In this work we have tried to evaluate the usefulness of 1-naphthol as an excited state proton transfer fluorescent probe for studying the ethanol-induced interdigitation in lipid bilayer membranes. When ethanol concentration in lipisome is progressively increased, the neutral form fluorescence of 1-naphthol is found to decrease with corresponding increase in the anionic form intensity. This behavior is in contrast to that observed in the absence of lipid where a reverse effect is noticed. Modification of lipid bilayer is known to occur in the presence of ethanol, which increases the packing density of the membrane. Due to this induction of interdigitated gel phase, redistribution of naphthol between the inner core and interfacial region of the lipid bilayer takes places, accounting for the reduction in neutral form fluorescence intensity. The partition coefficient values and the quenching studies also support the redistribution of 1-naphthol in the liposome membrane. The neutral form fluorescence of 1-naphthol successfully monitors the shift in phase transition temperature due to ethanol-induced interdigitation. It also explains the prevention of interdigitation in lipid bilayer at high cholesterol concentration.

M-DNA is a novel duplex conformation in which metal ions such as Co² , Ni² or Zn² replace the imino protons of every base pair. An ethidium fluorescence assay was used to estimate lesions in M-DNA induced by γ- and UV radiation. General damage to DNA was assessed from the loss of ethidium fluorescence after irradiation of calf thymus DNA. Crosslinks were measured from the return of ethidium fluorescence after a heating and cooling step. Strand breaks were estimated from the loss of fluorescence in covalently closed circular plasmid DNA after a heating and cooling step. For the Co² form of M-DNA, γ-radiation caused the very efficient formation of crosslinks which was not observed with B-DNA nor with the Ni² or Zn² forms of M-DNA. The crosslinks occurred in both A–T and G–C base pairs but did not form in the presence of a free radical scavenger. Crosslinks induced by UV radiation also formed at a faster rate in the Co² , Ni² and Zn² forms of M-DNA compared to B-DNA; crosslinking occurred in all DNA but was more prominent in AT-rich sequences and was not inhibited by a free radical scavenger. Therefore, the presence of certain metal ions may lead to large increases in the formation of radiation-induced crosslinks in DNA.

The absorption and fluorescence spectra, fluorescence quantum yields, lifetimes and time-resolved fluorescence spectra are reported for nine different fluorescent DNA-dyes. The work was initiated in search of a quantitative method to detect the ratio of single-to-double stranded DNA (ssDNA/dsDNA) in solution based on the photophysics of dye–DNA complexes; the result is a comprehensive study providing a vast amount of information for users of DNA stains. The dyes examined were the bisbenzimide or indole-derived stains (Hoechst 33342, Hoechst 33258 and 4′,6-diamidino-2-phenylindole), phenanthridinium stains (ethidium bromide and propidium iodide) and cyanine dyes (PicoGreen, YOYO-1 iodide, SYBR Green I and SYBR Gold). All were evaluated under the same experimental conditions in terms of ionic strength, pH and dye–DNA ratio. Among the photophysical properties evaluated only fluorescence lifetimes for the cyanine stilbene dyes allowed a convenient differentiation between ssDNA and dsDNA. The bisbenzimide dyes showed multiexponential decays when bound to either form of DNA, making lifetime-based analysis cumbersome with inherent errors. These dyes also presented biexponential decay when free in aqueous buffered solutions at different pH. A mechanism for their deactivation is proposed based on two different conformers decaying with different kinetics. The phenanthridinium dyes showed monoexponential decays with ssDNA and dsDNA, but there was no discrimination between them. High dye–DNA ratios (e.g. 1:1) resulted in multiexponential decays for cyanine dyes, resulting from energy transfer or self-quenching deactivation. Shifts in both absorption and fluorescence maxima for both ssDNA and dsDNA DNA–cyanine dye complexes were small. Broadening of dye–ssDNA absorption and fluorescence bands for the cyanine dyes relative to dye–dsDNA bands was detected and attributed to higher degrees of rotational freedom in the former.

