Interaction of potent photodynamic agents, sulfonated aluminum phthalocyanines (AlPcS_n where n is a number of sulfonic groups), with biological membranes was studied here using model systems: sensitized photoinactivation of gramicidin channels in planar lipid bilayers and adsorption on lipid monolayers. Fluoride anions known to form complexes with aluminum were found to inhibit both the adsorption of aluminum phthalocyanines on lipid monolayers, as measured with a Langmuir trough by surface pressure and surface potential changes, and photodynamic efficacy of the dyes, as studied by gramicidin channel photoinactivation. The similar effects were caused by the alkalinization of the medium. Fluoride anions appeared to be much more effective in the case of AlPcS₄ as compared to AlPcS₃. The suppression of the photodynamic potency of aluminum phthalocyanines was attributed to desorption of the dyes from lipid bilayers induced by fluoride or hydroxyl ions. With AlPcS₄ an enhancement of the dye aggregation leading to a decrease in the sensitizing activity was probably involved in the fluoride effect as revealed by absorption and fluorescence spectral measurements. Capillary electrophoresis was employed to understand the mechanism of the dye desorption. The results of these experiments indicated that the reduction in the membrane affinity was associated with an increase in the negative charge of the dye molecules due to the binding of fluoride or hydroxyl ions.

When exposed to the intracellular environment fluorescent probes sensitive to pH exhibit changes of photophysical characteristics as a result of an interaction of the dye molecule with cell constituents such as proteins, lipids or nucleic acids. This effect is reflected in calibration curves different from those found with the same dye in pure buffer solutions. To study an interaction of the probe 5′(and 6′)-carboxy-10-dimethylamino-3-hydroxy-spiro[7H-benzo[c]xanthene-7,1′(3H)-isobenzofuran]-3′-one (carboxy SNARF-1) with membrane lipids, we measured its fluorescence in model systems of large unilamellar vesicles (LUV) prepared by extrusion. When the dye was removed from the bulk solution by gel filtration the relative fluorescence intensity of the lipid-bound dye form was enhanced, showing a strong interaction of the dye molecule with LUV membrane lipids. Surprisingly, the dye molecules seem to be bound predominantly to the outer surface of the lipid bilayer. The same situation was found with small unilamellar vesicles prepared by sonication. This effect makes it difficult to use carboxy SNARF-1 for measurements of the internal pH in suspensions of liposomes.

The photodynamic activities of the free-base 5,10,15,20-tetrakis(4-methoxyphenyl)porphyrin (TMP) and their metal complexes with zinc(II) (ZnTMP), copper(II) (CuTMP) and cadmium(II) (CdTMP) have been compared in two systems: reverse micelle of n-heptane/sodium bis(2-ethylhexyl)sulfosuccinate/water bearing photooxidizable substrates and Hep-2 human larynx carcinoma cell line. The quantum yields of singlet molecular oxygen, O₂(¹Δ_g), production (Φ_Δ) of TMP, ZnTMP and CdTMP in tetrahydrofuran, were determined yielding values of 0.65, 0.73 and 0.73, respectively, while O₂(¹Δ_g) formation was not detected for CuTMP. In the reverse micellar system, the amino acid l-tryptophan (Trp) was used as biological substrate to analyze the O₂(¹Δ_g)-mediated photooxidation. The observed rate constants for Trp photooxidation (k_obs^Trp) were proportional to the sensitizer quantum yield of O₂(¹Δ_g). A value of ∼2 × 10⁷ s⁻¹ M⁻¹ was found for the second-order rate constant of Trp (k_r^Try) in this system. The response of Hep-2 cells to cytotoxicity photoinduced by these agents in a biological medium was studied. The Hep-2 cultures were treated with 1 μM of porphyrin for 24 h at 37°C and the cells exposed to visible light. The cell survival at different light exposure levels was dependent on Φ_Δ. Under these conditions, the cytotoxic effect increases in the order: CuTMP ≪ TMP < ZnTMP ∼ CdTMP, correlating with the production of O₂(¹Δ_g). A similar behavior was observed in both the chemical and biological media indicating that the O₂(¹Δ_g) mediation appears to be mainly responsible for the cell inactivation.

