bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of the Time‐resolved Absorption and Phosphorescence from the Tryptophan Triplet State in Proteins in Solution
Authors: Gershenson Anne , Gafni Ari , Steel Duncan
Published: January 2008

Measurement of the room temperature Trp triplet state lifetime in proteins by time‐resolved phosphorescence can provide valuable information on the structure and dynamics of proteins in solution. Our time‐resolved absorption measurements on the long‐lived states resulting from electronic excitation of the chromophore demonstrate the presence of more complex behavior than revealed by time‐resolved phosphorescence. To provide additional insight into this behavior, a comparative study of time‐resolved transient absorption and time‐resolved phosphorescence of proteins in solution was carried out. The results show that the time evolution of the long‐lived states observed through transient absorption often differs considerably from that observed in time‐resolved phosphorescence. In some proteins, the presence of competing reactions complicates the interpretation of the transient absorption measurements (which may affect the phosphorescence yield). A more complete characterization of these processes will likely prove useful in the study of protein structure and dynamics in solution.
Flavodoxin as an Electronic Donor in Photosynthetic Inorganic Nitrogen Assimilation by Iron‐deficient Chlorella fusca Cells
Authors: Vigara A. Javier , Inda Luis A. , Vega José M. , Gómez‐Moreno Carlos , Peleato M. Luisa
Published: January 2008

Two ferredoxin‐dependent enzymes involved in the photosynthetic assimilation of nitrogen, nitrite reductase (EC 1.7.7.1) and glutamate synthase (EC 1.4.7.1) from Chlorella fusca have been shown to use either ferredoxin or flavodoxin (from the same organism) as electron donor. Nitrite reductase showed Km, values of 13.8 ± 0.9 μM for ferredoxin and 30.4 ± 7.8 μM for flavodoxin. Glutamate synthase exhibited a Km of 6.6 ± 1.2 μM for ferredoxin compared to a calculated Km of 22.4 ± 6.4 μM for flavodoxin.
Sensitization of Peroxynitrite Chemiluminescence by the Triplet Carbonyl Sensitizer Coumarin‐525. Effect of CO2
Authors: Sharov Victor S. , Driomina Elena S. , Briviba Karlis , Sies Helmut
Published: January 2008

Abstract— The flash of spontaneous chemiluminescence (CL) that reflects the formation of electronically excited intermediates during the decay of peroxynitrite (ONOO) to nitrate was investigated. The half‐decay time of the CL flash (0.5 ± 0.1 s) was in agreement with the half‐life of peroxynitrite obtained in stopped‐flow experiments. The spontaneous CL intensity was linearly dependent on peroxynitrite concentration. The yield of spontaneous CL from peroxynitrite decay, 2 × 10‐9 photons/peroxynitrite at pH 9.5, was strongly enhanced by a sensitizer of triplet carbonyl CL, coumarin‐525 (C‐525). The maximal yield of sensitized CL was calculated to be 3 × 10‐6 photons/ peroxynitrite molecule for infinite concentration of C‐525. The dependence of both spontaneous and sensitized CL on pH has a maximum at about pH 9.5. Bubbling with CO2 or addition of NaHCO3 considerably enhanced the flash of CL, and it is concluded that the reaction of the peroxynitrite anion with CO2 is a major pathway leading to the formation of an electronically excited intermediate of peroxynitrite.
PUVA‐Induced Cell Mortality in NCTC 2544 Keratinocytes: Is It Related to the Microenvironmental Properties of the Excited States of Psoralens?
Authors: Sousa Cristina , Melo Teresa Sá e , Mazière Jean‐Claude , Santus René
Published: January 2008

The phototoxic effect of psoralen (PSO), 5‐methoxypsoralen (5MOP), 8‐methoxypsoralen (8MOP) and 4,5′,8‐trimethylpsoralen (TMP) has been compared on the NCTC 2544 keratinocyte cell line in terms of cell mortality and lipid peroxidation. The order of effectiveness for cell photokilling is TMP, 5MOP >> 8MOP, PSO, whereas a little lipid peroxidation is observed for the four psoralens under study. Oxygen‐independent membrane damage seem to play a key role in the lethal photodamage because the biological effectiveness of the most hydrophobic lipid‐soluble psoralens, TMP and 5MOP, is about an order of magnitude higher than that of the more water‐soluble 8MOP and PSO. In relation to this hypothesis, and in contrast to 8MOP, TMP is readily extracted from cells by ethyl acetate, a good membrane solvent, as shown by GC/MS analysis on cell extracts. The results are discussed in terms of the highly microenvironment‐dependent photophysical properties of psoralens. By the measure of the intracellular psoralen concentration, the neutral red uptake and the lipid peroxidation products, this work provides evidence that PUVA therapy‐mediated cell mortality is a lipid peroxidation‐independent phenomenon.
A Resin‐bound Photosensitizer for Aqueous Photooxidations
Authors: Prat Ferran , Foote Christopher S.
Published: January 2008

A new resin‐bound photosensitizer for aqueous photooxidations has been prepared. This product allows the sensitizer to be easily removed from the solution by a simple filtration. Its efficiency has been tested with two different substrates.
Protection Against UV‐Induced Reactive Intermediates in Human Cells and Mouse Skin by Glutathione Precursors: A Comparison of N‐Acetylcysteine and Glutathione Ethylester
Authors: Steenvoorden David P. T. , Hasselbaink Danny M. , van Henegouwen Gerard M. J. Beijersbergen
Published: January 2008

