bluuebunny/crossref_metadata_2025 huggingface dataset:
Study of Photochemical Reactions of Coniferyl Alcohol. II. Comparative Structural Study of a Photochemical and Enzymatic Polymer of Coniferyl Alcohol
Authors: Radotic Ksenija , Todorovic Smilja , Zakrzewska Joanna , Jeremic Milorad
Published: January 2008

The structure of the polymer synthesized by UV irradiation of coniferyl alcohol was studied, using UV‐visible, Raman, IR, H‐NMR and 13C‐NMR spectroscopy. The photochemical polymer was compared with the structure of the polymer obtained by peroxidase‐catalyzed polymerization of coniferyl alcohol. General similarity of the spectra of the two polymers was shown. However, differences in the fine structure of particular regions of the NMR spectra, as well as in certain bands in the Raman and IR spectra, could be explained through the various bond types and organization within the polymers. These results are consistent with molecular mass distribution of the polymers. Two fractions of enzymatic polymer correspond to the main two fractions of photochemical polymer. The later polymer has additional fractions that are probably the main reason for the observed spectral differences.
Human BCL‐2 Expression Delays Ultraviolet‐Induced Apoptosis in Marsupial Cells
Authors: Miyaji Eliane N. , Menck Carlos F. M.
Published: January 2008

We have introduced the human bcl‐2 gene under the control of the human metallothionein MTIIA promoter into the rat kangaroo PtK2 cell line. Two independent clones were obtained in which the levels of Bcl‐2 protein expression can be controlled by the addition of metals in the culture medium. These cell lines were employed to investigate the effects of this protein in UV‐induced apoptosis. Overexpression of Bcl‐2 in PtK2 cells resulted in a delay in the appearance of apoptosis markers, such as chromatin condensation and internucleosomal DNA fragmentation. However, colony survival after UV was not affected, suggesting that Bcl‐2 did not impose a definitive block for cell death. The elimination of cyclobutane py‐rimidine dimers through photoreactivation 24 h after irradiation in cells overexpressing Bcl‐2 did not affect apoptosis. This indicates that irreversible events in the signaling pathway of apoptosis occur in the period between irradiation and photoreactivation even in the presence of high levels of Bcl‐2 in the cell. Therefore, although the human Bcl‐2 protein can delay the onset of UV‐induced apoptosis in these marsupial cells, early events triggered by the pyrimidine dimers, upstream from the Bcl‐2 action, lead the cell to a state committed to die.
The Configuration of β‐Carotene in the Photosystem II Reaction Center
Authors: Yruela Inmaculada , Tomás Raquel , Sanjuán María L. , Torrado Elena , Aured Maria , Picorel Rafael
Published: January 2008

Different pigment extraction procedures and HPLC methods were tested to investigate the geometric configuration of the β‐carotene in two forms of the photosystem II reaction center (Dl‐D2‐cytochrome (Cyt) b559) complex containing one and two β‐carotene molecules per two pheophytin a. All the handling steps and HPLC analyses were done in darkness at room temperature and at 4°C. Two different pigment extracts were analyzed, a mixture of chlorophyll a, pheophytin a and 3‐carotene, and the isolated p‐carotene from that mixture. In both cases only the all‐frans‐β‐carotene was detected. The chromatographic profiles were similar at both temperatures only differing in the retention times that were longer at 4°C. This result was independent of the concentration of photosynthetic starting material. Furthermore, no differences were observed between Dl‐D2‐Cyt b559 complexes with one and two p‐carotene molecules per reaction center. The analysis of the β‐carotene chromatographic peak indicated no 15‐cis to all‐fraws isom‐erization occurred during the HPLC chromatography in our experimental conditions. Resonance Raman spectra were also recorded in the isolated Dl‐D2‐Cyt b559 complex at room and liquid nitrogen temperature with excitation at 514.5 nm from an Ar+ laser. Spectra of control preparations showed main bands at 1532, 1264, 1213, 1185, 1154 and 1003 cm−1 corresponding to the M‐trans isomer and confirm previous results. The presence in the reaction center suspension of artificial electron acceptors such as silicomolybdate or 2,5‐dibromo‐3‐methyl‐6‐iso‐propyl‐p‐benzoquinone that are able to quench 3P680 did not modify the resonance Raman spectra of the native Dl‐D2‐Cyt b559 complex. The results suggest that no isomerization takes place during the laser irradiation.
The N‐Terminal Amino Acid Sequences of the Firefly Luciferase Are Important for the Stability of the Enzyme
Authors: Sung Deukyong , Kang Hunseung
Published: January 2008

The structural and catalytic role of the N‐terminal amino acid sequence of Photinus pyralis luciferase was investigated by site‐directed mutagenesis. The firefly luciferase activity of a series of deletion and site‐directed mutants in the amino‐terminal region was investigated in vitro and in vivo. The mutant luciferases were produced either by in vitro transcription and translation or by expressing the cDNA encoding firefly luciferase in the yeast Saccha‐romyces cerevisiae as a fusion protein to the galactose DNA binding domain protein. Deleting the N‐terminal amino acid residues from 3 to 10 dramatically reduced the luciferase activity to less than 1% of the wild‐type activity. A marked decrease in the activity (to 5%) was also observed with the Lys8Glu mutant. The Glyl 0Arg and ProllAla mutants showed20–30% activity compared to the wild type. On the other hand, mutant Asn6Lys retained the wild‐type activity level. Randomizing3–11 N‐terminal amino acids also showed a marked decrease in activity (1%). The mutant luciferases with extremely low levels of enzymatic activity were thermally unstable. These mutational and stability data correlate with the crystal structure of firefly luciferase in which specific amino acids in the N‐terminal region form hydrogen bonds to the amino acids in the neighboring p‐stranded sheet. Because the N‐terminal region is not part of the active site, the present results suggest that the highly conserved N‐terminal amino acid sequences of the firefly luciferase are important for stabilizing the protein in its proper conformation for optimal enzyme activity.
UV Light‐induced Cross‐linking of Nucleosides, Nucleotides and a Dinucleotide to the Carboxy‐terminal Heptad Repeat Peptide of RNA Polymerase II as Studied by Mass Spectrometry
Authors: Connor Dallas A. , Falick Arnold M. , Shetlar Martin D.
Published: January 2008

