratio was observed in carotenoid-depleted chlorosomes relative to that in control chlorosomes, caused mainly by the decrease in the BChl a content. The steady-state fluorescence emission anisotropy in the BChl e region dropped from ∼0.24 for native chlorosomes to ∼0.14 for carotenoid-depleted ones, indicating reorganization of BChl e. The circular dichroism (CD) signal of the carotenoid-depleted chlorosomes was increased two times in the BChl e Q_y region. A simple model based on the structure proposed was used to explain the observed effects. Carotenoids might affect the angle between the direction of the BChl e Q_y transition and the axis of the rod. The orientation of BChl a in the baseplate remains unchanged in carotenoid-depleted chlorosomes, although there is a partial loss of BChl a as a consequence of a decrease in the baseplate size. The carotenoids are most likely rather close to the BChls and appear to be important for the aggregate structure in Chl. phaeobacteroides.

The aim of this prospective randomized study was to compare the clinical and cosmetic outcome of superficial basal cell carcinomas (BCC), using either laser or broadband halogen light, in photodynamic therapy with topical 5-aminolevulinic acid (ALA). A total of 83 patients with 245 superficial BCC were included in the study. Standard treatment involved 15 min of local pretreatment with 99% dimethylsulfoxide (DMSO) before topical application of 20% ALA with DMSO (2%) and ethylendiaminetetraacetic acid (2%) as cofactors for 3 h before light exposure with either laser or a broadband lamp (BL). A complete response was achieved in 95 lesions (86%) in the laser group and 110 lesions (82%) in the BL group 6 months after treatment. Of these, 80 lesions (84%) in the laser group and 101 lesions (92%) in the lamp group were independently evaluated to have an excellent or good cosmetic post-treatment score. No serious adverse events were reported. This study shows that there is no statistical significant difference in cure the rate (P = 0.49) and the cosmetic outcome (P = 0.075) with topical application of a modified ALA-cream between light exposure from a simple BL with continuous spectrum (570–740 nm) or from a red-light laser (monochromatic 630 nm). Cost and safety are further elements in favor of the BL in this setting.

Fluorescence spectroscopy has potential to improve cervical precancer detection. The relationship between tissue biochemistry and fluorescence is poorly understood. The goal of this study was to characterize normal cervical autofluorescence, using fresh tissue short-term tissue cultures and epithelial cell suspensions. Transverse, short-term tissue cultures were prepared from 31 cervical biopsies; autofluorescence images were obtained at 380 and 460 nm excitation. Fluorescence excitation–emission matrices were measured from normal, precancerous and cancerous cervical cell suspensions. Observed fluorescence patterns contrast those reported for frozen–thawed tissue, and were placed into groups with (1) bright epithelial and weak stromal fluorescence; (2) similar epithelial and stromal fluorescence; and (3) weak epithelial and bright stromal fluorescence. The average ages of women in the groups were 30.9, 38.0 and 49.2 years. Epithelial fluorescence intensity was similar in Groups 1 and 2, but weaker in Group 3. Stromal intensity was similar in Groups 2 and 3, but weaker in Group 1. The ratio of epithelial to stromal fluorescence intensity was significantly different for all groups. EEMs of cell suspensions showed peaks consistent with tryptophan, reduced form of nicotinamide adenine dinucleotide (phosphate) and flavin adenine dinucleotide. Short-term tissue cultures represent a novel, biologically appropriate model to understand cervical autofluorescence. Our results suggest a biological basis for the increased fluorescence seen in older, postmenopausal women.

Although histochemical and immunohistochemical methods are the standard procedures in diagnosis of lymphoproliferative disorders, useful improvements in evidencing histopathologic manifestations can be obtained with the introduction of tissue autofluorescence analyses. We used microspectrofluorometry and a Multispectral Imaging Autofluorescence Microscopy (MIAM) technique to analyze lymph-node biopsies from patients with lymphoadenopathy of different origins. Images of tissue autofluorescence were obtained by excitation at 365 nm of lymph-node sections and sequential detection with interference filters (50 nm bandwidth) peaked at 450, 550 and 658 nm. Monochrome images were combined together in a single red–green–blue color image. Most of the fluorescence was observed within the blue spectral band because of large contributions from extracellular collagen and elastin fibers as well as from reduced form of intracellular nicotinamide adenine dinucleotide (phosphate). Autofluorescence imaging shows morphological differences between neoplastic and non-neoplastic tissues. The reactive hyperplasia samples show the typical lymph-node organization with weak fluorescent follicles separated by high fluorescent connective trabeculae. In the neoplastic lymph nodes the loss of follicle organization is observed. Consequently, MIAM permits to discriminate between non-neoplastic and neoplastic tissues on the basis of their autofluorescence pattern. Multispectral imaging of tissue autofluorescence may present some advantages with respect to standard histochemical microscopy since it (1) does not require any chemical manipulation of samples; (2) gives real-time results performing the analysis immediately upon specimen resection; and (3) supplies a representation of the biological structure organization linked to endogenous fluorophores.