Because glutathione (GSH) plays a central part in the endogenous defense against UV radiation, an increase in GSH might provide photoprotection. Two agents that increase GSH levels were investigated. Cultured human cells and mouse skin were treated with N‐acetylcysteine (NAC) and glutathione ethylester (GSH‐Et). After 30 min, the GSH level was determined by HPLC. Photoprotection was assessed by testing the ability of the thiols to scavenge UV‐induced reactive intermediates in the same models. As compared to control cells, NAC and GSH‐Et increased intracellular GSH in vitro to maximally 144% and 174% respectively. In vitro protection (maximum 23% for NAC and 21% for GSH‐Et) did not correlate to the intracellular GSH level but to the concentration of the thiols in the medium. Invivo, epidermal GSH was increased to maximally 163% of the control level by NAC and 1234% by GSH‐Et. The maximum in vivo photoprotection provided by GSH‐Et was 55%, similar to what was found previously for NAC. Again, the protection seemed more closely correlated to the thiol dose than to the GSH level. The study showed that the protection against UV‐induced reactive intermediates depends on a general antioxidant action of these thiols, rather than only on their role as GSH precursors.
pH‐Dependent Excited‐State Properties of N,N‐di(2‐phosphonoethyl)‐1,4,5,8‐naphthalenediimide
Authors: Rodrigues Magali A. , Brochsztain Sérgio , Barros Teresa C. , Baptista Mauricio S. , Politi Mario J.
Published: January 2008

Abstract— The effect of the protonation state of a novel aqueous soluble N,N'‐di(2‐phosphonoethyl)‐l,4,5,8‐naphthalene‐diimide (DPN) on its spectroscopic properties is studied. In aqueous solution DPN depicts linear Lambert‐Beer plots up to the solubility limits at pH 1.0, 7.1 and 10.1, in accordance with its solubilization as a monomer. A small increase in the extinction coefficients with pH is observed. Conversely a strong decrease in the fluorescence emission yields are observed with the increase in pH. These results are shown to originate from the distinct efficiencies of the intersystem crossing of DPN as a function of the net charge on the phosphono moieties. Accordingly, from the decrease in fluorescence emission, titration curves were obtained resulting in two pKa of –1.5 and 6.7 in agreement with the potentiometric analysis. In parallel monitoring the amount of heat deposited in the system shows an inverse pattern, that is, increase in the thermal tensing time‐resolved signal with pH. Laser flash photolysis and thermal Iensing data in the absence and presence of oxygen show the interplay of an intersystem crossing process with the protonation state of the dye. The changes in protonation from neutral to two‐ and four‐fold charged species are accompanied by an enhancing effect on the naphthalenediimide ring toward spin inversion (triplet formation) and consequently in different fluorescence yields
Potentiation of Photodynamic Therapy Antitumor Activity in Mice by Nitric Oxide Synthase Inhibition Is Fluence Rate Dependent
Authors: Henderson Barbara W. , Sitnik‐Buscr Theresa M. , Vaughan Lurine A.
Published: January 2008

Abstract— The effects of systemic administration of the nitric oxide synthase (NOS) inhibitor NG‐nitro‐L‐arginine (L‐NNA) in combination with photodynamic therapy (PDT) on tumor response, tumor oxygenation and tumor and normal skin perfusion were studied in C3H mice bearing subcutaneous radiation‐induced fibrosarcoma tumors. Photodynamic therapy was carried out using the photosensitizer Photofrin®(5 mg/kg) in conjunction with a low fluence rate (30 mW/cm2) and a high fluence rate (150 raW/cm2) protocol at a total fluence of 100 J/cm2. Low fluence rate PDT produced –15% tumor cures, a response not significantly altered by administration of 20 mg/kg L‐NNA either 5 min before or after PDT. In contrast, high fluence rate PDT produced no tumor cures by itself, but addition of L‐NNA either pre‐ or post‐PDT resulted in –30% and ‐10% tumor cures, respectively. The L‐NNA by itself tended to decrease tumor pO2 levels and perfusion, but statistically significant differences were reached only at one time point (1 h) with one of the oxygenation parameters measured (% values < 2 mm Hg). Photodynamic therapy by itself decreased tumor oxygenation and perfusion more significantly. Addition of L‐NNA before PDT further potentiated this effect. The L‐NNA exerted its most striking effects on the PDT response of the normal skin microvasculature. Low fluence rate PDT caused severe and lasting shut‐down of skin microvascular perfusion. With high fluence rate PDT, skin perfusion was initially decreased but recovered to persistent normal levels within 1 h of treatment. Administration of L‐NNA reversed this response, converting it to complete and lasting vascular shut‐down identical to that achieved with low fluence rate PDT. This effect was somewhat L‐NNA dose dependent but was still marked at a dose of 1 mg/ kg. It occurred whether L‐NNA was given before or after PDT. The L‐NNA did not alter the long‐term vascular response of skin to low fluence rate PDT. The ability of L‐NNA to correspondingly improve tumor response and severely limit skin vascular perfusion following high fluence rate PDT, while providing no benefit for the low fluence rate protocol, suggests that vascular changes in the tumor surrounding normal tissue contribute to the enhanced tumor curability with adjuvant L‐NNA treatment.
Immunosuppressive Effects of Silicon Phthalocyanine Photodynamic Therapy
Authors: Reddan Julie C. , Anderson Cathy Young , Xu Hui , Hrabovsky Sharon , Freye Kristin , Fairchild Roberta , Tubesing Karen A. , Elmets Craig A.
Published: January 2008