Abstract— We report here the results of a study to assess the usefulness of mass spectrometry as a method for rapidly locating cross‐linking sites in peptides modified by UV irradiation in the presence of nucleic acid components. For this study, we selected two nucleosides (thymidine and 5‐bromo‐2′‐deoxyuridine), two nucleotides (thymi‐dine‐5′‐monophosphate and 5‐bromo‐2′‐deoxyuridine‐5‐monophosphate) and a dinucleotide (thymidylyl‐[3′→5′]‐2′‐deoxyadenosine). The peptide picked was SPSYSPT (l‐seryl‐l‐prolyl‐l‐seryl‐l‐tyrosyl‐l‐seryl‐l‐prolyl‐l‐threonine), the heptad repeat unit found in the largest subunit of the RNA polymerase II multiprotein complex. Modified peptides were isolated by reversed‐phase HPLC. Molecular mass measurements confirmed that covalent adducts had been formed. High‐energy tandem collision‐induced dissociation mass spectrometry pinpointed the location of cross‐linking in each modified peptide as being at the tyrosine residue. These results indicate that mass spectrometry is a potentially applicable technique for location of cross‐linking sites in peptides, modified by attachment of nucleosides, nucleotides and dinucleotides. Such modified peptides would be among the products expected after application of standard proteolytic and nucleolytic digestion protocols to digestion of cross‐linked DNA‐protein complexes.
Radiolysis‐Induced Oxidation of Bovine α‐Crystallin
Authors: Finley Eric L. , Dillon James , Crouch Rosalie K. , Schey Kevin L.
Published: January 2008

Abstract— Radiolysis of water by ionizing radiation results in the production of pure hydroxyl radicals. This technique, combined with analysis by tandem mass spectrometry (MS/MS), has been used to study the effect of hydroxyl radicals on the intact bovine α‐crystallin protein. After exposure to ‐γ‐irradiation, the oxidized α‐crystallin was digested with trypsin and the resulting peptides were fractionated by reverse‐phase HPLC. The isolated fractions were analyzed by matrix‐assisted laser desorption ionization and by MS/MS to determine the locations and identities of the modifications. Structural analysis revealed that methionine 1 of αA‐ and αB‐crystallin and methionine 68 of αB‐crystallin were oxidized to methionine sulfoxide. Hydroxytryptophan was formed from each tryptophan residue in α‐crystallin, although only tryptophan 9 of αA‐crystallin was converted into N‐for‐mylkynurenine. This study has, for the first time, identified the sites of modification and the structures produced in the intact α‐crystallin protein by exposure to hydroxyl radicals. By determining the consequences of in vitro exposure of α‐crystallin to pure hydroxyl radicals, the in vivo contribution of this reactive oxygen species to the overall oxidative stress of the lens will be achieved from the identification of the modifications to α‐crystallin purified from intact human lenses.
Derivatives of 5‐Aminolevulinic Acid for Photodynamic Therapy: Enzymatic Conversion into Protoporphyrin
Authors: Kloek Joris , Akkermans Willem , van Henegouwen Gerard M. J. Beijersbergen
Published: January 2008

In recent years, 5‐aminolevulinic acid (ALA) has become a widespread agent for photodynamic therapy (PDT). In nucleated cells, ALA is converted into the endogenous photosensitizer protoporphyrin IX (PpIX). A major drawback of ALA is its low bioavailability. As a result, high doses of ALA must be administered in order to reach clinically relevant levels of PpIX. Moreover, only superficially located lesions can be treated as a result of the poor penetration of ALA into tissues. A possible solution for this problem may be provided by the prod rug concept. In the present study, prodrugs of ALA have been synthesized. These ALA prodrugs are shown to result in higher PpIX levels in cells than does ALA itself. Of a range of ester prodrugs of ALA, the ALA‐pentyl ester elicits the highest fluorescence. Further‐more, the enzymatic conversion of the derivatives into ALA and PpIX has been studied in lysed cells. Under these circumstances, the esters with the shorter alkyl chains induce the highest fluorescence. The alcohols that arise as side products from enzymatic conversion of the prodrugs are shown to have no influence on the experiments.
Mutagenesis Mediated by Triple Helix–Forming Oligonucleotides Conjugated to Psoralen: Effects of Linker Arm Length and Sequence Context
Authors: Raha Manidipa , Lacroix Laurent , Glazer Peter M.
Published: January 2008

