bluuebunny/crossref_metadata_2025 huggingface dataset:
ANALYSIS OF VIRAL DNA, PROTEIN AND ENVELOPE DAMAGE AFTER METHYLENE BLUE, PHTHALOCYANINE DERIVATIVE OR MEROCYANINE 540 PHOTOSENSITIZATION
Authors: Abe Hideki , Wagner Stephen J.
Published: January 2008

Abstract— Although numerous photosensitizers have been used experimentally to decontaminate viruses in cellular blood components, little is known about their mechanisms of photoinactivation. Using M13 bacteriophage and vesicular stomatitis virus (VSV) as model viruses, we have investigated alteration of the viral genome, protein and envelope after phototreatment. Methylene blue (MB) and aluminum phthalocyanine tetrasulfonate (AlPcS4) phototreatment inactivated bacteriophage M13 and decreased the fraction of single‐stranded circular genomic DNA (sc‐DNA) by converting it to linear form. This conversion was enhanced by treating the extracted DNA with piperidine at 55°C. Piperidine‐labile breaks were well correlated to phage survival (5.1% sc‐DNA at 1.7% phage survival for MB) under conditions where only minor differences were seen in the relative abundance of M13 coat protein on sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS‐PAGE). Neither aluminum phthalocyanine (AlPc) nor merocyanine 540 (MC540) inactivated M13 nor were there significant changes observed in DNA and coat protein. Methylene blue, AlPcS4 and AlPc inactivated VSV and inhibited fusion of the virus envelope to Vero cells at pH 5.7 (i.e. with plasma membrane). However, the degree of this inhibition was small compared to the extent of virus inactivation (43% inhibition vs 4.7 log10 or 99.998% inactivation, for MB). In contrast, an antibody to VSV G‐spike protein inhibited fusion at pH 5.7 by 52% with a concomitant decline in VSV infectivity of 0.15 log10 (30%). Few changes were observed in the relative abundance of G protein for MB and AlPcS4 phototreated samples and no additional protein bands were observed on SDS‐PAGE. Phototreatment did not appreciably change the relative fusion ability at pH 7.2 (via the endocytotic pathway). These results collectively suggest that nucleic acid may be an important target for photoinactivation of these model viruses by MB and AlPcS4.
SENSITIVITY OF ACTIVATED MURINE PERITONEAL MACROPHAGES TO PHOTODYNAMIC KILLING WITH BENZOPORPHYRIN DERIVATIVE
Authors: Hunt David W. C. , Jiang Hui‐Jun , Levy Julia G. , Chan Agnes H.
Published: January 2008

Abstract— This study compared the ability of highly purified resting and activated DBA/2 mouse peritoneal macrophages to survive treatment with the photosensitizer benzoporphyrin derivative (BPD, verteporfin) and light. Culture of macrophages with recombinant murine interferon‐γ (rIFN‐γ, 100 U/mL) for 72 h imparted a phenotypic and functional activation by dramatically increasing cell surface expression of major histocompatibility complex Class II (Ia) molecules and the formation of nitric oxide. The rIFN‐γ‐activated macrophages were significantly (P < 0.05) more sensitive (lethal dose to cause a 50% reduction in cell survival, LD50= 14.4 ± 1.1 ng/mL) to photodynamic killing with BPD and light (10 J/cm2) than cells (LD50= 18.2 ± 2.0 ng/mL) cultured in medium alone. In contrast, macrophages treated with different concentrations of bacterial lipopolysaccharide (LPS) were as resistant or more resistant to photodynamic killing than cells cultured in medium alone. No cytotoxic effect of BPD was detected in cultures containing the drug but protected from light. Comparable amounts of BPD were taken up in vitro by unactivated and rIFN‐γ‐activated macrophages, as detected by flow cytometric analysis. However, cells cultured with LPS (10 μg/mL) took up more BPD than macrophages cultured in medium alone or with rIFN‐γ. The DBA/2 P815 mastocytoma cells took up greater amounts of the drug and were subsequently more vulnerable to treatment with BPD and light (LD50= 6.9 ng/mL) than macrophages cultured under any condition. The explanation for the increased vulnerability of rIFN‐γ‐activated macrophages and the greater resistance of LPS‐activated macrophages, relative to medium‐cultured macrophages, to photodynamic killing with BPD is uncertain. However, the increased susceptibility of macrophages, activated with the immunomodulatory cytokine IFN‐γ, to treatment with BPD and light might indicate how photodynamic therapy could interfere with the development of experimental autoimmune disease, conditions in which activated macrophages are known to be involved.
ANALYSIS OF LASER‐INDUCED FLUORESCENCE LINE SHAPE OF INTACT LEAVES: APPLICATION TO UV STRESS DETECTION
Authors: Subhash N. , Azzinghi Piero M , Agati Giovanni , Fusi Franco , Lercari Bartolomeo
Published: January 2008