Abstract— The purpose of this study was to determine if silicon phthalocyanine 4 (Pc 4), a second‐generation photosensitizer being evaluated for the photodynamic therapy (PDT) of solid tumors, was immunosuppressive. Mice treated with Pc 4 PDT 3 days before dinitrofluorobenzene sensitization showed significant suppression of their cell‐mediated immune response when compared to mice that were not exposed to PDT. The response was dose dependent, required both Pc 4 and light and occurred at a skin site remote from that exposed to the laser. The immunosuppression could not be reversed by in vivo pre‐treatment of mice with antibodies to tumor necrosis factor‐alpha or interleukin‐10. These results provide evidence that induction of cell‐mediated immunity is suppressed after Pc 4 PDT. Strategies that prevent PDT‐mediated immunosuppression may therefore enhance the efficacy of this therapeutic modality.
Computational Analysis of the Oxygen Addition at the C4a Site of Reduced Flavin in the Bacterial Luciferase Bioluminescence Reaction
Authors: Wada Naohisa , Sugimoto Tohru , Watanabe Haruo , Tu Shiao‐Chun
Published: January 2008

Abstract— The energetic characteristics of selected reaction steps in the bacterial luciferase‐catalyzed luminescence reaction were examined by computation using the MNDO‐PM3 method. Specifically, a three‐step model was proposed to account for the reaction between oxygen and reduced riboflavin 5'‐phosphate (1,5H2‐FMN) to generate first the 5‐hydroFMN‐4a‐peroxide (5H‐FMN‐4aOO−) and then the 5‐hydro‐4a‐hydroper‐oxyFMN (5H‐FMN‐4aOOH) intermediates. Lysine (Lys‐H+) and aspartate (Asp−) were chosen as representative catalytic residues involved in the protonation and deprotonation processes. Results show that deprotonation at the N, site of 1,5H2‐FMN by a basic amino acid residue at the luciferase active site would efficiently accelerate the reaction rate of O2 addition to form 5H‐FMN‐4aOO . The most favored site of oxygen attack is at the flavin C4a. With the aid of a catalytic acid group, the 5H‐FMN‐4aOO−so formed tends to undergo a spontaneous protonation reaction to yield the 5H‐FMN‐4aOOH.
The Effects of UV Radiation A and B on Diurnal Variation in Photosynthesis in Three Taxonomically and Ecologically Diverse Microbial Mats
Authors: Cockell Charles S. , Rothschild Lynn J.
Published: January 2008

Abstract— Photosynthetic primary production, the basis of most global food chains, is inhibited by UV radiation. Evaluating UV inhibition is therefore important for assessing the role of natural levels of UV radiation in regulating ecosystem behavior as well as the potential impact of stratospheric ozone depletion on global ecosystems. As both photosynthesis and UV fluxes are subject to diurnal variations, we examined the diurnal variability of the effect of UV radiation on photosynthesis in three diverse algal mats. In one of the mats (Cyanidium caldarium) a small mean decrease in primary productivity over the whole day occurred when both UVA and UVB were screened out. In two of the mats (Lyngbya aestuarii and Zygogonium sp.) we found a mean increase in the total primary productivity over the day when UVB alone was screened and a further increase when UVA and UVB were both screened out. Variations in the effects of UV radiation were found at different times of the day. This diurnal variability may be because even under the same solar radiation flux, there are different factors that may control photosynthetic rate, including nutritional status and other physiological processes in the cell. The results show the importance of assessing the complete diurnal productivity. For some of the time points the increase in the mean was still within the standard deviations in primary productivity, illustrating the difficulty in dissecting UV effects from other natural variations.
Effects of a High‐Affinity Antibody Fragment on DNA Polymerase Reactions Near a (6–4) Photoproduct Site
Authors: Kobayashi Hiroyuki , Sato Kousuke , Komatsu Yasuo , Morioka Hiroshi , Stewart Jon D. , Tsurimoto Toshiki , Ohtsuka Eiko
Published: January 2008

Abstract— Pyrimidine (6–4) pyrimidone photodimers are major photoproducts that have mutagenic and carcinogenic consequences. One major reason for these biological effects of (6–4) photoproducts may be base mispairing/DNA replication errors due to hydrogen bonding to bases opposite these damaged sites. We synthesized a modified 41‐mer DNA containing a (6–4) photoproduct using a preformed building block, then employed it as a template for primer extension reactions catalyzed by Klenow fragment and DNA polymerases α, β and δ (pol α, pol β and pol δ). None of these DNA polymerases were able to bypass the (6–4) photoproduct and elongation terminated at or near the 3′‐pyrimidone of the photoproduct, depending on the dNTP concentration. When a single‐chain Fv (scFv) with high affinity for the (6–4) photoproduct was included in the polymerization reaction, DNA synthesis was inhibited at base positions four, six, eight or eight nucleotides prior to the 3′‐pyrimidone by Klenow fragment, pol α, pol β or pol δ, respectively. These results suggest that the scFv can bind to the template DNA containing a (6–4) photoproduct and inhibit extension reactions by polymerases.
Electron Paramagnetic Resonance and Spin Trapping Investigations of the Photoreactivity of Human Lens Proteins
Authors: Dillon James , Ortwerth Beryl J. , Chignell Colin F. , Reszka Krzysztof J.
Published: January 2008

Abstract— Oxidation of cysteine, glutathione and ascorbate by photoexcited proteins from normal and cataractous lenses was investigated using electron paramagnetic resonance in combination with spin trapping. We report that illumination of these proteins in pH 7 buffer with light >300 nm in the presence of thiols (RSH) and a spin trap 5,5‐dimethyl‐1‐pyrroline N‐oxide (DMPO), afforded DMPO/S‐cysteine and DMPO/G adducts, suggesting the formation of the corresponding thiyl radicals. In a nonbufered aqueous solution, illumination of the proteins and glutathione also produced superoxide detected as a DMPO/O2H adduct. Irradiation of these proteins in the presence of ascorbate generated ascorbate radical. We conclude that chromophores present in the natural normal and cataractous lenses are capable of initiating photooxidative processes involving endogenous thiols and ascorbic acid. This observation may be pertinent to UV‐induced development of cataract.
Uptake of Topically Applied 5‐Aminolevulinic Acid and Production of Protoporphyrin IX in Normal Mouse Skin: Dependence on Skin Temperature
Authors: Juzenas Petras , Sørensen Roar , Lani Vladimir , Moan Johan
Published: January 2008