The objective of this study was to determine whether exposure of early suckling young of the opossum Monodelphis domestica to ultraviolet A (UVA) radiation (320–400 nm) can lead to the development of melanocytic lesions similar to those induced after exposure to ultraviolet B (UVB) radiation (280–320 nm) to total doses as low as 380 J/m². A total of 576 sucklings received nine exposures of 0.6, 2.6 or 15.5 kJ/m² per dose (total doses ∼ 6, 23 and 140 kJ/m² respectively) from a Blak Ray lamp source with a narrow range emission at 365 nm. A further 280 sucklings were exposed in the same way to doses of 2.6 kJ/m² per dose (total ∼ 23 kJ/m²) broad-band UVA with visible wavelengths from a Dermalight lamp. Frequency of litter loss following all of the UVA-exposure protocols was similar to that within the same stocks in the colony at large. Only one of the 856 UVA-exposed individuals possessed a melanocytic lesion at the 5 month assessment point. No radiation-induced lesions of any type were evident on the skin of the other animals exposed as sucklings. The affected male was from a group of 70 individuals exposed to the highest total dose (140 kJ/m²) from the Blak Ray light source. The melanocytic hyperplasia was provisionally identified as a potential melanoma but it slowly regressed as the animal aged. We conclude that in the opossum suckling exposure system, the potency of UVA for melanoma induction is extremely low compared with that of UVB. Possible explanations, amenable to further investigations, are given for the low UVA sensitivity of the suckling model compared to the adult exposure model of Ley (Ley, R. D. [1997] Cancer Res. 57, 3682–3684).

The relative contribution, to cell death, of photodynamic damage to respiratory proteins (known targets of photodynamic therapy with many photosensitizers) and other cellular sites was examined. The models were a human ovarian carcinoma cell line 2008, and its mitochondrial DNA-deficient derivative ET3, which lacks several key respiratory protein subunits. Phototoxicity was compared in the two cell lines with photosensitizers that localized to different cellular compartments. Photosensitizers included Victoria Blue BO (VBBO; mitochondria); Photofrin with a short incubation, (plasma membrane) or a long incubation (intracellular membranes including mitochondria); and Nile Blue A (NBA; lysosomes). Photosensitizer content and localization did not differ between the 2008 and ET3 cells. For sensitizers without a primary mitochondrial localization (NBA and Photofrin with a short incubation), there was no significant difference between 2008 and ET3 toxicity. Consistent with a mitochondrial localization of VBBO and independence from respiratory-chain damage, ET3 cells were less susceptible than 2008 to both dark- and light-activated VBBO-mediated damage. Statistical analysis of the data demonstrated minimal photobleaching of VBBO and a significant difference between the phototoxicity curves of ET3 and 2008. For Photofrin with a long incubation, dark- and phototoxicity effects were similar for both cell lines. Inhibition of respiratory enzymes is thus only a minor component of Photofrin-mediated (long incubation) phototoxicity in these cell lines and is overwhelmed by more significant damage elsewhere, whereas it is a major but not the exclusive element of death mediated by VBBO.

The survival of organisms depends on their ability to adapt to their environment, one important aspect of which is the daily cycle of day and night. During the day, organisms use a variety of strategies to protect themselves from deleterious ultraviolet (UV) wavelengths of sunlight. Among those strategies could be timing of UV-sensitive cellular processes to occur at night to avoid UV-induced damage. We tested whether the unicellular alga Chlamydomonas reinhardtii uses this strategy by measuring the survival of cells following exposure to UV radiation at different phases of the day. Chlamydomonas cells displayed a rhythm of survival from UV radiation where the most sensitive phases occurred during the end of the day and at the beginning of the night. This phase of sensitivity corresponds to the time of nuclear division. The rhythm continues in constant light indicating control by a circadian clock. The results presented here suggest a hypothesis of how circadian clocks may have evolved; a temporal program whereby light-sensitive processes are timed to avoid sunlight-induced damage would be advantageous and therefore selected.

The aim of this study was to compare the effects of polarized light versus nonpolarized light on melatonin secretion in healthy, humans (mean age, 25 years; N = 6). On separate evenings, each subject was exposed to four different light intensities (20, 40, 80 and 3200 lx) of both polarized and nonpolarized light, as well as to a control, dark exposure. Each evening experiment consisted of a 120 min dark exposure (0000–0200 h) followed by a 90 min light exposure (0200–0330 h). Subjects' pupils were dilated prior to exposures. Blood samples were drawn at the start and end of each light-exposure period and later assayed for melatonin by radioimmunoassay. When compared to control exposures, both polarized and nonpolarized light elicited significant suppression of plasma melatonin at each illuminance (P < 0.03 to P < 0.0001), There were no significant differences between the effects of polarized light and nonpolarized light at any illuminance. The two light stimuli modalities demonstrated very similar fluence–response relationships between illuminance and melatonin suppression. Thus, the human pineal gland is responsive to ocular exposure with polarized light in a dose-dependent manner similar to that of nonpolarized light, although no significant differences were detected between polarized and nonpolarized light on melatonin regulation.

The green fluorescent protein (GFP) has emerged, in recent years, as a powerful reporter molecule for monitoring gene expression, protein localization and protein–protein interaction. Several mutant variants are now available differing in absorption, emission spectra and quantum yield. Here we present a detailed study of the fluorescence properties of the Phe-64→Leu, Ser-65→Thr mutant down to the single molecule level in order to assess its use in quantitative fluorescence microscopy and single-protein trafficking. This enhanced GFP (EGFP) is being used extensively as it offers higher-intensity emission after blue-light excitation with respect to wild-type GFP. By means of fluorescence spectroscopy we demonstrate the absence of the neutral form of the chromophore and the lack of photobleaching recovery after ultraviolet light irradiation. Furthermore, we show that the EGFP spectral properties from isolated to densely packed molecules are highly conserved. From these experiments EGFP emerges as an ideal molecule for quantitative studies of intra and intercellular tagged-protein dynamics and fluorescence-activated cell sorting, but not for monitoring single-protein trafficking over extended periods of time.