Abstract— In vivo laser‐induced fluorescence spectra of intact leaves of healthy and UV‐irradiated Salvia splendens plants excited at 337 nm by a nitrogen laser were recorded using an optical multichannel analyzer system. The spectra showed the typical fluorescence bands centered around 450, 530, 685 and 730 nm. Exposure to UV radiation changed the relative intensity values of these bands and their peak positions. The analysis of the acquired spectra in terms of a linear combination of Gaussian bands was carried out to determine accurately the peak positions and the relative intensity contribution of the various bands to the laser‐induced fluorescence spectra on healthy and UV‐treated plants of different age.

The results indicate that a curve‐fitting analysis of the measured fluorescence spectra is a useful and sensitive method to discriminate the various band contribution to the whole leaf fluorescence spectrum. The comparison among blue‐green and red‐far‐red fluorescence of leaves was also confirmed as an effective indicator of UV stress in plants.
Excited States and Charge Separation in Membranes of the Green Sulfur Bacterium Prosthecochloris aestuarii
Authors: Kramer Hans , Aartsma Thijs J. , Amesz Jan
Published: January 2008

Time‐resolved absorbance changes were measured in isolated membranes, depleted of chlorosomes, and in the Fenna‐Matthews‐Olson (FMO) complex of the green sulfur bacterium Prosthecochloris aestuarri. The isolated FMO complex showed a biphasic decay of excited bacteriochlorophyll a (BChl a) with time constants of about 80 and 1400 ps. Approximately the same time constants were observed upon excitation of isolated membranes together with a component of about 30 ps. It is concluded that the efficiency of energy transfer from the FMO to the core complex is very low, in agreement with earlier measurements of the efficiency of charge separation. The 30 ps decay component is ascribed to trapping of the excitation energy from the core BChl a by the reaction center.
Characterization of Carotenoid Triplet States in the Light‐Harvesting Complex B800–850from the Purple Bacterium Rubrivivax gelationsus
Authors: Jirsakova Vladimira , Reiss‐Husson Françoise , Dijk Bas van , Owen Gabrielle , Hoff Arnold J.
Published: January 2008

The carotenoid triplet states in the light‐harvesting complex B800–850from purple bacterium Rubrivivax gelatinosus were characterized by absorption‐detected magnetic resonance in zero magnetic field (ADMR) spectroscopy. Detailed HPLC analysis of carotenoids from B800–850demonstrated the presence of several carotenoids bound to the complex: the major ones are hydroxyspheroidene and spheroidene (together 80%), followed by neurosporene and hydroxyneurosporene (7%), spheroidenone and hydroxyspheroidenone (7.5%) and two other minor carotenoids that could be 3,4‐dihydrospheroidenone and 3,4‐dihydrohydroxyspheroidenone (5.5%). Three triplet states originating from carotenoids present in the B800–850were observed. The identical T‐S spectra recorded at selectively chosen 2|E| transitions of carotenoids indicated that all these triplet states can be attributed to three different populations of one carotenoid family, probably to spheroidene and to hydroxyspheroidene, with different out‐of‐plane distortions of their polyene chain due to a different protein environment. Triplet states of the neurosporene and the spheroidenone families are probably not observed because of the low signal amplitude.
Book Review
Authors: Bocian David F.
Published: January 2008