Abstract— The temperature dependence of the uptake phase of 5‐aminolevulinic acid (ALA) and the following production phase of protoporphyrin IX (PpIX) in normal mouse skin was investigated. A cream containing 20% ALA was topically applied on the skin for 10 min. The amount of ALA‐induced PpIX was evaluated by measuring the fluorescence of PpIX from the treated skin. No measurable amount of PpIX was found in the skin immediately after 10 min application of ALA. The penetration of ALA into the skin was almost temperature independent while the following production of PpIX was found to be a strongly temperature‐dependent process. Practically no PpIX was formed in the skin as long as skin temperature was kept low (12°C).
Analysis of Pulmonary Microvasculature Changes after Photodynamic Therapy Delivered to Distant Sites
Authors: ten Tije Albert J. , Wieman T. Jeffery , Taber Scott W. , Tseng Michael T. , Cerrito Patricia B. , Jansen Jeroen M. , Quo Hai Hong , Fingar Victor H.
Published: January 2008

Abstract— Photodynamic therapy (PDT) can exert local damage by direct tumor cytotoxicity, by disruption of the microvas‐culature or by a combination of these effects. Although systemic effects after PDT of small tissue areas (< 1% total body surface area) are unlikely, treatment of larger areas may result in an accumulated effect leading to toxicity. Several investigators have described animal death after high dose PDT to tumors on the hind limb of animals and hypothesized that a toxic shock syndrome caused by vasoactive agents released after PDT is responsible. Because one of the most vulnerable organs to toxic shock injury is the lung, we studied the systemic effects of local PDT to this organ by intravital microscopy using a pulmonary window chamber. The PDT treatment conditions (25 mg/kg Photofrin®, 24 h, 150 J/cm2 630 nm, maximum area 6.28 cm2) were chosen that produce systemic toxicity and lethality in rats. Adhesion of leukocytes in the lung was monitored in vivo using anti‐CD‐13‐labeled microspheres. The progression of pulmonary edema was assessed by monitoring the leakage of rhodamine‐labeled albumin and by wet‐to‐dry lung weight ratios. Although an increased leukocyte adherence was observed and a significant number of animals died after the extensive PDT treatment, no biologically significant lung edema could be demonstrated. These data indicate that lung edema and acute respiratory distress syndrome is not the cause of death in these animals and that the toxicity is related to other mechanisms including circulatory shock after extensive muscle damage.
Determination of Fluorescence Yields, Singlet Lifetimes and Singlet Oxygen Yields of Water‐Insoluble Porphyrins and Metalloporphyrins in Organic Solvents and in Aqueous Media
Authors: Figueiredo Telmo L. C. , Johnstone Robert A. W. , Sørensen Alexandra M. P. SantAna , Burget Dominique , Jacques Patrice
Published: January 2008

Abstract— Fluorescence quantum yields and singlet lifetimes for a wide range of hydrophilic to hydrophobic porphyrins and metalloporphyrins have been determined in toluene, methanol or acetone. Photosensitized singlet oxygen yields have been determined in the same solvents. For some porphyrins, the same quantities were determined in an aqueous medium, through use of an amphiphilic polymer to solubilize the porphyrin sensitizer and target molecule, 1, 3‐diphenylisobenzofuran. Because rate constants for the deactivation of singlet oxygen (kd) and for its reaction with a target molecule (ka) are unknown in such aqueous polymer systems, a new method was developed for evaluating yields of singlet oxygen formation that also provides a value for the ratio kd/ka. A variation observed in quantum yield of singlet oxygen production for the aqueous polymer system with variation in initial concentration of the target molecule is discussed.
Rapid Communication: Stability of the Fluorescence Measurement of Foscan® in the Normal Human Oral Cavity as an Indicator of its Content in Early Cancers of the Esophagus and the Bronchi
Authors: Zellweger Matthieu , Grosjean Pierre , Monnier Philippe , van den Bergh Hubert , Wagnieres Georges
Published: January 2008

Abstract— Photodynamic therapy (PDT) with Foscan® (mTHPC) is used to cure early cancers of the esophagus or the tracheobronchial tree. However, fixed PDT parameters (drug dose, light dose, etc.) do not permit an accurate prediction of the tissue damage. Large interpatient fluctuations in tissue drug level, at the time of light application, suggest that the light dose must be adjusted to the drug dose shortly before the PDT. This drug dose can be measured endoscopically by light‐induced fluorescence spectroscopy, but this measurement is inconvenient and somewhat difficult. A better test site, yielding comparable information, is needed. The oral cavity seems ideal. However, it first had to be established to what extent the estimation of the drug dose was dependent upon the location of the measurement and the pressure applied to the probe. These measurements prove to be not only correlated to similar measurements in the esophagus or the bronchi but also more consistent and less sensitive to the location and the applied pressure. The buccal mucosa is therefore recommended as a test site for measuring the Foscan(r) fluorescence signal at the time of PDT in the esophagus or the bronchi. This measurement is accurate enough for use in light‐dose adjustment.
Analysis of Phosphorescence Decay in Heterogeneous Systems: Consequences of Finite Excitation Flash Duration
Authors: Golub Aleksander S. , Popel Aleksander S. , Zheng Lei , Pittman Roland N.
Published: January 2008