Targeted mutagenesis and gene knock‐out can be mediated by triple helix‐forming oligonucleotides (TFO) linked to mutagenic agents, such as psoralen. However, this strategy is limited by the availability of homopurine/ homopyrimidine stretches at or near the target site because such sequences are required for high‐affinity triplex formation. To overcome this limitation, we have tested TFO conjugated to psoralen via linker arms of lengths varying from 2 to 86 bonds, thereby designed to deliver the psoralen at varying distances from the third strand binding site present at the 3’end of the supFG1 mutation reporter gene. Following triplex formation and UVA irradiation, mutations were detected using an SV40‐based shuttle vector assay in human cells. The frequency and distribution of mutations depended on the length of the linker arm. Precise targeting was observed only for linker arms of length 2 and 6, which also yielded the highest mutation frequencies (3 and 14%, respectively). Psoralen–TFO with longer tethers yielded mutations at multiple sites, with the maximum distance from the triplex site limited by the linker length but with the distribution within that range influenced by the propensity for psoralen intercalation at A:T base‐pair‐rich sites. Thus, gene modification can be extended beyond the site of third strand binding but with a decrease in the precision of the targeting.
Tryptophan Quenching of Zinc‐Phthalocyanine and Porphycene Fluorescence in Micellar CTAC
Authors: Daraio Marta E. , Völker Axel , Aramendia Pedro F. , Román Enrique San
Published: January 2008

Singlet excited state deactivation of a zinc phthalocyanine (ZnPc), porphycene (Po) and tetrapropyl‐porphycene (PrPo) by anionic tryptophan (Trp−) were investigated in cetyltrimethylammonium chloride (CTAC) micelles at pH 9.2 ± 0.1, regulated by a Tris buffer. Data obtained from steady‐state experiments over a wide range of detergent and added NaCl concentrations were analyzed by using a pseudophase ion‐exchange model (Abuin et al., J. Phys. Chem. 87, 5166–5172, 1983). The model was applied to derive singlet quenching rate constants for ZnPc and the porphycenes by Trp− and the selectivity coefficient for Trp/Cl exchange at the micellar surface. The results point to an electron transfer quenching. Neutral tryptophan also quenches efficiently ZnPc fluorescence in CTAC without added buffer and Trp− does not deactivate the triplet state of these dyes. By flash photolysis, only the absorption of the triplet species was detected.
UV Light‐induced Duplex‐to‐duplex Crosslinking of DNA Molecules in Aqueous Ethanol Solutions
Authors: PospíŠlová Šárka , Kypr Jaroslav
Published: January 2008

Ultraviolet light is known to generate crosslinks between the complementary strands of DNA and between DNA and proteins. Here we demonstrate that the UV light also crosslinks DNA duplexes to other DNA duplexes. However, the duplex‐to‐duplex crosslinks only appear in the presence of about 75% (vol/vol) ethanol plus a millimolar or submillimolar concentration of monovalent or divalent cations, e.g. 2 mM Na+ Methanol or formamide are ineffective. The present observations provide a direct means to detect physical contacts of DNA molecules or their parts, e.g. during recombination. It is remarkable that the solution conditions leading to the duplex‐to‐duplex UV light‐induced crosslink formation are the same as those inducing the B‐to‐A conformational transition of DNA.
On the Apparent Lack of Oxygen Quenching of Triplet Excited States
Authors: White Emil H. , Roswell David F.
Published: January 2008

The early observation that excited triplet states obtained chemically from trimethyldioxetane (and subsequent energy transfers) appeared not to be quenched by oxygen is now shown to be an artifact; acetaldehyde is a product of the reaction and via chemical reactions it scavenges oxygen present in the medium; the attendant extended lifetime of the triplet states then permits their employment in photochemical‐type reactions. The effect of the aldehyde can be over‐ridden through an efficient introduction of an excess of oxygen into the liquid reaction phase.
The Induction and Analysis of Micronuclei and Cell Killing by Ultraviolet‐B Radiation in Human Peripheral Blood Lymphocytes
Authors: Keulers Rob A. C. , de Roon Angelique R. , Roode Saskia de , Tates Ad D.
Published: January 2008

The DNA‐damaging potential of ultraviolet‐B (UVB) radiation was investigated by analyzing the frequency and origin of micronuclei (MN) in cytokinesis‐blocked, binucleated (BN) peripheral blood lymphocytes (PBL) and cloning efficiencies (CE) of PBL after exposure to different fluences of UVB. In total, PBL obtained from five normal donors were investigated. The PBL from all donors showed a dose‐related, linear‐quadratic increase in the frequency of MN per 1000 BN cells and in the frequency of micronucleated BN cells. In two experiments the origin of UVB‐induced MN was studied by analyzing MN for the presence or absence of centromeres by applying the MN assay in combination with a centromeric probe and fluorescence in situ hybridization. This revealed, for the first time, that UVB‐induced MN were centromere negative, indicating that UVB acted exclusively as a clastogenic agent in the tested dose range. The PBL from all donors showed a clear dose‐dependent decrease in CE, after UVB exposure. The UVB‐exposed PBL from all donors showed an inverse relationship between the induction of MN and the decrease in CE, but regression analysis revealed no correlation between the induction of MN and the decrease in cell survival. It is concluded that UVB has a clastogenic and cytotoxic effect on PBL.
Photoactivated Psoralens Elicit Defense Genes and Phytoalexin Production in the Pea Plant
Authors: Parsons M. Angela , Hadwiger Lee A.
Published: January 2008