Infrared Spectroscopy of Biological Molecules. Edited by Henry H. Mantsch and Dennis Chapman
Experimental Correspondence between Spore Dosimetry and Spectral Photometry of Solar Ultraviolet Radiation
Authors: Munakata Nobuo , Morohoshi Fumiko , Hieda Kotaro , Suzuki Keiji , Furusawa Yoshiya , Shimura Hidehiro , Ito Tomoyuki
Published: January 2008

Abstract— The biologically effective dose of solar UV radiation was estimated from the inactivation of UV‐sensitive Bacillus subtilis spores. Two types of independent measurements were carried out concurrently at the Aerological Observatory in Tsukuba: one was the direct measurement of colony‐forming survival that provided the inactivation dose per minute (ID/min) and the other was the measurement of the spectral irradiance by a Brewer spectrophotometer. To obtain the effective spectrum, the irradiance for each 1 nm wavelength interval from 290 to 400 nm was multiplied with the efficiency for inactivation derived from the inactivation action spectrum of identically prepared spore samples. Integration of the effective spectrum provided the estimate for ID/min. The observed values of ID/min were closely concordant with the calculated values for the data obtained in four afternoons in 1993. The average ratio (±SD) between them was 1.24 (±0.16) for 14 data points showing high inactivation rates (<0.05 ID/min). Considering difficulties in the absolute dosimetry of UV radiation, the concordance was satisfactory and improved credibility of the two types of monitoring systems of biologically effective dose of solar UV radiation.
Effects of Solar UV Radiation on Diatom Assemblages of the Mediterranean
Authors: Santas Regas , Häder Donat‐P. , Lianou Charalambia
Published: January 2008

Abstract— Three UV treatments (PAR; PAR + UVA; PAR + UVA + UVB) were performed by placing different UV‐ab‐sorbing Alters over communities developing on ceramic tiles in a natural marine habitat near Korinthos, Greece. The experiment was repeated at three depths (0.5 m, 1 m, 1.5 m) below the surface of the sea. Differences in community structure due to UV radiation exposure were more pronounced during the early stages of community development. After the first 3 weeks of growth, the productivity of the PAR + UVA + UVB treatment was significantly lower than the PAR + UVA but not than the PAR treatment. This difference did not persist thereafter. At 5 weeks of growth, the productivity at 0.5 m was significantly lower that at 1.0 m. No other significant differences were observed. The findings of the present study suggest that periphytic communities occurring at the upper layers of the euphotic zone may be capable of adjusting to changes in environmental stresses such as by increased solar UVB irradiance.
A Monoclonal Antibody Affects Chromophore Apoprotein Interactions of Phytochrome A
Authors: Nakazawa Miki , Moriuchi Naoko , Wada Naoko , Hirose Tetsuya , Tokutomi Satoru , Manabe Katsushi
Published: January 2008

Abstract— A monoclonal antibody designated Mep‐1 was raised against phytochrome A from pea (Pisum sativum L.). The binding of this antibody (class IgG1) to partially degraded phytochrome (114 kDa) caused a considerable increase in the far‐red peak at the red‐light‐induced stationary state. The effect reached a plateau value when the antibody and phytochrome were present in approximately equimolar amounts. The dark transformation of the far‐red‐light‐absorbing form to the red‐light‐absorbing form of the 114 kDa phytochrome was inhibited by the addition of the antibody. However, binding of the antibody to the undegraded 121 kDa phytochrome had no effects on the spectrum of the red‐light‐induced steady state. The site at which the antibody bound to phytochrome was determined to be between amino acid residues 256 and 383 of pea phytochrome A. This is the first report of a monoclonal antibody that enhances the far‐red absorption of phytochrome in the red‐light‐induced photostationary state.
Book Review
Authors: Sandison David R.
Published: January 2008