Photoinduced electron-transfer processes in the systems of chlorophylls (Chl) (chlorophyll-a [Chl-a] and chlorophyll-b) and fullerenes (C₆₀/C₇₀) in both polar and nonpolar solvents have been investigated with nanosecond laser photolysis technique, observing the transient spectra in the visible/near-IR regions. By the excitation of Chl in benzonitrile (BN) it has been proved that electron transfer takes place from the triplet excited states of Chl to the ground states of C₆₀/C₇₀. By the excitation of C₇₀ in BN electron transfer takes place from the ground states of Chl to the triplet excited state of C₇₀. In both Chl the rate constants and quantum yields for the electron-transfer processes are as high as those of zinc porphyrins and zinc phthalocyanines, indicating that the long alkyl chains of Chl play no role in retarding the electron transfer. The rate constant for the electron-mediating process from the radical anion of C₇₀ to octylviologen dication yielding the octylviologen radical cation was evaluated. The back electron-transfer process from the viologen radical cation to the radical cation of Chl-a takes place in a longer time-scale, indicating that a photosensitized electron-transfer/electron-mediating cycle is achieved.

The steady-state and time-resolved emission properties of aqueous solutions containing different aggregation state distributions of eumelanin are reported. Excitation spectra of the size-selected samples reveal, for the first time, differences in absorption bands due to varying levels of aggregation. These size-dependent absorption properties result in size-dependent emission band shapes and quantum yields. For all size fractions, absorption and emission overlap significantly. The emission yield for small eumelanin aggregates is 5.7 times greater than that for large eumelanin aggregates. Time-resolved population decays reveal that small eumelanin aggregates are responsible for long-lived emission dynamics (lifetimes greater than 1 ns), while large eumelanin aggregates are the source of short emission decay (lifetimes less than 1 ns). Polarized emission decays for the large and small aggregates reveal that energy transfer occurs both within the same and between the separate fundamental building blocks of eumelanin. The observed energy transfer dynamics can be accounted for using Förster theory.

Absorption and reflectance spectra of maple (Acer platanoides), cotoneaster (Cotoneaster alaunica), dogwood (Cornus alba) and pelargonium (Pelargonium zonale) leaves with a wide range of pigment content and composition were studied in visible and near-infrared spectra in order to reveal specific anthocyanin (Anth) spectral features in leaves. Comparing absorption spectra of Anth-containing and Anth-free leaves with the same chlorophyll (Chl) content, absorption spectra of Anth in leaves were derived. The main spectral feature of Anth absorption in vivo was a peak around 550 nm; the peak magnitude was closely related to Anth content. A quantitative nondestructive technique was developed to subtract Chl contribution to reflectance in this spectral region and retrieve Anth content from reflectance over a wide range of pigment content and composition. Anth reflectance index in the form ARI = (R₅₅₀)⁻¹ − (R₇₀₀)⁻¹, where (R₅₅₀)⁻¹ and (R₇₀₀)⁻¹ are inverse reflectances at 550 and 700 nm, respectively, allowed an accurate estimation of Anth accumulation, even in minute amounts, in intact senescing and stressed leaves.