In the pea plant (Pisum sativum), compounds that intercalate into DNA induce the production of ∼20 major proteins similar to the pattern induced during nonhost disease resistance to the bean fungal pathogen, Fusarium solani f.sp. phaseoli. The pea phytoalexin, pisatin, as well as RNA homologous to several disease‐resistance response (DRR) genes accumulate following treatment with these compounds. Psoralen was chosen to characterize this interaction further because it intercalates into DNA and, following irradiation with 365 nm UV light (UV365), forms covalent bonds with pyrimidines on either or both strands of DNA. This produces monoadducts or cross‐links, respectively. Dose experiments showed that 60 μg/mL 4′‐aminomethyl‐4,5′,8‐trimethylpsoralen followed by 18 J/cm2 UV365 was sufficient to produce an accumulation of pisatin similar to that produced in response to the fungus. Under these inducing conditions, there was an average of 0.19 adducts per kb of pea genomic DNA. The accumulation of pisatin and the RNA of several DRR genes by psoralen required photoactivation, which suggests that covalent binding to DNA was necessary for induction. As the promoters of several putative fungal‐induced pea genes contain long stretches of d(AT)n, which is the preferred psoralen photobinding site, restriction fragments spanning DRR genes were examined after in vivo psoralen treatment. The rate of crosslinking was compared between fungal‐induced and noninduced genes using a modified Southern blot analysis. Implications of the induction of the DRR due to psoralen binding are discussed.
Protection of UV‐induced Suppression of Skin Contact Hypersensitivity: A Common Feature of Flavonoids after Oral Administration?
Authors: Steerenberg P. A. , Garssen J. , Dortant P. , Hollman P. C. , Alink G. M. , Dekker M. , Bueno‐de‐Mesquita H. B. , Van Loveren H.
Published: January 2008

In this study we investigated the effect of the dietary ingredients fruit and vegetable, green tea phenol extract (GTP) and the specific flavonoid components quercetin and chrysin on the UV‐induced suppression of the con‐tact hypersensitivity (CHS) response to picryl chloride (PCl). The SKH‐1 mice were fed with test diet from 2 or 4 weeks before and during the UV irradiation (daily, 95 mJ/cm2) and tested for the CHS ear‐swelling response 10 weeks after the onset of the irradiation. For the CHS, mice were immunized with PCl by epicutaneous application on nonirradiated sites. Four days after sensitization all mice were challenged on both sides of each ear by topical application of one drop PCl. In addition, from mice fed with the fruit and vegetable mixture the number of Langerhans cells (LC) were scored in the skin and from mice fed with quercetin, quercetin levels in plasma were measured at week 11 after the start of UV irradiation. It was found that fruit and vegetable (19% in the diet), GTP (0.1% and 0.01% in the drinking water), quercetin (1% in the diet) and chrysin (1% and 0.1% in the diet), prevented statistically significantly the UV‐induced suppression of CHS to PCl. In the skin of mice fed with fruit and vegetables combined with UV irradiation the number of LC were comparable to the control mice, whereas the number of LC were significantly diminished in mice treated with UV only. This protective effect on the presence of LC in the epidermis after UV irradiation, which was also observed in a previous study with quercetin, may play a role in the prevention of UV‐induced immunosuppression by the flavonoids tested. In conclusion, we found protection of flavonoids against UV‐induced effects on CHS, which may be a common feature of most flavonoids.
The Correct Use of “Average” Fluorescence Parameters
Authors: Sillen Alain , Engelborghs Yves
Published: January 2008

When more than one fluorophore is present or when one fluorophore displays a multiple exponential decay “average” fluorescence parameters are derived, which can be combined with “average” lifetimes for further interpretation. However, two kinds of average lifetimes are used in this context: the intensity and the amplitude average lifetime. In this paper the different average parameters are carefully analyzed and their “best” combinations are derived. These average parameters are analyzed in the context of external and internal dynamic and static quenching, Föster energy transfer and the calculation of the radiative rate constant. The use of the amplitude average lifetime for the analysis of multiple fluorophore‐containing systems and the detection of interactions is discussed.
Probing for the Threshold Energy for Visual Transduction: Red‐Shifted Visual Pigment Analogs from 3‐Methoxy‐3‐Dehydroretinal and Related Compounds
Authors: Imai Hiroo , Hirano Takahiro , Terakita Akihisa , Shichida Yoshinori , Muthyala Rajeev S. , Chen Rong‐Liang , Colmenares Leticia U. , Liu Robert S. H.
Published: January 2008

Abstract— While azulenic retinal analogs failed to yield a red‐shifted visual pigment analog, the 9‐cis isomers of the push‐pull polyenals 3‐methoxy‐3‐dehydroretinal and 14F‐3‐me‐thoxy‐3‐dehydroretinal yielded iodopsin pigment analogs with absorption maxima at, respectively, 663 and 720 nm. The former gave a relatively stable batho product (700 nm) and was able to activate transducin. A lower activity was observed for the latter. One possible explanation for the combined results is that the excitation energies of these red‐shifted pigments are approaching the threshold energy for visual transduction (although at this time we cannot rigorously exclude a role of the added F‐atom in reducing the transducin activity).
The Anatomic Site of Photodynamic Therapy is a Determinant for Immunosuppression in a Murine Model
Authors: Musser David A. , Camacho Susan H. , Manderscheid Patricia A. , Oseroff Allan R.
Published: January 2008

