Studies on the electron transfer (ET) interaction of 1,4-dihydroxy-9,10-anthraquinone and 6,11-dihydroxy-5,12-naphthacenequinone with aliphatic and aromatic amine (AlA and ArA, respectively) donors have been investigated in acetonitrile solutions. Steady-state (SS) measurements show quenching of the quinone fluorescence by amines, without indicating any change in the shape of the fluorescence spectra. No significant change in the absorption spectra of the quinones is also observed in the presence of the amines. For all the quinone–amine pairs, the bimolecular quenching constants (k_q) estimated from SS and time-resolved measurements are found to be similar. Variation in the k_q values with the oxidation potentials of the amines indicates the involvement of the ET mechanism for the quenching process. A reasonably good correlation between the k_q values and the free energy changes (ΔG⁰) for the ET reactions following Marcus' outer-sphere ET theory also supports this mechanism. It is seen that for both the quinone–ArA and quinone–AlA systems, the k_q values initially increase and then get saturated at some diffusion-controlled limiting values (k_q^DC) as ΔG⁰ values gradually become more negative. Interestingly, however, it is seen that the k_q^DC value for the quinone–AlA systems is substantially lower than that for quinone–ArA systems. Such a large difference in the k_q^DC values between quinone–AlA and quinone–ArA systems is quite unusual. Present results have been rationalized based on the assumption that an orientational restriction is imposed for the encounter complexes in quinone–AlA systems to undergo ET reactions, which arises because of the localized (at amino nitrogen) shapes of the highest-occupied molecular orbitals (HOMO) of AlA in comparison to the π-like HOMO of the ArA.

Polarized steady-state fluorescence and fluorescence excitation spectra as well as time-resolved fluorescence for B-phycoerythrin (B-PE) from red algae, Porphyridium cruentum, embedded in polyvinyl stretched films were measured. The lifetimes of polarized fluorescence were analyzed using exponential components and fractal models. The interactions between various chromophores of the pigment–protein complexes investigated were discussed. The anisotropy of fluorescence excitation spectra differs from the anisotropy of absorption spectra and depends on the wavelength of observation. This shows that differently oriented chromophores take part in various paths of excitation energy transfer (ET) or change their excitation into heat with various efficiencies (or both). Also, analysis of time-resolved fluorescence measured in various spectral regions gives different polarized components of emission. Fractal analysis of lifetimes, done under supposition of the Foerster resonance ET mechanism, suggests different arrangements of energy donors and acceptors for molecules absorbing in different spectral regions. It shows that several fractions of differently oriented “forms” of chromophores exhibiting different spectral properties occur in B-PE complexes. Small changes in the orientation of the chromophores can be followed by modification of the path of excitation energy migration. Based on the results obtained a new reorientational mechanism of the State 1 → State 2 transition was proposed: Even small conformational modifications of biliproteins, which could be caused in vivo by the change in the conditions of preillumination of bacteria, are able to modify the path of excitation ET. Such a reorientation may be responsible for the change in the partition of biliprotein excitation energy between photosystem II (PSII) and PSI (State 1 → State 2 transition). The proposed mechanism needs further verification by the investigation of whole bacteria cells.

Lipofuscin is a yellow-brown, highly fluorescent pigment that undergoes an age-related progressive accumulation in animal cells, mainly in postmitotic cells. It is a heterogeneous, high–molecular weight material associated with proteins, lipids and nucleic acids. Lipofuscin is implicated in many aspects of human health, including aging, oxidative stress, macular degeneration, lipid peroxidation, atherosclerosis, dementia (Alzheimer's Disease) and diseases associated with prions. Although the fluorescent properties of lipofuscin have long been recognized, neither histologists nor chemists have yet isolated the pigments themselves or characterized their optical properties. We have prepared lipofuscinlike species by reacting malondialdehyde (MDA) with cysteine (Cys). MDA: Cys adducts 3:2 and 2:2 are two of those that have been identified among the many that were present in the reaction. Whereas previous attempts to synthesize lipofuscinlike species resulted in compounds that were either nonfluorescent or emitted principally in the blue, the MDA:Cys adducts reported in this study are not only fluorescent but also emit over a broader range.