Fluorescence Imaging Spectroscopy and Microscopy. Edited by Xue Feng Wang and Brian Herman.
Kinetics of Repair of UV‐induced DNA Damage in Repair‐proficient and ‐deficient Cells as Determined by Quantitative Polymerase Chain Reaction
Authors: McCarthy Michael J. , Rosenblatt Judah I. , Lloyd R. Stephen
Published: January 2008

Abstract— Advances in methodologies to monitor gene‐specific repair in human cells have facilitated a detailed understanding of the complexity of the nucleotide excision repair system. One of these procedures, quantitative polymerase chain reaction (QPCR), holds significant promise for dissecting the fine structure of the repair of UV‐induced DNA damage. This assay was used to study the repair of UV photoproducts in both actively transcribed and nontranscribed genes from human cells that were capable of (1) repair of both cyclobutane pyrimidine dimers and 6‐4 photoproducts; (2) removal of neither dinners nor 6‐4 photoproducts; (3) strong preferential repair of 6‐4 photoproducts relative to dimers; and (4) severely depressed rates of 6‐4 photoproducts and dimers. Detailed kinetic analyses revealed that repair of both active and inactive genes can be studied with a very fine degree of precision and that the repair status of the cells can easily be detected by use of the procedures described.
Enzyme‐assisted Cell Photosensitization: A Proposal for an Efficient Approach to Tumor Therapy and Diagnosis. The Rose Bengal Fluorogenic Substrate
Authors: Bottiroli G. , Croce A. C. , Balzarini P. , Locatelli D. , Baglioni P. , Nostro P. Lo , Monici M. , Pratesi R.
Published: January 2008

Abstract— Rose bengal, a xanthene derivative among the most efficient producer of singlet oxygen, was submitted to a chemical modification consisting in the introduction of an acetate group into the aromatic ring fluorophore structure. The acetate group acts as a quencher, thus inactivating both fluorescence and photosensitization properties of the molecule. In the modified structure, rose bengal acts as a fluorogenic substrate giving rise to the cellular reaction termed fluorochromasia. The acetate group is recognized by a carboxylic esterase activity that splits it. Removal of the quencher group results in restoring the native structure of photosensitizer inside the cells. The intracellular turnover of rose bengal acetate was studied in rat glioma‐derived cultured cells, in terms of the balance of the processes of influx and enzyme hydrolysis of the fl0075 orogenic substrate, and of the efflux of the fluorescent product. A large intracellular accumulation of photosensitizer is obtained when treatments are performed with the fluorogenic substrate, even at the drug concentration at which rose bengal does not enter the cells. The intracellular localization allows rose bengal to exert a more effective photosensitization effect. Provided that the quencher group is selected according to the metabolic properties of the tumor cells, the use of fluorogenic substrates as photosensitizer precursors could improve fluorescence diagnosis and the photodynamic therapy of tumors, exploiting the biological properties that distinguish pathological from normal conditions.
Stepwise Two‐Photon Excitation of 1,5‐Dihydroflavin Mononucleotide: Study of Flavosemiquinone Properties
Authors: Hanine‐Lmoumene Chaibia El , Lindqvist Lars
Published: January 2008