Sunscreens penetrate human epidermis and modify the biology of proliferating cells. This study addressed the question whether the UV response of cultured human cells is affected by direct treatment with nontoxic levels of sunscreens. Cell survival following exposure to UVC or unfiltered UVB was not altered by preincubation with 25 μg/mL of octyl p-dimethylaminobenzoate (o-PABA), 2-ethylhexyl p-methoxycinnamate (EHMC) or oxybenzone. However, UVA or UVB filtered to reproduce the solar UV spectrum penetrating to the basal layer of the epidermis, highly sensitized cells to killing by o-PABA but not by its hydrolysis product, 4-dimethylaminobenzoic acid. Sensitization was found in all cell types tested, except normal keratinocytes, and could be prevented by certain antioxidants particularly pyruvate and the hydroxyl radical scavenger mannitol. o-PABA and EHMC applied without UV reduced the adherence of cells. The results indicate that sunscreens may increase cell mobility and the combination of o-PABA with solar UV may selectively damage melanocytes in the skin.

The inhibitory processes operating when a 2(-2′-hydroxyphenyl) benzotriazole-based ultraviolet absorber (UVA) and a hindered nitroxide free radical are applied to high-yield pulps were studied using milled wood lignin and filter paper as a model. Using quantitative ³¹P NMR it was determined that the UVA is almost completely photostable during irradiation, suggesting that its protective mechanism is based primarily on ultraviolet absorption. Furthermore, the nitroxide was found to protect the UVA from photodegradation. Analysis of irradiated lignin samples involved derivatization followed by reductive cleavage of β-arylether groups. The phenolic-OH groups thus released were quantified using ³¹P NMR. The benzotriazole/nitroxide stabilizing system was found to cooperatively inhibit the cleavage of β-arylether groups. Furthermore, this system was found to have a synergistic inhibitory effect on the formation of catechol structures. These results suggest that the synergism observed between benzotriazole-based UV screens and nitroxyl radicals on the inhibition of yellowing could be a result of their ability to slow the formation of catechol structures and β-O-4 cleavage in addition to the possibility of a UVA-regeneration mechanism.

The alga Chlamydomonas nivalis lives in a high-light, cold environment: persistent alpine snowfields. Since the algae in snow receive light from all angles, the photon fluence rate is the critical parameter for photosynthesis, but it is rarely measured. We measured photon irradiance and photon fluence rate in the snow that contained blooms of C. nivalis. On a cloudless day the photon fluence rate at the snow surface was nearly twice the photon irradiance, and it can be many times greater than the photon irradiance when the solar angle is low or the light is diffuse. Beneath the surface the photon fluence rate can be five times the photon irradiance. Photon irradiance and photon fluence rate declined exponentially with depth, approximating the Bouguer–Lambert relationship. We used an integrating sphere to measure the spectral characteristics of a monolayer of cells and microscopic techniques to examine the spectral characteristics of individual cells. Astaxanthin blocked blue light and unknown absorbers blocked UV radiation; the penetration of these wavelengths through whole cells was negligible. We extracted astaxanthin, measured absorbance on a per-cell basis and estimated that the layer of astaxanthin within cells would allow only a small percentage of the blue light to reach the chloroplast, potentially protecting the chloroplast from excessive light.

The UV doses of Americans were never measured, but are needed for assessing the risks of UV-related health effects. We calculated these doses using a novel approach. The Environmental Protection Agency's (EPA) National Human Activity Pattern Survey (NHAPS) recorded the activity profiles of 9386 Americans over 24 months to assess their exposure to environmental pollutants, one of which is UV radiation. NHAPS used randomized telephone interviews to get their previous day's minute-by-minute activities. From NHAPS we extracted only the outdoor-daylight data of the northern and southern indoor workers (95%), stratifying by season, sex and age (0–21, 22–40, 41–59 and 60 years) to find the average time Americans spend outdoors. Knowing the total daylight time and that while outdoors Americans are exposed to about 30% of the available solar UV (on a horizontal plane), we calculated their percent ambients. The average American's percent ambients are 2.6 and 2.5% for northern and southern females, respectively, and 3.5 and 3.6% for northern and southern males, respectively. Men over 40 years of age have the highest ambients (4%). From their ambients we calculated their annual doses using seasonal averages of UV measurements taken daily for over 2 years by EPA Brewer spectrophotometers located in four quadrants of the United States: Atlanta, GA; Boston, MA; Bozeman, MT and Riverside, CA. The average erythemal UV doses of Americans are about 25 000 J/m²/year, 22 000 for females and 28 000 for males, or 33 000 J/m²/year including a conservative continental U.S. vacation (7800 J/m²). Thus, we can now assess the risks of UV-related health effects for Americans.