During the spring, when ozone depletion at the polar regions is at its maximum and consequently the environmental UV exposure is potentially high, many terrestrial communities are covered in snow and heterogeneous snow-encrusted ice that form near the edges of snowpack. Using field measurements and a theoretical radiative transfer model, we calculated the thicknesses of these covers that are necessary to reduce DNA-weighted dose to levels equal to or lower than those received later in the season in the absence of covers when there is no ozone depletion. This depth is approximately 4 cm for a 60% depletion of the ozone column, suggesting that even thin snow–ice covers are enough to completely cancel the biological effects of ozone depletion. Loss of snow–ice covers during early summer can be rapid. The maximum rate of retreat of snow cover measured during November at Mars Oasis, Antarctica (71.9°S, 68.2°W), was 44.1 cm/day, with a mean retreat of 15.4 cm/day. Climate warming might increase UV-radiation damage by melting UV-protecting terrestrial snow–ice covers earlier in the season, when ozone depletion is more severe. Conversely, climate cooling could increase UV-protection afforded to terrestrial communities by increasing the extent of snow and ice covers. Even if anthropogenic ozone depletion is eventually reversed, these data suggest the importance of climate forcing in determining UV exposures of terrestrial microbial communities in snow- and ice-covered environments.

The 2002 revision of the UV index (UVI) issued by the World Health Organisation (WHO), the World Meteorological Office (WMO), the United Nations Environment Programme (UNEP) and the International Commission on Non-Ionizing Radiation Protection (ICNIRP) (World Health Organization [2002] Global Solar UV Index: A Practical Guide. WHO, Geneva) was motivated by the need to further standardize the use and presentation of the UVI. Awareness of the hazards of solar UV radiation (UVR) is generally high in Australia, but more effective use of the UVI will assist in promoting further changes to the population's sun exposure behavior. UVI levels for a number of cities around Australia as measured by the Australian Radiation Protection and Nuclear Safety Agency (ARPANSA), covering the time period 1996–2000, are presented. Also shown are UVI forecasts from the Australian Bureau of Meteorology (BOM). Agreement between the BOM data and the measurements varies depending on the location but is within 2 UVI units approximately 75% of the time. UVI levels are supplied to the media, and in summer values in excess of 12–14 are regularly recorded, although the more northerly locations occasionally reach 16 and 17. The factors affecting the solar UVR environment and the measured UVI are also discussed and compared against measurements from the UK.

One of the major technical challenges in calculating solar irradiance on the human form has been the complexity of the surface geometry (i.e. the surface-normal vis-a-vis the incident radiation). Over 80% of skin cancers occur on the face, head, neck and back of the hands. The quantification, as well as the mapping of the anatomical distribution of solar radiation on the human form, is essential if we are to study the etiology of skin cancers or cataracts or immune system suppression. Using advances in computer graphics, including high-resolution three-dimensional mathematical representations of the human form, the calculation of irradiance has been attained to subcentimeter precision. Lighting detail included partitioning of direct beam and diffuse skylight, shadowing effects and gradations of model surface illumination depending on model surface geometry and incident light angle. With the incorporation of ray-tracing and irradiance algorithms, the results are not only realistic renderings but also accurate representations of the distribution of light on the subject model. The calculation of light illumination at various receptor points across the anatomy provides information about differential radiant exposure as a function of subject posture, orientation relative to the sun and sun elevation. The integration of a geodesic sun-tracking model into the lighting module enabled simulation of specific sun exposure scenarios, with instantaneous irradiance, as well as the cumulative radiant exposure, calculated for a given latitude, date, time of day and duration. Illustration of instantaneous irradiance or cumulative radiant exposure is achieved using a false-color rendering—mapping light intensity to color—creating irradiance or exposure isopleths. This approach may find application in the determination of the reduction in exposure that one achieves by wearing a hat, shirt or sunglasses. More fundamentally, such an analysis tool could provide improved estimates of scenario-specific dose (i.e. absorbed radiant exposure) needed to develop dose–response functions for sunlight-induced disease.