Abstract— The role of the irradiation site in the induction of suppression of the contact hypersensitivity (CHS) response following photodynamic therapy (PDT) was examined in a murine model. Laser irradiation on the flanks of nontumor‐bearing Photofrin®‐injected mice caused suppression of the CHS response. If the irradiation was conducted on a subcutaneously implanted foil disc on the flank no immunosuppression occurred, indicating that no suppressive factor (s) of sufficient quantity to cause suppression was released from the skin, but rather irradiation of internal organs was the cause. Irradiation of tumors implanted on the flanks of mice reduced the suppression, suggesting an immunopotentiating effect of PDT. Irradiation on the thigh in the presence or absence of a tumor gave no immunosuppression. These results suggest that the anatomic site of irradiation is one determinant for the elicitation of suppression of the CHS response.
Photoinduced Structural Volume Changes in Aqueous Solutions of Blepharismin
Authors: Losi Aba , Vecll Arnaldo , Viappiani Cristiano
Published: January 2008

Abstract— Employing time‐resolved photoacoustics we measured the structural volume changes (ΔVri) occurring after photo‐excitation of blepharismin (BPR) aqueous solutions; an expansion occurring in the subnanosecond timescale is followed by a back contraction within some hundreds of nanoseconds. The magnitude of the ΔVri strongly depends on pH, allowing the determination of the pKa of BPR with this method. The values so measured are very close to those found by means of UV‐visible absorption spectroscopy. The presence of water‐soluble electron acceptors or donors (hexacyanoferrate [III] hexacyanofer‐rate [II]) as well as the concentration of oxygen do not affect the magnitude or the kinetics of the structural volume changes. On the contrary, we detect a strong deuterium effect; this suggests that the observed ΔVri are related to an altered hydrogen bond pattern of the excited state of the pigment with respect to the ground state. Comparative measurements with the parent compound hypericin are also reported, suggesting that the photo‐induced expansion‐contraction pattern is a general characteristic of polyhydroxylated quinones.
Photoinactivation of Deinococcus radiodurans: An Unusual Gram‐Positive Microorganism
Authors: Nitzan Yeshayahu , Ashkenazi Helena
Published: January 2008

Abstract— Deinococcus radiodurans is an extremely resistant bacteria to both the mutagenic and lethal effects of ionizing radiation. In the present study, we examined photoinactivation of D. radiodurans by two photosensitizers, the hydrophilic cationic 5, 10, 15, 20‐tetra(4‐N‐methylpyridyl)porphine (TMPyP) and deuteroporphyrin (Dp), which is more hydrophobic. The effects of illumination with various lights at different wavelengths were also examined. Efficient photoinactivation was obtained when D. radiodurans cells were irradiated by blue light (400–450 nm) and treated with TMPyP at a concentration of 0.73 μM. Under these conditions the viability of the culture was found to be decreased by almost seven orders of magnitude after 2 min of treatment at an intensity of only 2 J/cm2. Treatment of the bacterial cells by TMPyP or Dp resulted in an efflux of potassium and magnesium from the cells as well as phosphate loss as a function of light dose. These results can be regarded as a consequence of membrane damage, because transmission electron microscopy revealed cell membrane damage shortly after exposure of the treated bacteria to light. In addition, asymmetric septation and filamentous chromosomal DNA were seen. It seems that the cytoplasmic membrane damage in this bacterial species plays a more important role than in other gram‐positive or even gram‐negative bacteria, rendering this radiation‐resistant bacterium susceptible to photosensitization.
Evaluation of UVA‐Mediated Oxidative Damage to Proteins and Lipids in Extracorporeal Photoimmunotherapy
Authors: Reinheckel Thomas , Bohne Marisela , Halangk Walter , Augustin Wolfgang , Gollnick Harald
Published: January 2008

Abstract— The combination of UVA and 8‐methoxypsoralen (8‐MOP) is known for the ability to produce reactive oxygen species (ROS) that react subsequently with DNA, lipids and proteins. In most studies concerned with UVA effects mediated by free radicals, UVA doses higher than those exhibiting beneficial clinical results in extracorporeal photoimmunotherapy (ECPI) were used. The present study was undertaken to determine markers of oxidative stress in plasma and cells from the buffy coat using conditions relevant for ECPI (cumulative UVA dose at the sample level ≤ 2 J/cm2). Plasma exposed to UVA of 20 J/cm2 resulted in protein oxidation as well in crosslinking and fragmentation revealed by electrophoresis. Exposure of the buffy coat and plasma to considerably lower doses of UVA (up to 2 J/cm2) combined with various 8‐MOP concentrations resulted neither in an increase of malon‐dialdehyde as a marker of Hpid peroxidation nor in a changed electrophoretic protein pattern. In these same experiments the total antioxidative capacity decreased to 65% of the initial value, suggesting that the antioxidative defense of plasma is able to cope with oxidative stress under ECPI conditions. These results were confirmed by data from 10 patients with scleroderma or cutaneous T‐cell lymphoma during ECPI treatment. The present results suggest that, although ROS are formed during ECPI, gross oxidative damage does not occur. It is, however, possible, that specific effects mediated by oxygen radicals may co‐trigger the photoimmunomodulatory effects of ECPI.
Cell Cycle Delay, Mitochondrial Stress and Uptake of Hydrophobic Cations Induced by Sunscreens in Cultured Human Cells
Authors: Xu Chunxia , Parsons Peter G.
Published: January 2008