A study of the photocatalytic production of molecular hydrogen from platinized photosystem I (PSI) reaction centers is reported. At pH 7 and room temperature metallic platinum was photoprecipitated at the reducing end of PSI according to the reaction, [PtCl₆]²⁻ 4e⁻ hν → Pt↓ 6Cl⁻, where it interacted with photogenerated PSI electrons and catalyzed the evolution of molecular hydrogen. The reaction mixture included purified spinach PSI reaction centers, sodium ascorbate and spinach plastocyanin. Experimental data on real-time catalytic platinum formation as measured by the onset and rates of hydrogen photoevolution as a function of time are presented. The key objective of the experiments was demonstration of functional nanoscale surface metalization at the reducing end of isolated PSI by substituting negatively charged [PtCl₆]²⁻ for negatively charged ferredoxin, the naturally occurring water-soluble electron carrier in photosynthesis. The data are interpreted in terms of electrostatic interactions between [PtCl₆]²⁻ and the positively charged surface of psaD, the ferredoxin docking site situated at the stromal interface of the photosynthetic membrane and which is presumably retained in our PSI preparation. A discussion of the rates of hydrogen evolution in terms of the structural components of the various PSI preparations as well as of those of the intact thylakoid membranes is presented.

Fluorescence spectroscopy offers an effective, noninvasive approach to the detection of precancers in multiple organ sites. Clinical studies have demonstrated that fluorescence spectroscopy can provide highly sensitive, specific and cost-effective diagnosis of cervical precancers. However, the underlying biochemical mechanisms responsible for differences in the fluorescence spectra of normal and dysplastic tissue are not fully understood. We designed a study to assess the differences in autofluorescence of normal and dysplastic cervical tissue. Transverse, fresh tissue sections were prepared from colposcopically normal and abnormal biopsies in a 34-patient study. Autofluorescence images were acquired at 380 and 460 nm excitation. Results showed statistically significant increases in epithelial fluorescence intensity (arbitrary units) at 380 nm excitation in dysplastic tissue (106 ± 39) relative to normal tissue (85 ± 30). The fluorophore responsible for this increase is possibly reduced nicotinamide adenine dinucleotide. Stromal fluorescence intensities in the dysplastic samples decreased at both 380 nm (102 ± 34 [dysplasia] vs 151 ± 44 [normal]) and 460 nm excitation (93 ± 35 [dysplasia] vs 137 ± 49 [normal]), wavelengths at which collagen is excited. Decreased redox ratio (17–40% reduction) in dysplastic tissue sections, indicative of increased metabolic activity, was observed in one-third of the paired samples. These results provide valuable insight into the biological basis of the differences in fluorescence of normal and precancerous cervical tissue.

A novel laser system has been developed to study the effects of multiple laser pulses of differing wavelengths on cutaneous blood vessels in vivo, using the hamster dorsal skin flap preparation and in vitro, using cuvettes of whole or diluted blood. The system permits sequenced irradiation with well-defined intrapulse spacing at 532 nm, using a long-pulse frequency-doubled Nd:YAG laser, and at 1064 nm, using a long-pulse Nd:YAG laser. Using this system, we have identified a parameter space where two pulses of different wavelengths act in a synergistic manner to effect permanent vessel damage at radiant exposures where the two pulses individually have little or no effect. Using a two-color pump–probe technique in vitro, we have identified a phenomenon we call green-light–induced infrared absorption, where a pulse of green light causes photochemical and photothermal modifications to the chemical constituents of blood and results in enhanced infrared absorption. We identify a new chemical species, met-hemoglobin, not normally present in healthy human blood but formed during laser photocoagulation which we believe is implicated in the enhanced near-infrared absorption.