The mechanisms of ultraviolet-B (UV-B)–induced apoptosis and the role of p38 mitogen-activated protein kinase (MAPK) were investigated in murine peritoneal macrophages. Exposure of murine peritoneal macrophages to UV-B irradiation induced rapid apoptosis concurrent with DNA fragmentation and activation of caspase-3 but did not activate caspase-1. UV-B irradiation (100 mJ/cm²) also induced expression of phospho-p38 and –c-Jun N-terminal kinase (JNK) MAPK; however, no significant expression of phospho-p42/44 was observed 120 min after exposure. Pretreatment of macrophages with a p38 MAPK inhibitor, 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole (SB202190), and a caspase-3 inhibitor, N-acetyl-Asp-Glu-Val-Asp-CHO, suppressed UV-B irradiation–induced apoptosis as observed by DNA laddering and DNA fragmentation estimation quantitatively. Pretreatment with caspase-1 inhibitor, N-acetyl-Tyr-Val-Ala-Asp-CHO, had no effect. UV-B–induced caspase-3 activation resulted in the cleavage of poly-(ADP-ribose) polymerase (PARP), which was inhibited by the caspase-3 inhibitor. SB202190 pretreatment also prevented activation of caspase-3 and the cleavage of PARP. However, the caspase-3 and -1 inhibitors did not affect UV-B–induced expression of phospho-p38 and -JNK. These results suggest that activation of p38 MAPK upstream of caspases might play an important role in the apoptotic process of macrophages exposed to UV-B irradiation.

3-Deacetyl-3-(1-hydroxyethyl)bacteriopyrochlorophyll-a (1), 7,8-dihydrobacteriochlorophyll-d possessing 8-ethyl, 12-methyl and 17⁴-phytyl groups, was prepared by modifying naturally occurring bacteriochlorophyll-a. The synthetic 3¹-epimers were separated by high-performance liquid chromotagraphy, and the absolute configuration at the 3¹-position was determined by derivatization of 1 to a structurally determined chlorin. A dichloromethane solution of 3¹R-1 or 3¹S-1 was diluted with a 1000-fold volume of cyclohexane to give self-aggregation species absorbing light at a near-infrared (NIR) region (<910 nm). The resulting Q_y maximum in 3¹R-1 was 860 nm and redshifted by 2170 cm⁻¹ from the monomeric one, whereas epimeric 3¹S-1 showed a less redshifted peak at ca 800 nm, with a small dimeric band around 740 nm. Such visible spectra indicated that 3¹R/S-1 formed different supramolecular structures in the self-aggregates. In contrast, self-aggregation of the 7,8-dehydro-compound 2, bacteriochlorophyll-d_P, found in natural antennas of photosynthetic green bacteria showed much smaller diastereomeric control. The self-aggregates of 3¹R-1 absorbing light in the NIR region would be models of intrinsic membranous light-harvesting complexes 1 in photosynthetic purple bacteria as well as extramembranous antennas in green bacteria.