Abstract— Reports of systemic absorption of sunscreens prompted a study of the effects of emulsions of 3 commonly used sunscreens on cultured human cells; vegetable oil and paraffin oil were used as controls. Ethylhexyl p‐methoxycinnamate (EHMC), octyl p‐dimethylaminobenzoate (PABA) and oxybenzone (OB) inhibited cell growth and DNA synthesis and retarded cycle progression from G1 in the dose range 25–100 μg/mL. An extended period of exposure (up to 24 h) was required for maximum uptake of sunscreens and for inhibition of cell growth. Melanocytes and fibroblasts tended to be more resistant than tumor cell lines (melanoma, cervical carcinoma). Sunscreens had no major effects on the transcription of certain genes, as judged by the activity of reporter constructs driven by the p53, c‐fos and metal response (sheep metallothionein Ia promoter) elements and transfected into a human melanoma cell line (MM96L). The activity of the cytomegalovirus promoter was also not affected. A cell line (CI80–13S) with mitochondrial dysfunction was significantly more sensitive to growth inhibition by EHMC and PABA than the other cell lines tested. Treatment of MM96L with the mitochondrial inhibitor ethidium bromide sensitized the cells to killing by cotreatment with sunscreens, in association with increased cellular uptake of ethidium bromide. These results established conditions for studying the action of sunscreens on cultured human cells. Further studies are required to determine whether the mitochondrial stress and changes in drug uptake associated with sunscreens in the above cell lines are relevant to their action in vivo.
Time‐Resolved Studies of the Excited‐State Dynamics of meso‐Tetra(hydroxylphenyl)chlorin in Solution
Authors: Howe Lori , Suchetat Artur , Einarsdóttir Ólof , Zhang Jin Z.
Published: January 2008

Abstract— Meso‐tetra(hydroxyphenyl)chlorin (m‐THPC) is a new photosensitizer developed for potential use in photodynamic therapy (PDT) for cancer treatment. In PDT, the accepted mechanism of tumor destruction involves the formation of excited singlet oxygen via intermolecular energy transfer from the excited triplet‐state dye to the ground triplet‐state oxygen. Femtosecond transient absorption measurements are reported here for the excited singlet state dynamics of m‐THPC in solution. The observed early time kinetics were best fit using a triple exponential function with time constants of 350 fs, 80 ps and 3.3 ns. The fastest decay (350 fs) was attributed to either internal conversion from S2 to S1 or vibrational relaxation in S2. Multichannel time‐resolved absorption and emission spectroscopies were also used to characterize the excited singlet and triplet states of the dye on nanosecond to microsecond time scales at varying concentrations of oxygen. The nanosecond time‐resolved absorption data were fit with a double exponential with time constants of 14 ns and 250 ns in ambient air, corresponding to lifetimes of the S1 and T1 states, respectively. The decay of the T1 state varied linearly with oxygen concentration, from which the intrinsic decay rate constant, ki, of 1.5 × 106 s−1 and the bimolecular collisional quenching constant, kc, of 1.7 × 109M−1 s−1 were determined. The lifetime of the S1 state of 10 ns was confirmed by fluorescence measurements. It was found to be independent of oxygen concentration and longer than lifetimes of other photosensitizers.
The Human Melanocyte as a Particular Target for UVA Radiation and an Endpoint for Photoprotection Assessment
Authors: Marrot Laurent , Belaidi Jean‐Philippe , Meunier Jean‐Roch , Perez Philippe , Agapakis‐Causse Catherine
Published: January 2008

Abstract— The induction of DNA breaks by UVA (320–400 nm) in the nucleus of normal human melanocytes in culture was investigated using single cell gel electrophoresis, also called the comet assay. Endogenous pigment and/or melanin‐related molecules were found to enhance DNA breakage: comets were more intense in melanocytes than in fibroblasts, in cells with high melanin content or after stimulation of melanogenesis by supplying tyrosine in the culture medium. After UVA doses where strong comets were observed, neither cytotoxicity nor stimulation of tyrosinase activity were detected. However, the accumulation of p53 protein suggested that cells reacted to genotoxic stress under these experimental conditions. The same approach was used to compare two sunscreens with identical sun protection factors but different UVA protection factors. The results presented in this paper suggest that human melanocytes may be used as a target cell to evidence broadspectrum photoprotection. Moreover, these data appear to be helpful in getting a better understanding of the role of sunlight in the initiating steps of melanocyte transformation.
Differences in Sensitivity to UVC, UVB and UVA Radiation of a Multidrug‐Resistant Cell Line Overexpressing P‐Glycoprotein
Authors: Trindade G. S. , Capella M. A. M. , Capella L. S. , Affonso‐Mitidieri O. R. , Rumjanek V. M.
Published: January 2008

Abstract— Multidrug resistance (MDR) is the phenomenon in which cultured tumor cells, selected for resistance to one chemotherapeutic agent, simultaneously acquire resistance to several apparently unrelated drugs. The MDR phenotype is multifactorial. The best‐studied mechanism involves the expression of a membrane protein that acts as an energy‐dependent efflux pump, known as P‐glycoprotein (Pgp), capable of extruding toxic materials from the cell. In this work, resistance to UVA radiation, but not to UVC nor UVB, was observed in an MDR leukemia cell line. This cell line overexpresses Pgp. To study the role of Pgp in the resistance to UVA radiation, two MDR modulators or reversing agents (verapamil and cyclosporin A) capable of blocking Pgp activity were used. Cell viability was assessed and the techniques of flow cytometry and fluorescence microscopy were employed to measure the extrusion of rhodamine 123 by the efflux pump. The results show that MDR modulators did not modify the resistance to UVA radiation. Furthermore, although cell viability was not significantly altered, Pgp function was impaired after UVA treatment, suggesting that this glycoprotein may be a physical target for oxidative damage, and that other factors may be responsible for the UVA resistance. In agreement with this, it was found that the resistant cell line presented a higher catalase activity than the parental (non‐MDR) cell line.
Fluorescence Analysis of Oat phyA Deletion Mutants Expressed in Tobacco Suggests that the N‐Terminal Domain Determines the Photochemical and Spectroscopic Distinctions between phyA′ and phyA″†
Authors: Sineshchekov Vitally A. , Clough Richard C. , Jordan‐Beebe Emily T. , Vierstra Richard D.
Published: January 2008