The green anole (Anolis carolinensis) is the most northerly distributed of its Neotropical genus. This lizard avoids a winter hibernation phase by the use of sun basking behaviors. Inevitably, this species is exposed to high doses of ambient solar ultraviolet radiation (UVR). Increases in terrestrial ultraviolet-B (UV-B) radiation secondary to stratospheric ozone depletion and habitat perturbation potentially place this species at risk of UVR-induced immunosuppression. Daily exposure to subinflammatory UVR (8 kJ/m²/day UV-B, 85 kJ/m²/day ultraviolet A [UV-A]), 6 days per week for 4 weeks (total cumulative doses of 192 kJ/m² UV-B, 2.04 × 10³ kJ/m² UV-A) did not suppress the anole's acute or delayed type hypersensitivity (DTH) response to horseshoe crab hemocyanin. In comparison with the available literature UV-B doses as low as 0.1 and 15.9 kJ/m² induced suppression of DTH responses in mice and humans, respectively. Exposure of anoles to UVR did not result in the inhibition of ex vivo splenocyte phagocytosis of fluorescein labeled Escherichia coli or ex vivo splenocyte nitric oxide production. Doses of UV-B ranging from 0.35 to 45 kJ/m² have been reported to suppress murine splenic/peritoneal macrophage phagocytosis and nitric oxide production. These preliminary studies demonstrate the resistance of green anoles to UVR-induced immunosuppression. Methanol extracts of anole skin contained two peaks in the ultraviolet wavelength range that could be indicative of photoprotective substances. However, the resistance of green anoles to UVR is probably not completely attributable to absorption by UVR photoprotective substances in the skin but more likely results from a combination of other factors including absorption by the cutis and absorption and reflectance by various components of the dermis.

The single-cell gel/comet assay is an electrophoretic technique used to detect single-strand breaks in DNA. Damage is assessed examining individual cells under an epifluorescent microscope. UV-induced DNA damage consists mostly of the formation of pyrimidine dimers; therefore, most of the damage cannot be detected using a standard comet assay. The enzyme T4 endonuclease V breaks DNA strands at sites of pyrimidine dimers. The main objective of this work is to evaluate the comet assay to detect UV-induced damage in DNA after an initial treatment of cells with T4 endonuclease V. This work was conducted on Rhodomonas sp. (Cryptophyta), a marine unicellular flagellate. Cells of Rhodomonas sp. were exposed to 12 h visible ultraviolet-A ultraviolet-B (VIS UVA UVB) and VIS (control), with and without T4 endonuclease V. Cells exposed to VIS UVA UVB showed approximately 200% more damage than control if these were treated with T4 endonuclease V. Rhodomonas sp. were exposed to 3, 6, 9 and 12 h of VIS, VIS UVA and VIS UVA UVB. Damage induced by VIS UVA UVB as detected by the comet assay increased along with exposure time. However, damage caused by VIS and VIS UVA remained relatively constant at all times. Results of this study indicate that the comet assay is more sensitive to UV radiation damage when used in conjunction with T4 endonuclease V. This modification of the comet assay can be used as an alternative technique to detect DNA damage in single cells caused by UV radiation.

The ultraviolet A (320–400 nm) (UVA) exposure of sunscreen-protected skin depends not just on the absorption characteristics of the product but also on a number of other factors. These include the amount of sunscreen applied and how it is spread over the skin. The importance of the spectral absorption of a sunscreen compared with these other two variables in controlling cutaneous UVA exposure is examined here using an analysis of variance approach. The results demonstrate that by far the most important factor is the application of a liberal quantity of sunscreen. Less important is to spread it uniformly, and least important is the precise shape of the sunscreen-absorption spectrum, providing, of course, the spectrum extends into the UVA region.

Carotenoids are well-known physical quenchers of chlorophyll excited states and reactive oxygen species both in vivo and in vitro. They may also be involved in chemical quenching undergoing, e.g. isomerizations or oxidations. We have found that β-carotene (Car) in aerobic acetone is rapidly oxygenated under strong illumination with red light (λ_exc ≥ 630 nm) in the presence of bacteriopheophytin a. At the same time the photosensitizer undergoes only slight (<10%) photodegradation. By preparative high-performance liquid chromatography as many as seven major products of oxygen attachment to Car have been isolated. Their molecular masses show that Car sequentially accumulates up to six oxygen atoms while its C₄₀-skeleton remains intact.