Abstract— Photoionization of 1,5‐dihydroflavin mononucleotide in aqueous solution, leading to formation of hydrated electrons and flavosemiquinone radicals, was studied by nanosecond laser photolysis (excitation at LD = 354.7 nm). Analysis of the laser fluence dependence of the photoionization yield indicates that the reaction is a stepwise two‐photon reaction with the fluorescent state of the flavin as intermediate step. Extinction coefficients of the neutral and anionic flavosemiquinone were obtained from transient absorbance measurements at 310‐660 nm. The pro‐tolytic dissociation constant of the neutral radical was determined from a study at pH 7.3‐10.3 of the spectral evolution in the submicrosecond time range due to de‐protonation of this radical. The value pKa= 8.7 0.1 was obtained. The formation of tautomeric species of the neutral flavosemiquinone was suggested based on the existence of a spectral evolution of this species during the first microseconds after excitation.
One‐ and Two‐Photon‐Induced Photochemistry of Modified Palladium Porphyrazines Involving Molecular Oxygen
Authors: Freyer W. , Stiel H. , Hild M. , Teuchner K. , Leupold D.
Published: January 2008

Abstract— Octaphenyltetraanthraporphyrazinato palladium undergoes a self‐sensitized photoreaction in the presence of oxygen to form a substituted palladium phthalocyanine with four endoperoxide bridges. This compound exhibits photophysical behavior similiar to palladium tetra‐tert‐butyl‐phthalocyanine. The phthalocyanine‐palladium complex with four endoperoxide bridges ejects molecular oxygen when excited by consecutive two‐photon absorption in the Q‐band region at 662 nm. This photocyclo‐reversion, which produces palladium porphyrazines bearing a diminished number of endoperoxide bridges, can occur up to four times per initial molecule. Irradiation of these photoproducts in the presence of oxygen produces substituted palladium phthalocyanine containing four endoperoxide groups.
High Resolution Structure of Bacteriorhodopsin Determined by Electron Crystallography
Authors: Kimura Yoshiaki , Vassylyev Dmitry G. , Miyazawa Atsuo , Kidera Akinori , Matsushima Masaaki , Mitsuoka Kaoru , Murata Kazuyoshi , Hirai Teruhisa , Fujiyoshi Yoshinori
Published: January 2008

Abstract— Bacteriorhodopsin pumps protons from the cytoplasm to the outside of halobacteria, Halobacterium salinarium, by using absorbed light energy. The newly observed density map at 3 Å resolution clarified nearly the entire structure; the resolution in the direction perpendicular to the membrane surface is 3.2 Å. The new structure clearly indicates the proton transfer pathway in bacteriorhodopsin. In particular, the location of key aspartic acid and glutamic acid residues in the derived structural model suggested funneling structures with different designs for input and output of protons on the cytoplasmic and extracellular sides, respectively, of the protein. This paper describes the major differences between the model based on the new observation and the former model obtained through crystallographic refinement by Grigorieff et al. (J. Mol. Biol 259; 393‐421, 1996).
Magnetic Resonance Imaging Evaluation of Photodynamic Therapy‐Induced Hemorrhagic Necrosis in the Murine M1 Tumor Model
Authors: Winsborrow BeAtrice G. , Grondey Hiltrud , Savoie Huguette , Fyfe Colin A. , Dolphin David
Published: January 2008

Abstract— Proton magnetic resonance imaging (MRI) and histological methods were used to evaluate photodynamic therapy (PDT)‐induced hemorrhagic necrosis in the murine Ml tumor within 72 h of treatment of male DBA/2 mice. The effects of three photosensitizing drugs were investigated: Photofrin (n = 4), Zn (II) phthalocyanine (n = 7) and benzoporphyrin derivative monoacid ring A (n = 11). As noted in previous studies of PDT using MRI, MRI makes possible serial, noninvasive, in vivo observation of tissue response to PDT. Our serial study of MRI and histological data confirms that tumors responded in the same way to PDT treatment using the three photosensitizing drugs: vascular damage followed by hemorrhagic necrosis. Most importantly and unlike previous MRI studies of PDT, we used a very high field magnet that enhanced the effect of magnetic susceptibility on image signal when blood is processed by the body after PDT‐induced hemorrhagic necrosis. This last finding demonstrates the utility of high field magnets and the importance of localized, serial experiments in future magnetic resonance studies of PDT.
Multiphoton‐Excited Visible Emission by Serotonin Solutions
Authors: Shear Jason B. , Xu Chris , Webb Watt W.
Published: January 2008