In this work, absorption and fluorescence spectra of protochlorophyllide (Pchlide), as well as its fluorescence lifetime, were investigated in organic solvents having different physical properties. The obtained Pchlide spectral features are discussed in relation to the parameters describing solvent properties (refractive index and dielectric constant) and taking into account the specific solvent–Pchlide interaction. The correlation of Pchlide Q_y and Soret absorption bands with solvent polarizability function ((n² − 1)/(n² 2)) has been found; however, the dispersion of the observed points was rather high. A small Stokes shift of a magnitude between 50 and 300 cm⁻¹ was found, which indicates low sensitivity of Pchlide to nonspecific solvation. The fluorescence decay of Pchlide was single exponential in all the investigated solvents, with the lifetime value ranging from 5.2 ns for dioxane to 3.5 ns for methanol. Dependence of the obtained fluorescence lifetimes on the solvent orientation polarizability, a parameter being the function of both refractive index and dielectric constant, was discussed. In water–methanol mixtures, a further decrease of the fluorescence lifetime was observed, giving values of 2.9 ns for 25% methanol. Double-exponential decay of Pchlide fluorescence was found for Pchlide in a solution of 15% methanol with the lifetimes of 4.5 ± 0.5 ns and 1.2 ± 0.3 ns and in pure water with the lifetimes of 2.5 ± 0.5 ns and 0.4 ± 0.1 ns. The obtained results are discussed in relation to spectroscopic properties of Pchlide in vivo.

Multichannel flash spectroscopy (with microsecond time resolution) has been applied to carotenoid (Car)-containing and Car-less reaction centers (RC) of Rhodobacter sphaeroides with a view to investigate the interaction between the Car and its neighboring pigments at room temperature. Under neutral redox potential conditions, where the primary quinone acceptor (Q_A) is oxidized, the light-induced spectral changes in the 350–1000 nm region are attributed to the photochemical oxidation of the special pair (denoted here as P₈₇₀), the generation of P ₈₇₀Q⁻_A, and the attendant electrochromism of adjacent chromophores. A bathochromic shift of <1 nm in the visible absorption region of Car reveals the sensitivity of Car to the P₈₇₀ photooxidation. Under low redox potential conditions, where Q_A is reduced, P₈₇₀ triplets (P^†₈₇₀) are formed. The time-resolved triplet-minus-singlet (TmS) spectrum of Car-less RC shows a deep bleaching at 870 nm, which belongs to P^†₈₇₀, and additional (but smaller) bleaching at 800 nm; the entire spectrum decays at the same rate (with a lifetime of about 50 μs). The bleaching at 800 nm arises from the pigment interaction between P^†₈₇₀ and the accessory bacteriochlorophylls on A and B branches (B_A,B). In Car-containing RC, the TmS spectra of Car are accompanied by two smaller, negative signals—a sharp peak at 809 ± 2 nm and a broad band at 870 nm—which decay at the same rate as the TmS spectrum of Car (ca 10 μs). The former is ascribed to the perturbation, by Car^†, of the absorption spectrum of B_B; the latter, to the TmS spectrum of P^†₈₇₀, a species that appears to be in approximate thermal equilibrium with Car^†. These assignments are consistent with the absorption-detected magnetic resonance spectra obtained by other workers at low temperatures.

Assessment of laser-induced tissue damage is not complete without an investigation into the resulting cellular and molecular changes. In the past, tissue damage was quantified macroscopically by visual effects such as tissue mass removal, carbonization and melting. Microscopically, assessment of tissue damage has been typically limited to histological analysis of excised tissue samples. In this research, we used heat shock protein (hsp70) transcription to track cellular response to laser-induced injury. A stable cell line (NIH-3T3) was generated containing the firefly luciferase (luc) reporter gene attached to the hsp promoter (murine hsp70a1). After thermal injury with a pulsed holmium–yttrium aluminum garnet laser (λ = 2.1 μm, τ_p = 250 μs, 30 pulses, 3 Hz), luciferase is produced on hsp70 activation and emits broad-spectrum bioluminescence over a range of 500–700 nm, with a peak at 563 nm. The onset of bioluminescence can be seen as early as 2 h after treatment and usually peaks at 8–12 h depending on the severity of heat shock. The luminescence was quantified in live cells using bioluminescence imaging. A minimum pulse energy (65 mJ/pulse [total energy 1.95 J; total radiant exposure = 6 J/cm²]) was needed to activate the hsp70 response, and a higher energy (103 mJ/pulse [total energy 3.09 J; total radiant exposure = 9.6 J/cm²]) was associated with a reduction in hsp70 response and cell death. Bioluminescence levels correlated well with actual hsp70 protein concentrations as determined by enzyme-linked immunosorbent assay. Photon counts were normalized to the percentage of live cells by means of a flow cytometry cell viability assay. Within a relatively small range between a lower activation threshold and an upper threshold that leads to cell death, the hsp70 response followed an Arrhenius relationship when constant-temperature water bath and laser experiments were carried out.