Our approach to examine the mechanism(s) of action for photodynamic therapy (PDT) has been via the generation of PDT-resistant cell lines. In this study we used three human cell lines, namely, human colon adenocarcinoma (HT29), human bladder carcinoma and human neuroblastoma. The three photosensitizers used were Photofrin, Nile Blue A and aluminum phthalocyanine tetrasulfonate. The protocol for inducing resistance consisted of repeated in vitro photodynamic treatments with a photosensitizer to the 1–10%-survival level followed by regrowth of single surviving colonies. Varying degrees of resistance were observed. The three induced variants of the HT29 cell line were the most extensively studied. Their ratios of increased survival at the LD90 level range between 1.5- and 2.62-fold more resistant.

Repeated exposure to solar ultraviolet radiation results in premature skin aging due, in part, to the degradation of dermal collagen by fibroblast collagenase (matrix metalloproteinase 1 [MMP-1]). We have established TaqMan^™ reverse transcription (RT) polymerase chain reaction (PCR) systems to quantify the messenger RNA (mRNA) expression of MMP-1 and its specific inhibitor TIMP-1 in human buttock skin exposed in vivo to solar simulated radiation (SSR). A time-course study (n = 6) with two minimal erythema doses (MED) of SSR showed maximal induction of MMP-1 and TIMP-1 at 24 h. A dose–response study (n = 6) sampled at 24 h revealed that doses of about 1 MED were necessary to induce expression of MMP-1 mRNA, and our data suggest that the response is saturated at about 2 MED. We also investigated SSR-induced gene expression in the dermis and epidermis separately (n = 5). MMP-1 was present in both tissues, but TIMP-1 was only detected in the dermis. In general, we could only measure MMP-1 mRNA in the nonirradiated control skin of volunteers who were smokers. We hypothesize very large interpersonal variation with MMP-1 induction compared with TIMP-1 which was detected in all the control sites. This suggests a lack of relationship between MMP-1 and TIMP-1 mRNA expression. The large donor variability for MMP-1 in all the studies demonstrates that it is important to analyze gene expression individually.

Clinical interest in laser-induced fluorescence (LIF) spectroscopy and photodynamic therapy (PDT) is growing rapidly and may ultimately lead to close parallel use of these techniques. However, variations in LIF due to photosensitizer retention as well as tissue damage and healing processes may interfere with autofluorescence-based diagnostic methods. We have investigated the compatibility of these two techniques by quantifying PDT-induced changes in LIF in the human esophagus. Fluorescence spectra were collected endoscopically at excitation wavelengths (λ_ex) of 337, 400 and 410 nm in 32 patients. Measurements were performed immediately before and after PDT treatment with porfimer sodium and during follow-up procedures. In the months following PDT regions of reepithelialized squamous showed reduced autofluorescence in comparison with untreated squamous regions (P = 0.0007). Photosensitizer fluorescence was undetectable with λ_ex = 337 nm during follow-up procedures, whereas for λ_ex = 400 and 410 nm porfimer sodium fluorescence was noted for nearly a year after treatment. Therefore, residual photosensitizer fluorescence is likely to affect certain LIF-based diagnostic techniques during a period when patients are at high risk for tumor recurrence. Modification of LIF systems and/or the use of alternative photosensitizers may be required to optimize the detection of lesions in the post-PDT patient. Given the potential of LIF as a method for surveillance following cancer therapy, further investigation of the compatibility of specific LIF approaches with cancer pharmaceuticals may be warranted.