Abstract— In vivo low‐temperature (85 K) fluorescence spectroscopy has defined two phytochrome A (phyA) subpopulations, designated phyA′ and phyA″, in etiolated seedlings (V. A. Sineshchekov, J. Photochem. Photobiol. 28, 53–55, 1995). Phytochrome A' is the more abundant but light‐labile species characterized by longer wavelength emission/absorption maxima (687/673 nm) and by a higher extent of the photoconversion of its red‐absorbing form (Pr) into photoproduct (lumi‐R) at 85 K (γ1≈ 0.5). Phytochrome A″ is the minor but relatively light‐stable species, characterized by shorter wavelength maxima (682/668 nm) and by a lower γ1 (<0.05). To help define domains within phyA responsible for these differences, the low‐temperature spectral properties of transgenic tobacco expressing full‐length (FL) oat phyA and C‐and N‐terminally truncated versions (CD [Δ786–1129] and NA [Δ7–69], respectively) were compared. Oat phytochrome expression was more pronounced than that of tobacco in the basal section of etiolated seedlings following 2 h irradiation with white light. Seedlings expressing FL and CD phyA had spectral properties for phyA′ and phyA″ that were indistinguishable from that of wild‐type tobacco. Conversely, expression of NA phyA generated an abundant phy species that behaved like phyA″. From this we conclude that the N‐terminal domain of phyA is involved in determining the photochemical and spectroscopic distinctions between the native phyA′ and phyA″ species.
Absorption Spectra of Planarian Visual Pigments and Two States of the Metarhodopsin Intermediates
Authors: Azuma K. , Iwasaki N. , Ohtsu K.
Published: January 2008

Abstract— Absorption spectra measurements of isolated planarian ocelli by a microspectrophotometer (MSP) and intra‐ocellar recordings of the early receptor potential (ERP) were carried out in order to characterize in situ planarian rhodopsin (pRh) and its photoproducts. The MSP spectra of the isolated ocelli revealed Λmax at about 500 nm. The ERP evoked by a test stimulus was a positive monophasic waveform in the dark but became negative during exposure to violet light. During subsequent darkness, the ERP rapidly reverted to a positive waveform but with a smaller amplitude than before exposure. The ERP amplitude recovered to its initial level upon exposure to red light. The ERP experiments suggest that pRh produces two metarhodopsin intermediates, with Λmax longer than that of pRh: the metastate responsible for the negative ERP converts to another metastate that results in a smaller ERP in the dark‐adapted ocellus.
Epidemiological Support for an Hypothesis for Melanoma Induction Indicating a Role for UVA Radiation
Authors: Moan J. , Dahlback A. , Setlow R. B.
Published: January 2008

Abstract. An hypothesis for melanoma induction is presented: UV radiation absorbed by melanin in melanocytes generates products that may activate the carcinogenic process. Products formed by UV absorption in the upper layers of the epidermis cannot diffuse down as far as to the melanocytes. Thus, melanin in the upper layer of the skin may be protective, while that in melanocytes may be pho‐tocarcinogenic. Observations that support this hypothesis include: (1) Africans with dark skin have a reduced risk of getting all types of skin cancer as compared with Caucasians, but the ratio of their incidence rates of cutaneous malignant melanoma to that of squamous cell carcinoma is larger than the corresponding ratio for Caucasians. (2) Albino Africans, as compared with normally pigmented Africans, seem to have a relatively small risk of getting cutaneous malignant melanomas compared to nonmela‐nomas. This is probably also true for albino and normally pigmented Caucasians. (3) Among sun‐sensitive, poorly tanning persons, frequent UV exposures are associated with increased risk of melanoma, wherease among sun‐resistant, well‐tanning persons, increased frequency of exposure is associated with decreased melanoma risk. (4) It is likely that UVA, being absorbed by melanin, might have a melanoma‐inducing effect. This is in agreement with some epidemiological investigations which indicate that sun‐screen lotions may not protect sufficiently against melanoma induction. The relative latitude gradient for UVA is much smaller than that for UVB. The same is true for the relative latitude gradient of cutaneous malignant melanoma as compared with squamous cell carcinoma and basal cell carcinoma. Under the assumption that the average slopes of the curves relating incidence rates with fluences of carcinogenic UV radiation are similar for melanomas and nonmelanomas, these facts are in agreement with the assumption that UVA plays a significant role in the induction of melano.
Dosimetry of Room‐Air Germicidal (254 nm) Radiation Using Spherical Actinometry
Authors: Rahn Ronald O. , Xu Peng , Miller Shelly L.
Published: January 2008