Synthesis of extracellular matrix (ECM) proteins and their degradation by matrix metalloproteinases (MMP) are part of the dermal remodeling resulting from chronic exposure of skin to ultraviolet radiation (UVR). We have compared two alternative mechanisms for these responses, namely, a direct mechanism in which UV-B or UV-A is absorbed by fibroblasts and an indirect mechanism in which cytokines, produced in skin in response to UVR, stimulate production of the ECM proteins and MMP. These studies were carried out on human dermal fibroblasts grown in contracted, free-floating 9 day old collagen gels as a dermal equivalent. Synthesis of tropoelastin, collagen, fibrillin, MMP-1, -2, -3 and -9 and tissue inhibitors of metalloproteinases (TIMP)-1 and -2 were measured. Tropoelastin, collagen and fibrillin levels were stable between days 4 and 10, and MMP and TIMP decreased by day 10. Neither UV-B (2.5–50 mJ/cm²) nor UV-A (2–12 J/cm²) altered synthesis of ECM proteins, but UV-A increased MMP-1 and -3 production. Tropoelastin synthesis increased in response to transforming growth factor-β1 (5 ng/mL) treatment. Both interleukin-1β and tumor necrosis factor-α (10 ng/mL) decreased fibrillin messenger RNA levels but increased MMP-1, -3 and -9 synthesis markedly. Collagen synthesis was not modulated by UV-B, UV-A or cytokine treatment. These results indicate that certain cytokines may have greater effects on production of ECM proteins and MMP than absorption of UV-B and UV-A by fibroblasts grown in dermal equivalents and suggest that the former pathway may play a role in the dermal remodeling in photoaged skin.

We examined the apoptotic effects of photodynamic therapy (PDT) in leukemia cells (HL60) and lymphoma cells (Raji). Moreover, we also investigated the relationship of apoptosis induced by PDT to heat shock protein (HSP) expression. To induce 80% of cell death by PDT, HL60 cells required 6 μg/mL and Raji cells required 9 μg/mL of Photofrin®. PDT induced apoptosis in 77.2% of HL60 and in 0.4% of Raji at lethal dose (LD80) conditions. The cell line in which apoptosis is predisposed may be more susceptible to PDT compared with the cell line in which necrosis is predisposed. Furthermore, HSP-70 was expressed constitutively in Raji cells but not in HL60 cells. Heat treatment of HL60 cells induced expression of HSP-70 and resulted in significant reduction of PDT-mediated apoptosis. From the results of this experiment, it is suggestive that HSP-70 contributes to inhibition of apoptosis mediated by PDT.

Halogenated squaraine dyes 1 and 2 possess favorable photophysical and in vitro photobiological properties that make these new class of molecules interesting for photodynamic therapeutic applications. For a better understanding of the mechanism of their photobiological activity, we have analyzed the DNA damage and the cytotoxicity induced by these photosensitizers in mammalian cells and cell-free systems in the presence and absence of various additives and scavengers. Both photoactivated squaraines were found to be similar efficient in inducing single-strand breaks (SSB) in cell-free DNA when compared with the cellular DNA. Superoxide dismutase and catalase did not show any influence. However, the presence of tert-butanol and glutathione inhibited the formation of the DNA SSB, indicating an indirect (possibly squaraine radical mediated) mechanism under cell-free conditions. Replacing H₂O in the buffer by D₂O resulted in a five- to six-fold increase in the number of the SSB in cell-free DNA and a significant enhancement of the photocytotoxicity in mouse lymphoma cells. The results demonstrate that singlet oxygen is the major reactive species under cell-free and cellular conditions and confirm that squaraine-based sensitizers 1 and 2 can have potential applications in photodynamic therapy.