Benzoporphyrin-derivative (BPD)–monoacid-ring A photodynamic therapy (PDT) was performed on subcutaneous tumor implants in a rat ovarian cancer model. In order to assess PDT efficacy the tumor and normal tissue optical properties were measured noninvasively prior to and during PDT using frequency-domain photon migration (FDPM). FDPM data were used to quantify tissue absorption and reduced scattering properties (given by the parameters μ_a and μ′_s, respectively) at four near-infrared (NIR) wavelengths (674, 811, 849 and 956 nm). Tissue physiologic properties, including the in vivo concentration of BPD, deoxy-hemoglobin (Hb), oxy-hemoglobin (HbO₂), total hemoglobin (TotHb), water (H₂O) and percent tissue hemoglobin oxygen saturation (%S_tO₂), were calculated from optical property data. PDT efficacy was also determined from morphometric analysis of tumor necrosis in histologic specimens. All the measured tumor properties changed significantly during PDT. [Hb] increased by 9%, while [HbO₂], [TotHb] and %S_tO₂ decreased by 18, 7 and 12%, respectively. Using histologic data we show that long-term PDT efficacy is highly correlated to mean BPD concentration in tumor and PDT-induced acute changes in [HbO₂], [TotHb] and %S_tO₂ (correlation coefficients of 0.829, 0.817 and 0.953, respectively). Overall, our results indicate that NIR FDPM spectroscopy is able to quantify noninvasively and dynamically the PDT-induced physiological effects in vivo that are highly correlated with therapeutic efficacy.

Many plant species are able to acclimate to changes in ultraviolet-B radiation (UVB) (290–320 nm) exposure. Due to the wide range of targets of UVB, plants have evolved diverse repair and protection mechanisms. These include increased biosynthesis of UVB screening compounds, elevated antioxidant activity and increased rates of DNA repair. We have shown previously that Brassica napus L. cv Topas plants can acclimate quite effectively to environmentally relevant increases in UVB through the accumulation of specific flavonoids in the leaf epidermis. However, B. napus was found to lose other flavonoids when plants are exposed to ultraviolet-A radiation (UVA) (320–400 nm) and/or UVB (Wilson et al. [1998] Photochem. Photobiol. 67, 547–553). In this study we demonstrate that the levels of all the extractable flavonoids in the leaves of B. napus plants are decreased in a dose-dependent manner in response to UVA exposure. Additionally, the accumulation of the extractable flavonoids was examined following a shift from photosynthetically active radiation (PAR) UVA to PAR UVB to assess if preexposure to UVA affected UVB-induced flavonoid accumulation. UVA preexposures were found to impede UVB-induced accumulation of some flavonoids. This down regulation was particularly evident for quercetin-3-O-sophoroside and quercetin-3-O-sophoroside-7-O-glucoside, which is interesting because quercetins have been demonstrated to be induced by UVB and correlated with UVB tolerance in some plant species. The photobiological nature of these UVA-mediated effects on flavonoid accumulation implies complex interactions between UVA and UVB responses.

The purpose of this study was to quantitatively study the changes that occur upon irradiation of 3-hydroxykynurenine (3-HK) in the presence of α-crystallin under conditions similar to those in the lens. The samples were prepared in 10 mM phosphate buffer at pH 7.4, bubbled with O₂ or Ar and irradiated with 300–400 nm light. The amount of light absorbed by the samples (I_abs) was measured using azobenzene as an actinometer. Modifications to α-crystallin were monitored by ultraviolet–visible and fluorescence spectroscopy. Aerobic samples had increased absorption around 320 nm and above 400 nm while the 3-HK maximum at 368 nm decreased. The isolated modified protein showed that there was increased absorption throughout the spectrum. Changes in the anaerobic samples were similar to those of the aerobic but occurred more slowly. As irradiation time increased fluorescence emission of the isolated protein red shifted and quantum yields of fluorescence (Φ_f) were calculated at different irradiation time intervals by comparison to 3-HK. By comparing OD₃₂₀/OD₃₆₅ for the model system to values from primate lenses, I_abs can be correlated with age and transmission of the sample in the blue region of the spectrum and thus allows lenticular aging to be quantitated.