Abstract A method is demonstrated for measuring germicidal (254 nm) ultraviolet radiation (GUVR) in an omnidirectional manner. This method has application for those situations in which GUVR from more than one direction or source impinges on a three‐dimensional object, and when it is of interest to determine the radiant fluence experienced by the object. For this purpose spherical quartz vessels were constructed varying from 0.8 to 1.25 cm in outside diameter. The vessels were filled with an actinometric solution consisting of KI and KIO3. This solution is optically opaque at 254 nm and is insensitive to room light. Upon exposure to GUVR this solution, which counts all of the 254 nm photons, forms triiodide. Following radiation, the contents of the vessel are removed, the absor‐bance of triiodide measured and the fluence rate determined based on a relationship that takes the volume to cross‐sectional area of the actinometer into account It is demonstrated that, in accordance with theory, the area of the sphere that intercepts the radiant energy is equal to the cross‐sectional area of the sphere. A demonstration of the utility of this method was carried out in a test facility 90 m3 equipped with five GUVR lamps, one in each corner and one in the center of the room, arranged so as to irradiate the air in the upper portion of the room. Twenty spherical actinometers were evenly distributed around the room and exposed for either 30 or 90 min. The fluence rate varied from one part of the room to another, peaking near the GUVR lamps at 65–85 uW per cm2. By averaging over all 20 points, a pseudoaverage fluence rate for the room was found to be 42 and 44 u.W per cm2 for the 30 and 90 min exposures, respectively. The similarities in the values for this metric demonstrates that the actinometric response is linear over this exposure range.
Singlet Oxygen‐mediated Photobleaching of the Prosthetic Group in Hemoglobins and C‐Phycocyanin
Authors: Tapia Guillermina , Galetovic Alexandra , Lemp Else , Pino Eduardo , Lissi Eduardo
Published: January 2008

Proteins bearing colored prosthetic groups, such as the heme group in hemoglobin or the bilin group in c‐phy‐cocyanin, quench singlet oxygen by interactions at the apoprotein and the prosthetic group levels. In both proteins, chemical modification of the chromophore constitutes only a minor reaction pathway. While total deactivation of singlet oxygen takes place with rate constants of 4.0 times 109and 4.2 times 108M‐ls‐1for hemoglobin and phycocyanin, respectively, the bleaching of the chromophore takes place with rate constants of 3.2 times 106and ˜1 times 107M‐1s‐1. Irradiation of phycocyanin with red light bleaches the chromophore with low yields (˜0.8 times 10‐4). Part of this bleaching is mediated by singlet oxygen produced by the irradiation of the bilin group. The low relevance of the singlet oxygen pathway is compatible with a low quantum yield (˜10‐3) of free singlet oxygen production after irradiation of the protein.
A Model for the Interaction of 6‐Lauroyl‐2‐(N,N‐dimethylamino)naphthalene with Lipid Environments: Implications for Spectral Properties
Authors: Bagatoll Luis A. , Parasassi Tiziana , Fidelio Gerardo D. , Gratton Enrico
Published: January 2008

Although 6‐lauroyl‐2‐(N,N‐dimethylamino)naphthalene (LAURDAN) is now widely used as a probe for lipid systems, most studies focus on the effect of the lipid environment on its emission properties but not on the excitation properties. The present study is intended to investigate the excitation properties of LAURDAN in diverse lipid environments. To this end, the fluorescence properties of LAURDAN were studied in synthetic ester and ether phosphatidylcholines and sphingomyelin vesicles below, at and above the corresponding lipid main phase‐transition temperature. The excitation spectra of LAURDAN in these environments always show at least two well‐resolved bands. In the different lipid vesicles the behavior of the red band in the LAURDAN excitation spectra is sensitive to the lipid chemical environment near the probe fluorescent moiety and to the packing of the different lipid phases (gel and liquid crystalline). We propose that the interaction between the LAURDAN di‐methylamino group and the ester linkage of ester phospholipids is responsible for the strong stabilization of LAURDAN's red excitation band in the gel phase of ester phospholipid vesicles. We discuss the consequence of these proposed ground‐state interactions on LAURDAN's emission generalized polarization function. In the context of variable excitation wavelengths, information concerning solvent dipolar relaxation through excitation generalized polarization function is also discussed.
UVA and UVB Radiation Differentially Regulate Vascular Endothelial Growth Factor Expression in Keratinocyte‐derived Cell Lines and in Human Keratinocytes
Authors: Mildner Michael , Weninger Wolfgang , Trautinger Franz , Ban Jozef , Tschachler Erwin
Published: January 2008

Vascular endothelial growth factor (VEGF) is a central regulator of neoangiogenesis in inflammatory and neoplastic conditions. Ultraviolet irradiation is one of the mainstays of dermatological therapy for various inflammatory skin diseases. In the present study we have compared the effects of UV irradiation on the production of VEGF by keratinocytes (KC) and by the KC‐derived cell lines A431 and HaCaT. Irradiation of A431 and HaCaT cells with both UVA (10 J/cm2and 20 J/cm2) and UVB (8 mj/cm2and 16 mj/cm2) led to strong upregulation of VEGF mRNA and protein. Induction of VEGF by UVA and UVB in these cells was mediated by different pathways, i.e. the generation of free radicals and the secretion of (a) soluble factor(s), respectively. Unlike KC‐derived cell lines, no increase in VEGF production was observed in KC in primary culture after irradiation with the same UV doses. Increasing the irradiation dose in these cells of UVA to 40 J/cm2led to a marked decrease in soluble VEGF, whereas doses as high as 32 mj/cm2UVB only minimally affected VEGF levels. Reduction of VEGF production by KC might contribute to the effect of UVA irradiation in inflammatory skin diseases. The differential response of primary KC and autonomously growing KC‐derived cell lines to the induction of VEGF by UV light could favor neoangiogenesis in the vicinity of epidermal tumor cells in vivo, thereby endowing them with a growth advantage over normal cells.