Photodynamic therapy (PDT) kills cells via the production of singlet oxygen and other reactive oxygen species. PDT causes chromosomal damage and mutation to cultured cells. However, DNA damage does not contribute to the phototoxic effect. To study the effect of Photofrin-PDT–induced DNA damage, we used the comet assay in combination with endonuclease III and formamidopyrimidine DNA glycosylase and a human keratinocyte cell line to investigate photogenotoxicity and its prevention by tocopherol (TOC). This study shows that PDT induced DNA damage in HaCaT cells at doses allowing cells to survive 7 days after irradiation. α-TOC did not prevent the acute cell lysis caused by Photofrin-PDT but did prevent Photofrin-PDT–induced DNA damage. However, the concentration of TOC that conferred protection (100 μM) was higher than is detected in human serum. Base oxidation was also measured using the comet assay. Although TOC could prevent frank DNA strand breaks caused by PDT, it was unable to decrease the level of base oxidation as revealed by enzyme-sensitive sites. It is suggested that the potential genotoxic risk from laser-PDT could be low, and that topical α-TOC at a high concentration may be useful in preventing some types of DNA damage without preventing acute photolysis after Photofrin-PDT.

The unicellular cyanobacterium Synechocystis sp. PCC 6803 (Syn6803) exhibits photomovement through gliding motility. For a better understanding of photomovement in Syn6803, we examined the effects of Ca² on photoorientation and motility using a computer-assisted videomicroscope motion analysis system. When calcium ion was chelated from the basic motility medium by adding 0.5 mM ethylene glycol-bis-(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), the photoorientation was completely inhibited, whereas the gliding motility remained approximately 70% of the control. Photoorientation impaired by EGTA was nearly recovered within 30 min upon addition of 1 mM Ca² . The recovery of photoorientation by Ca² was mimicked by either Mn² or Mg² but not by Ba² or Sr² . Lanthanum ion at 10 μM completely inhibited both phototactic orientation and gliding motility of Syn6803. Furthermore, pimozide (voltage-gated L-type calcium channel inhibitor), orthovanadate (calcium efflux blocker) and A23187 (calcium ionophore) partially inhibited phototactic orientation and gliding motility. Interestingly, photoorientation was prevented with increasing concentrations of calmodulin antagonist such as trifluoperazine (TFP) and chlorpromazine, but gliding motility was inhibited in proportion to the concentration of TFP. The results we present strongly indicate that Ca² plays a significant role in regulating the photomovement of Syn6803.

Yellow-emitting Vibrio fischeri Y1 modulates its bioluminescence (BL) depending on the dissolved O₂ concentration. On supplying O₂ to the cells under anaerobiosis, the cells begin to emit striking yellow BL peaking around 535 nm. The enhanced yellow emission reverts reversibly to the original level after O₂ is consumed. Moreover, the reversible rise and fall of the yellow emission occurs repeatedly in accord with the repeating cycles of aeration on and off. This indicates that an increase in the cellular amount of yellow fluorescent protein (YFP) is not an immediate cause of the yellow emission enhancement. One suggested explanation is that the activity of YFP originating from its highly fluorescent property is altered by redox interaction with the respiratory components, including the soluble cytochrome c. Under the O₂-limited conditions, the cellular YFP molecules, in part, seem to lose the fluorescent property possibly because of being reduced via redox interaction with some respiratory components in reduced form. On stimulating aerobic respiration with O₂ supply, the reduced YFP seems to retrieve its fluorescent property via oxidation possibly with both O₂, diffused across the cell membrane, and ferricytochrome c, generated during the respiratory turnover. The suggested redox interactions seem primarily to cause the reversible BL modulation.