Previously, two pools of phytochrome A (phyA′ and phyA″) have been detected by in situ low-temperature fluorescence spectroscopy and photochemistry; it was suggested that they might differ in the nature of their posttranslational modification. In order to verify this possibility Arabidopsis and rice (Oryza) phyA were expressed in yeast and the pigments were assembled in vivo with phycocyanobilin (PCB) and phytochromobilin (PΦB). The resulting recombinant phytochromes in the red-light–absorbing form (Pr) were characterized in the yeast cell by (1) the fluorescence emission spectra; (2) the temperature dependence of Pr fluorescence intensity and activation energy of fluorescence decay; and (3) the extent of photoconversion of Pr into photoproduct lumi-R (γ₁) or far-red–light absorbing form (Pfr) (γ₂). Both Arabidopsis phyA/PCB and Oryza phyA/PΦB had low γ₁ of ca 0.05, allowing their attribution to the Pr″ phenomenological type of phytochrome comprising phyA″, phyB and cryptogam phytochromes. The spectroscopic properties of Oryza phyA/PΦB were also very close to phyA″. However, both investigated holoproteins differed from phyA″, both with respect to the character of temperature dependence of the fluorescence yield and activation energy. Thus, recombinant Oryza phyA/PΦB is similar but not identical to phyA″. The data demonstrate that the low-abundance–fraction plant phyA (phyA″) comes from the same gene as the major (phyA′) fraction. Because both endogenous phyA fractions differ from the phytochrome expressed in yeast, they appear to be posttranslationally modified and/or bound to partner proteins or cellular substructures. However, the character of the presumed chemical modification is different in phyA′ and phyA″ and its extent is more profound in the case of the former.

Sporulation of the true slime mold Physarum polycephalum (Myxomycetales) can be triggered by the far-red/red reversible Physarum phytochrome. Physarum plasmodia were analyzed with a purpose-built dual-wavelength photometer that is designed for phytochrome measurements. A photoreversible absorbance change at 670 nm was monitored after actinic red (R) and far-red (FR) irradiation of starved plasmodia, confirming the occurrence of a phytochrome-like photoreceptor in Physarum spectroscopically. These signals were not found in growing plasmodia, suggesting the Physarum phytochrome to be synthesized during starvation, which makes the cells competent for the photoinduction of sporulation. The photoconversion rates by R and FR light were similar in the phytochromes of Physarum and etiolated oat shoots. In dark-grown Physarum plasmodia that had not been preexposed to any light only R induced a detectable absorbance change while FR did not. This indicates that most (at least 90%) of the photoreversible pigment occurs in the red-absorbing form. Since the effectiveness of FR in triggering sporulation was enhanced by preirradiation with R, it is concluded that at least part of the P_r can be photoconverted to the active P_fr photoreceptor species. We propose a kinetic mechanism for the photocontrol of sporulation by photoconversion of P_fr, which may also hold for the high-irradiance response to FR in Arabidopsis and Cuscuta.

Remarkable rates of oxygen consumption are observed via microelectrode measurements immediately upon the onset of 325 nm irradiation of multicell tumor spheroids. Consumption is irradiance dependent over the range 20–200 mW cm⁻², and its magnitude is comparable to that observed previously in the same system using exogenous photosensitizers. Oscillations in the oxygen concentrations suggest that oxygen is also being evolved during irradiation. Oxygen evolution is likely the result of enzymatic dissociation of hydrogen peroxide, which is formed through UV-induced photochemistry. Irradiation of spheroids at 442 and at 514 nm produces a much more modest but detectable oxygen consumption. The dynamics of oxygen concentration changes are quite different at these wavelengths, suggesting a different photochemical mechanism. In these cases, initial oxygen depletion is followed immediately by a more gradual, monotonic increase in the oxygen concentration, consistent with irreversible photobleaching. No oscillations in the oxygen concentration are detectable. At 662 nm, no oxygen consumption was observed over the range of irradiances studied. Fluorescence spectra of cells prior to irradiation include contributions from anthranilic acid and reduced nicotinamide adenine dinucleotide (NADH). During 325 nm irradiation, anthranilic acid is rapidly and irreversibly bleached, while NADH emission undergoes only modest reduction.