Abstract— Analysis of phosphorescence lifetimes using the Stern‐Volmer equation is a reliable means of determining quencher concentration for a uniform sample. Methods of analysis for heterogeneous systems are based on the assumption that the excitation is produced by a momentary flash. This condition is an idealization because a real flash has a finite duration and a complex time profile. In the case of a heterogeneous quencher concentration, an excitation flash produces different initial intensities and different times of peak intensity from compartments having different concentrations of quencher. We formulated a model to explore the effects of flash duration on the shape of the emission curve obtained from systems in which the heterogeneity is continuous. We developed mathematical models that can be used to recover fitting parameters of continuous distributions of reciprocal lifetimes approximated as rectangular or Gaussian distributions, or an arbitrary histogram. We also formulated a procedure to convert the distribution of reciprocal lifetimes into a volume distribution of quencher concentration. We found that (1) the Stern‐Volmer ratio of phosphorescence intensities cannot be employed for interpretation of pulse phosphorometric data in terms of a volume distribution of quencher; (2) shortening the flash duration decreases the difference of initial intensities between compartments having high and low quencher concentration; (3) the parameters of the volume distribution of quencher concentration can be recovered correctly only after taking account of the difference in initial intensities; and (4) calibration of the initial intensities for a given fitting delay and flash function is necessary.
Evidence for [2+2] Photoreaction of α‐Methylene‐γ‐Butyrolactones with Thymine: An Explanation for Chronic Actinic Dermatitis to Sesquiterpene Lactones?
Authors: Berl Valérie , Lepoittevin Jean‐Pierre
Published: January 2008

Abstract— The photoreactivity of isoalantolactone, a natural sesquiterpene lactone, toward thymine was studied. After 313 nm irradiation of a deoxygenated acetone solution of isoalantolactone (2 × 10–3M) and thymine (4 × 10–3 M), two intermolecular [2+2] photoadducts, 3 and 4, were isolated with respective yields of 30% and 18%. The structures of these two photoadducts were determined by a combination of NMR experiments. Compound 3 was identified as a cis‐syn‐exo intermolecular [2+2] photoadduct involving the 5,6 double bond of thymine and the exomethylenic double bond of the lactone, while compound 4 was identified as an intermolecular [2+2] photoadduct involving the same bonds but with the cis‐synendo conformation. This high photoreactivity of sesquiterpene lactones toward thymine could be an explanation of the progressive evolution of allergic contact dermatitis toward chronic actinic dermatitis.
Dynamics of Flavin in Flavocytochrome b2: A Fluorescence Study
Authors: Albani J. R. , Sillen A. , Engelborghs Y. , Gervais M.
Published: January 2008

Abstract— The dynamics of the flavin bound to the flavocytochrome b2 from Hansenula anomala were studied by fluorescence intensity quenching and quenching emission anisotropy with iodide. The fluorescence intensity of bound flavin is decreased 13‐fold as compared to the free molecule. The remaining fluorescence decays with two lifetimes equal to 0.963 ± 0.040 and 4.635 ± 0.008 ns and fractional intensities of 0.036 ± 0.002 and 0.964 ± 0.002, respectively. The bimolecular diffusion constant was found to be 3.33 × 109M‐1 s‐1 when the flavin is bound to the enzyme and 8.3 × 109Mv s‐1 when the flavin is free in solution. Thus, the flavin in flavocytochrome b2 is accessible to the solvent, but the amino acid residues of the binding site inhibit the diffusion of iodide. The rotational correlation time of bound flavin was found to be 2.015 ± 0.365 ns, a value higher than that (155 ps) of free flavin in solution. Our results are discussed on the basis of local dynamics of the flavin.
Chronology of the Apoptotic Events Induced in the K562 Cell Line by Photodynamic Treatment with Hematoporphyrin and Monoglucosylporphyrin
Authors: Carré Vincent , Jayat Chantal , Granet Robert , Krausz Pierre , Guilloton Michel
Published: January 2008

Abstract— Photodynamic treatment of promyelocytic K562 cells in the presence of a monoglucosylporphyrin or hematoporphyrin leads to a sequence of events recognized as hallmarks of apoptosis: a drop in mitochondrial potential, concurrent with a drop in ATP level and a decrease in cell respiration, translocation of phosphatidylserine of the plasma membrane, DNA fragmentation, appearance of apoptotic bodies and eventually loss of plasma membrane integrity. The chronology of these events is in accordance with sequential events induced by other known proapoptotic agents; in contrast to these agents that induce apoptosis in a restricted part of the cell population, we observed that the entire cell population (apart from a small percentage of cells that endured rapid necrosis during phototreatment) underwent apoptosis after irradiation in the presence of porphyrins. It appears that photodynamic treatment allows the bypass of early apoptotic signals in K562 cells that are otherwise renowned for their resistance to drug‐induced apoptosis (A. McGahon, R. Bissonnette, M. Schmitt, K. M. Cotter, D. R. Green and T. G. Cotter, Blood 83, 1179–1187, 1994). Singlet oxygen is believed to be the proximate reactive species generated by porphyrin illumination. Because this molecule reacts with almost every cellular constituent, the way that singlet oxygen or its reactive oxygen species byproducts trigger apoptosis remains to be elucidated.
Action Spectra for UVB Impacts on Blepharisma japonicum Motility and Photobehavior
Authors: Checcucci Giovanni , Sgarbossa Antonella , Gioffré Domenico , Lenci Francesco , Ghetti Francesco
Published: January 2008