Self-aggregation of naturally occurring bacteriochlorophyll (BChl)-e in nonpolar organic solvents was investigated by visible absorption, fluorescence emission and circular dichroism spectra. Cultured brown-colored photosynthetic bacteria have several BChl-e as light-harvesting antenna pigments. Three major BChl-e homologs were separated from the extracts of the culture by reverse-phase high-performance liquid chromatography (HPLC) and characterized by ¹H-NMR and fast-atom bombardment mass spectroscopy: 8-ethyl-12-ethyl ([E,E])-, 8-propyl-12-ethyl- and 8-isobutyl-12-ethyl-BChl-e farnesyl esters. All the homologs consisted of a mixture of the 3¹-epimers, and epimerically pure BChl-e were also given by HPLC separation. All the separated BChl-e epimers, the epimeric mixtures and the homologous mixtures formed self-aggregates in 2% dichloromethane/hexane, giving visible absorption spectra similar to that of the whole cells, which showed two peaks (or shoulders) around 430–450 and 520 nm at the Soret region as well as a red-shifted Q_y band relative to the monomeric. The spectral properties of the Soret band were basically unchanged among the epimers or epimeric/homologous mixtures. In contrast, the Q_y band of aggregates of epimeric mixtures (except [E,E]) and homologous mixtures red-shifted and broadened compared with the epimerically pure. The red-shift and broadening of the Q_y band are advantageous for efficient energy transfer from BChl-e aggregates to BChl-a in a baseplate in chlorosomes because their spectral overlap increases.

Photoacoustic measurements using a broad frequency band hydrophone were carried out in photosynthetic reaction centers (RC) isolated from Rhodobacter sphaeroides R-26 purple bacteria. Data were extracted on enthalpy and volume changes accompanying the primary steps after excitation in the range of 0–500 μs aimed at further characterizing the thermodynamic properties of the RC. Quinone titration showed that the volume contraction accompanying the electron transport is sensitive to the molecular species occupying the secondary quinone site. ΔV_M = 14.4, 7.7 and 4.3 cm³ molar volume contractions were calculated from the measured parameters for 1, 2 and 0.07 quinone/RC after light excitation. Comparing the enthalpy changes (ΔH) to the Gibbs free energy data in the literature, a rather large (26%) entropic contribution to the free energy changes (ΔG) is estimated for the P*Q_AQ_B → P Q_A⁻Q_B electron transport (where Q_A and Q_B represent primary and secondary quinones, respectively). This is in contrast to previous estimations that ΔG = ΔH in these processes. On the other hand, only a small (4%) entropic contribution to the ΔG of the P*Q_AQ_B → P Q_AQ_B⁻ process is estimated, in agreement with the literature data. Our results are in good agreement with the data obtained earlier (Edens et al. [2000] J. Am. Chem. Soc. 122, 1479–1485).

Ultraviolet radiation (UVR) is known to suppress immune responses in human subjects. The purpose of this study was to develop dose responses across a broad range of skin pigmentation in order to facilitate risk assessment. UVR was administered using FS 20 bulbs. Skin pigmentation and UVR sensitivity were evaluated using Fitzpatrick classifications, minimal erythemal dose (MED), slope of the erythemal dose response curve (sED), baseline pigmentation and tanning response. To assess immune responses dinitrochlorobenzene (DNCB) was applied to irradiated buttock skin 72 h after irradiation. Two weeks later DNCB was applied to the inside upper arm. Skin thickness was measured before and after challenge. Dose response was modeled (to obtain a regression line) for the entire group of 185 subjects. With the exception of sED none of the above-mentioned pigmentation indicators contributed significantly to variability around the regression line. Thus, differences in sensitivity for multiple skin types based on Fitzpatrick classification or MED were not observed. However, differences in immune sensitivity to UVR were detected between subjects with steep erythemal dose response curves and those with moderate or flat responses. For subjects with steep erythemal responses the dose calculated to suppress the immune response by 50% was 114 mJ/cm². This group included individuals with Fitzpatrick skin types I–V, MED for these subjects ranged from 30 to 80 mJ/cm². The 50% suppression dose for subjects with weak or no erythemal response could not be computed (the dose response was flat). This resistant group included subjects with skin types IV–VI and MED for these subjects ranged from 41 to >105 mJ/cm². This study provides a human dose response for UVR suppression of contact sensitivity that will be useful in risk assessment. It is the first study to provide this information using the FS sun lamp and is the first study to include people of color. The sED appears to be a new variable for identifying sensitive subjects at risk of UVR-induced immune suppression.