Abstract— The ciliate Blepharisma japonicum was exposed to artificial polychromatic and monochromatic UV radiation to evaluate the relative roles of UVB (280–315 nm UV radiation) and UVA (315–400 nm UV radiation) in altering its motility and photobehavior and to determine absolute weighting coefficients for these effects in the UVB range. Under polychromatic UV irradiation B. japonicum cells showed a severe reduction of cell speed and of the capability to respond to light stimuli. At low doses, however, UV caused a significant increase in the average velocity of a cell population. The UVB exclusion experiments indicated that UVA does not significantly alter motility and photoresponsiveness. The increase and the subsequent decrease in cell velocity was observed also under monochromatic irradiation at 281, 290 and 300 nm, whereas at 310 nm cells swim faster up to the highest photon flux density used. The cell capability of reacting to photic stimuli, conversely, steadily declined with increasing photon flux density at all the tested UVB wavelengths. The action spectra for the alteration of cell velocity and the impairment of photoresponsiveness show that the lower the irradiation wavelength, the more remarkable are the UVB effects and suggest different targets for the increase and the decrease in cell velocity.
Isolation and Characterization of Two Furan‐side Photoadducts of 7‐Methylpyrido[3,4‐c] Psoralen to the Sugar Moiety of 2‐Deoxyadenosine†
Authors: Voituriez Lucienne , Cadet Jean
Published: January 2008

Abstract. The UVA‐mediated photoreaction of the monofunctional 7‐methylpyrido[3,4‐c]psoralen (MePyPs) with 2′‐deoxy‐adenosine was investigated in the dry state. Two main 7‐methylpyrido[3,4‐c]psoralen monoadducts to 2′‐deoxy‐adenosine were isolated by HPLC and characterized by soft ionization mass spectrometry (fast atom bombardment) and extensive 1H NMR analyses including correlation spectroscopy, total correlation spectroscopy and nuclear Overhauser effect measurements. The two unusual photoadducts exhibit a covalent bond between the C‐4′ of the furan moiety of the psoralen on one hand and either the C‐l' or the C‐4′ of the furanose ring of the purine nucleoside on the other hand. The furan‐side monoadducts appear to be specific for both 2′‐deoxy‐adenosine and MePyPs. They are likely to result from the recombination of the 4′,5′‐dihydrofuran‐4′‐yl radical of MePyPs with 2′‐deoxyadenosyl carbon‐centered sugar radicals at either the 1′ or the 5′ position.
A New Series of Proton/Charge Transfer Molecules: Synthesis and Spectral Studies of 2‐(5‐Aryl‐1‐carbomethoxy‐1H‐pyrazol‐3‐yl)phenols
Authors: Rampey Mary E. , Halkyard Carrie E. , Williams Angela R. , Angel April J. , Hurst Douglas R. , Townsend Jessica D. , Finefrock Anne E. , Beam Charles F. , Studer‐Martinez Shannon L.
Published: January 2008

Abstract. The carbomethoxyhydrazone of 2′‐hydroxyacetophenone was trilithiated with excess lithium diisopropylamide and C‐acylated with a variety of benzoate esters followed by acid cyclization of the intermediates to 2‐(5‐aryl‐l‐car‐bomethoxy‐lHpyrazol‐3‐yl)phenols [3‐(2‐hydroxyphen‐yl)‐lH‐pyrazoles]. The products were characterized by Fourier transform‐IR, lH NMR, 13C NMR, UV‐visible absorption and fluorescence. All the derivatives in n ‐heptane have an absorption maximum at ˜304 nm and an extremely weak (φr= 10 4) fluorescence with maxima in the range of 335–460 nm. The broad range of fluorescence maxima and fluorescence quantum yields is attributed to varying contributions of charge transfer that are dependent on both the identity of the substituent and solvent polarity. A phenomenally large Stokes‐shifted fluorescence maximum at 620 nm was observed for 2‐(l‐car‐bomethoxy‐5‐[4‐dimethylaminophenyl]‐lff‐pyrazol‐3‐y1)phenol in n‐heptane and attributed to excited‐state intramolecular proton transfer. As a result, competitive excited‐state proton/charge transfer properties have been observed in the pyrazoles studied, of which the spectral properties can be fine tuned by substituent as well as solvent effects.
Quantitative Characterization and Study of the Relationship between Constitutive‐Facultative Skin Color and Phototype in Caucasians
Authors: Rubegni Pietro , Cevenini Gabriele , Barbini Paolo , Flori Maria Laura , Fimiani Michele , Andreassi Lucio
Published: January 2008

Abstract In this study constitutive and facultative colorimetric values were quantified to determine the physiologic changes in Caucasian skin color and to define the correlation between skin color and phototype assessed according to the Fitzpatrick method. Our population consisted of 401 subjects ranging in age from 24 to 28 years with similar life styles. Skin color was measured with a Minolta CR‐200 colorimeter on the upper medial quarter of the buttock (constitutive color) and on the cheek (facultative color). Advanced multivariate statistical analysis allowed differentiation between constitutive and facultative skin color in relation to the phototype to be quantified. Moreover, Kullback divergence showed that the probability of correctly determining a subject's phototype is high when the variables of constitutive and facultative skin color are considered together. This interesting result makes it possible, in the future, to use colorimetric values of exposed and nonexposed skin, together with determination of Fitzpatrick phototype and of other phenotypic characters, to better predict cutaneous sun reactivity.
Effect of 5‐AmJnolevulinic Acid on Protoporphyrin IX Accumulation in Tumor Cells Transfected with Plasmids Containing Porphobilinogen Deaminase DNA
Authors: Hilf Russell , Havens James J. , Gibson Scott L.
Published: January 2008