Fluorescence excitation spectroscopy was used to assess cellular turnover in human skin by monitoring changes of endogenous fluorescence. Epidermal proliferation was induced with α-hydroxy acids. Commercially available glycolic acid creams (8 and 4% wt/wt concentration) and a vehicle cream (placebo) were applied in a randomized double blinded fashion on subjects' forearms, twice daily for 21 days. Excitation spectra were recorded (excitation 250–360 nm, emission 380 nm) at days 0, 1, 3, 7, 10, 11, 14, 17 and 21. The 295 nm excitation band (assigned to tryptophan moieties) was used in this study as a marker for cellular proliferation. To further reduce the day-to-day variability of the skin fluorescence the intensity of the 295 nm band was normalized to the 334 nm band (assigned to collagen crosslinks). The fluorescence emission intensity from placebo-treated skin remained practically unchanged over the period of the measurements while the fluorescence intensity measured from the glycolic acid–treated skin increased monotonically with treatment. The rate of increase of the excitation intensity with treatment was found to be dose dependent. The epidermal 295 nm band may be used as a quantitative marker to monitor the rate of proliferation of epidermal keratinocytes noninvasively.

Strawberry (Fragaria ananassa) fruit size and flavor are important to both growers and consumers. Plastic mulches are frequently used in raised-bed culture to conserve water, control weeds with less herbicides, keep fruit clean and produce ripe berries earlier in the season. The most commonly used plastic mulch color is black. We hypothesized that changing mulch color to reflect more far-red (FR) and red light (R) and a higher FR/R photon ratio would keep those benefits and improve berry size and flavor by altering phytochrome-mediated regulation of pathways in ripening berries. Size and chemical composition of berries developed in sunlight over a specially formulated red plastic were compared with those that developed over standard black plastic mulch. Berries that ripened over red were about 20% larger, had higher sugar to organic acid ratios and emitted higher concentrations of favorable aroma compounds. We conclude that FR and the FR/R ratio in light reflected from the red mulch on the soil surface acted through the natural phytochrome system within the growing plants to modify gene expression enough to result in increased fruit size and improved concentrations of phytonutrient, flavor and aroma compounds.

We have developed a model of cutaneous herpes simplex virus-1 (HSV-1) reactivation in SKH-1 hairless mice which closely mimics the condition in humans. Sixty plaque-forming units of HSV-1 strain 17 syn were applied to a superficially abraded area on the lateral body wall. More than 85% of mice developed primary HSV-1 infection characterized by a zosteriform pattern of cutaneous vesiculation and ulceration. Approximately one-third of mice with primary skin lesions succumbed to neurologic disease and in the remaining mice cutaneous lesions healed completely. Subsequent exposure of healed areas to two minimal inflammatory doses of UV resulted in recrudescence of skin lesions in the irradiated areas in almost 60% of mice. Lesions appeared approximately 4 days after irradiation, persisted for 3–5 days and then resolved completely. Reactivation rarely resulted in death due to neurologic disease. Primary lesions had a histologic appearance typical of cutaneous HSV-1 infection with vesicles and focal epithelial necrosis accompanied by the formation of epithelial syncytial cells and the presence of herpetic intranuclear inclusion bodies. In primary lesions HSV-1 was demonstrated by immunohistochemistry, polymerase chain reaction and culture. In reactivated lesions epithelial syncytia and inclusion bodies were not seen; however, virus was demonstrable by polymerase chain reaction and culture. Exposure of the uninfected side to UV did not stimulate disease recurrence suggesting that local effects of UV rather than systemic immunosuppression were responsible for reactivation. Reactivation could also be obtained with two minimal inflammatory doses of UV from a UV-340 light source which emits light approximating the solar spectrum.