Abstract Recently, we reported that the δ‐aminolevulinic acid (δ‐ALA)‐induced increase in porphobilinogen deaminase (PBGD) activity was closely correlated with an increase in the accumulation of protoporphyrin IX (PPIX), resulting in augmented phototoxicity. In this report, we asked whether increasing the cellular expression of PBGD by use of gene transection techniques in vitro would further enhance δ‐ALA‐induced PPIX accumulation and hence, phototoxicity. For these experiments we constructed plasmid vectors containing the PBGD‐DNA, using a reverse transcription‐polymerase chain reaction‐generated cDNA fragment encoded from its published sequence. Subsequently, transfection of the human mammary tumor cell line, MCF‐7, and the human mesothelioma cell line, H‐MESO‐1, with the PBGD‐DNA‐con‐taining plasmids was shown to produce a 2.5‐2.7‐fold increase in enzyme activity. Twenty‐four hours after completion of the transfection procedure, transfectants were exposed for 3 h to 0.5 niM δ‐ALA. Exposure of either wild type or transfectants to δ‐ALA led to measurable levels of PPIX. Although this produced a modest but significant increase in intracellular PPIX content in H‐MESO‐1 cells compared to wild‐type cells incubated with δ‐ALA alone, the increase above the transfection control did not reach statistical significance. Likewise, a significant increase in PPIX was not observed in transfected MCF‐7 cells subsequently exposed to δ‐ALA. These data demonstrate that transient transfection of cells with the cDNA of PBGD was successful in elevating enzyme activity in both tumor cell lines, but this did not result in a comparable difference in the levels of PPIX. Such an approach for the study of other enzymes in the heme pathway should provide a model to better define rate‐limiting steps in the 8‐ALA induction of PPIX, and ultimately, to enhance the effectiveness of photodynamic therapy.
Effect of Self‐association and Protein Binding on the Photochemical Reactivity of Triarylmethanes. Implications of Noncovalent Interactions on the Competition between Photosensitization Mechanisms Type I and Type II
Authors: Bartlett Jeremy A. , Indig Guilherme L.
Published: January 2008

We have explored the photochemical behavior of cationic triarylmethane dye monomers and dimers free in solution and noncovalently bound to bovine serum albumin (BSA) and examined how self‐association and the formation of host‐guest complexes involving biopolymers and photosensitizers affect the competition between the photosensitization type I and type II mechanisms. Our results have clearly indicated that tri‐para ‐substituted triarylmethane dyes bind efficiently to albumin as monomers and dimers and, interestingly, that the formation of dye aggregates in aqueous solutions is actually assisted by the protein. Protein‐assisted dye aggregation takes place under conditions of high biopolymer loading (high [dye]/[protein] ratios), as attested by the appearance of a hypsochromically shifted absorption band (H‐band) that overlaps with the spectral shoulder of the respective dye monomer. As predicted by the molecular exciton theory, the intersystem crossing efficiency in H‐type dimers is expected to be higher than in the respective dye monomers, and photoinduced electron transfer events are intrinsically favored in dye aggregates as a result of the physical contact between donor and acceptor. We have found that when triarylmethanes are noncovalently bound to BSA their photoreactivity undergoes a remarkable enhancement, and that the photooxidation mechanism type I is particularly favored in the macromolecular environment. A comparative examination of the behavior of triarylmethane dyes with that of methylene blue have shown that in the case of methylene blue the binding phenomenon also favor the type I mechanism.
Oxidation of Chiral 1,4‐Cyclohexadienes: A Comparison of Chemically and Photochemically Generated Singlet Oxygen1O2(1Δg)
Authors: Nardello V. , Aubry J. M. , Linker T.
Published: January 2008

Chiral alkyl‐substituted 2,5‐cyclohexadiene‐l‐carboxyIic acids la‐c have been oxidized in water and in methanol with singlet oxygen, 1O2 (1Δg), generated either photochemically or chemically from the catalytic system hydrogen peroxide/sodium molybdate. These methods were compared in terms of chemo‐, regio‐ and diastereoselec‐tivities and the chemical (kT) and physical (kq) quenching rate constants of 1O2 were determined. The ratio of the cis and trans isomers of the hydroperoxides 2a‐c is not influenced by the source of 1O2 but, on the other hand, it depends slightly on the solvent and greatly on the steric hindrance of the substituents linked to the chiral carbon. The results may be interpreted on the basis of the successive formation of an exciplex and a perepoxide that evolves either by giving the final allylic hydroperoxide or by dissociating into the starting substrate and singlet or triplet oxygen.
Long‐Range Electron Transfer in Rigid 310‐Helical Oligopeptides Containing Redox Cyclic a‐Amino Acids
Authors: Lang Kamil , Kuki Atsuo
Published: January 2008

Intrahelical photoinduced electron transfer processes (ET) in conformationally restricted oligopeptides have been studied by nanosecond time‐resolved transient spectroscopy. The helical peptides were constructed from ste‐rically hindered a‐aminoisobutyric acid (Aib) and two cyclic a‐amino acids (Aib class) bearing electron acceptor and donor side chains (DkNap, ThQx). This helical backbone design provides high conformation stability, as previously demonstrated, and yields reliable 310‐helical architectures in solution. The forward ET between ThQx and 3DkNap is followed by a slow back ET thus giving rise to an accumulation of the charge‐separated ion pairs for hundreds of nanoseconds. We demonstrate the modulation of electronic interactions by the number of intervening Aib residues separating acceptor‐donor side chains and propose modifications of the peptide framework by inclusion of a non‐Aib amino acid residue. These well‐defined and sterically stable frameworks are suited for the precise evaluation of intrahelical electron transfer processes mediated by peptides.
Lifetime Quenching of the Fluorophore: A Signature of Magnetic Field Maximization
Authors: Aichf Sanjukta , Basu Samita
Published: January 2008

The fluorescence lifetime quenching of the fluorophores in the presence of quenchers were measured with the variation of solvent polarity (e) for the two donor‐acceptor (D‐A) pair, JV‐ethyl carbazole‐1,4 dicyanobenzene and pyrene‐N,N‐dimethylaniline. We observed that maximum quenching of lifetimes occurred at the ε where maximum magnetic field effects had been achieved for both the pairs. The importance of the nature of the ex‐ciplex and the distance between D and A in magnetic field maximization has also been elucidated.
Chirality and Spectroscopic Changes Induced by the Recognition of Ethyl 5‐Amino‐2‐methyl‐1,2‐dihydro‐3‐phenylpyrido[3,4‐b]pyrazin‐7‐yl Carbamate Analogs by Tubulin
Authors: Peyrot V. , Barbier P. , Sarrazin M. , Briand C. , Andreu J. M.
Published: January 2008

The two chiral isomers of ethyl 5‐amino‐2‐methyl‐l,2‐di‐hydro‐3‐phenylpyrido[3,4‐b]pyrazin‐7‐yl carbamate, NSC 613863 (R‐isomer)‐(+) and NSC 613862 (S‐isomer)‐(‐) (CI980) and the three achiral analogs NSC 330770 (2‐de‐methylated analog A), NSC 337238 and C179 are potent microtubule inhibitors. These ligands interact with tubulin overlapping the colchicine binding site. This study addresses the effects of recognition by tubulin on the conformational properties of the ligands. The near‐UV CD (circular dichroism) band of the R‐isomer was suppressed, while that of the S‐isomer displayed a more intense negative band when these compounds were bound to tubulin. Interestingly, the three other initially achiral compounds became optically active upon binding to tubulin; particularly, analog A exhibited a negative CD band on the order of magnitude of chiral compounds. The CD changes are reversible, highly specific and actually permit measuring the binding of the ligands by tubulin. These CD changes are compatible with the deformation of the bound ligands. Fluorescence emission is strongly enhanced and blue shifted upon binding to tubulin. Water among a solvent series had a specific solvent effect, except on the 1,2‐dehydro analogs NSC 337238 and C179, suggesting hydrogen bonding to Nl. The emission of tubulin‐bound R‐isomer, S‐isomer and analog A could be mimicked by solvent viscosity, supporting the notion that the intramolecular rotation between the pyridopyrazine and phenyl rings is frozen upon binding.
The Effects of pH and Ionic Strength on the Aggregation of Bacteriochlorophyll c in Aqueous Organic Media: The Possibility of Two Kinds of Aggregates
Authors: Ishii Takasada , Uehara Kaku , Ozaki Yukihiro , Mimuro Mamoru
Published: January 2008

The aggregation behavior of two homologs of bacteriochlorophyll c (BChl c) in various media was investigated for the effects of pH and salt, and the corresponding structures were analyzed by Fourier transform (FT)‐IR spectroscopy. R‐[P, E] BChl cF (31‐R‐form of BChl c with a propyl group at the C‐8 position and an ethyl group at the C‐12 position) and R‐[E, E] BChl cF (31‐R‐form of BChl c with two ethyl groups at positions C‐8 and C‐12) were isolated from the green sulfur bacterium Chloro‐bium limicola. Aggregates of each homolog showed a pH‐dependent shift of the absorption maximum; at low pH, the peak moved to the red. This tendency was also revealed by circular dichroic spectra. A similar red shift of the peak was also induced by a high concentration of salt (NaCl) or buffer for both homologs. The FT‐IR spectrum indicates that at low pH, both homologs formed a rather amorphous aggregate. On the other hand, a regular structure of R‐[P, E] BChl cF was indicated in an acetone‐water mixture. This structure was stabilized by a triangular interaction among three pigment molecules through the Mg‐OH (3>) O = C (131) linkage. This structure was not found for R‐[E, E] BChl cF. These results indicate that the replacement of the side chain at the C‐8 position on the macrocycle induces a change in aggregation behavior. A possible heterogeneity of the in vivo rod structure of chlorosomes in green sulfur bacteria is discussed based on the above results.
Selective Photocleavage of DNA and RNA by Anthraquinone Derivatives: Targeting the Single‐Strand Region of Hairpin Structures
Authors: Fernandez‐Saiz Maria , Henderson Paul T. , Wilson W. David , Schuster Gary B.
Published: January 2008

A tetracationic anthraquinone derivative (27AQS2) binds to hairpin DNA and RNA. Ultraviolet irradiation of the bound quinone causes cleavage in the loop region of both oligonucleotides and at guanines in the stem region of the DNA hairpin. The absence of observable strand cleavage at guanines in the RNA hairpin suggests that either aniline treatment does not cause cleavage at damaged guanines in RNA or that radical cation migration does not occur readily in RNA duplexes. The ability to target the single‐stranded regions of DNA and RNA structures is an important property of this photonuclease.
Enhanced Responsiveness to Photodynamic Therapy‐Induced Apoptosis after Mitochondrial DNA Depletion
Authors: Kessel David , Sun Hai Hao
Published: January 2008

Several lines of evidence indicate that mitochondria are an especially sensitive target for photodamage. Reports of cross resistance between photodynamic therapy (PDT) and the drug cisplatin, along with evidence that depletion of mitochondrial DNA (mtDNA) sensitized cells to cisplatin suggested a study of the photodynamic responsiveness of murine leukemia control L1210 cells versus cells depleted of mtDNA. Loss of mtDNA led to an increased sensitivity to mitochondrial photodamage, while the cytotoxic effects of lysosomal photodamage were not affected. Cells depleted of mtDNA showed an enhanced ap‐optotic response to PDT involving a mitochondrial target, compared with control cells.