Nonlinear excitation of the neurotransmitter serotonin (5HT) in aqueous solution is shown to generate a blue‐green‐emitting photoproduct in addition to UV fluorescence characteristic of native 5HT. The visible emission rate in diffusional steady‐state measurements scales as the sixth power of excitation intensity, demonstrating that absorption of six near‐IR photons is required to generate emission of one visible photon. Transient measurements reveal that this process is composed of two sequential nonlinear steps, the first excited by four photons and the second by two photons. These results, in combination with measurements of multiphoton‐excited serotonin UV fluorescence, support a model in which 5HT is photochemically transformed as a consequence of four‐photon absorption (Etot≅6 eV) to a photoproduct that then emits in the visible region via two‐photon excitation. A minimum bound of ˜10‐51 cm4 s photon‐1 is observed for the two‐photon emission action cross section at 830 nm. Photoionization, rather than reaction with a dissolved oxygen species, appears to be the primary mechanism for generation of the blue‐green‐emitting photoproduct. The peak intensities required to generate significant blue‐green emission (˜5 times 1011 W cm‐2 from 80 MHz 150 fs titanium: sapphire laser pulses) are approximately five‐fold higher than are typically used in two‐photon laser scanning microscopy but are still substantially lower than the estimated intensity needed to induce dielectric breakdown of water.
Photoreactivating Enzyme for (6–4) Photoproducts in Cultured Goldfish Cells
Authors: Uchida Nobuhiro , Mitani Hiroshi , Todo Takeshi , Ikenaga Mituo , Shima Akihiro
Published: January 2008

We previously reported that when cultured goldfish cells are illuminated with fluorescent light, photorepair ability for both cyclobutane pyrimidine dimers and (6–4) photoproducts increased. In the present study, it was found that the duration of the induced photorepair ability for cyclobutane pyrimidine dimers was longer than that for (6–4) photoproducts, suggesting the presence of different photolyases for repair of these two major forms of DNA damage. A gel shift assay was then performed to show the presence of protein(s) binding to (6–4) photoproducts and its dissociation from (6–4) photoproducts under fluorescent light illumination. In addition, at 8 h after fluorescent light illumination of the cell, the binding of pro‐tein(s) to (6–4) photoproducts increased. The restriction enzymes that have recognition sites containing TT or TC sequences failed to digest the UV‐irradiated DNA pho‐toreactivated by using Escherichia coli photolyase for cyclobutane pyrimidine dimers, indicating that restriction enzymes could not function because (6–4) photoproducts remained in recognition sites. But, when UV‐irradiated DNA depleted of cyclobutane pyrimidine dimers was incubated with extract of cultured goldfish cells under fluorescent light illumination, it was digested with those restriction enzymes. These results suggested the presence of (6–4) photolyase in cultured goldfish cells as in Dro‐sophila, Xenopus and Crotalus.
In vivo Laser‐induced Fluorescence Imaging of a Rat Pancreatic Cancer with Pheophorbide‐a
Authors: Tassetti V. , Hajri A. , Sowinska M. , Evrard S. , Heisel F. , Cheng L. Q. , Miehe J. A. , Marescaux J. , Aprahamian M.
Published: January 2008

Laser‐induced fluorescence (LIF) of pheophorbide‐a (Ph‐a) was used for imaging of a rat pancreatic tumor. Using a dimensionless function (the ratio of Ph‐a fluorescence by bluish autofluorescence), the fluorescence contrasts between excised tumors and their paired pancreas were investigated up to 48 h after a 9 mg kg‐1 Ph‐a intravenous administration. Among five tested excitation wavelengths, 355 and 610 nm excitations gave the best distinctive contrasts, both 48 h after dye injection. The LIF imaging of six intrapancreatic tumors and six healthy pancreas was carried out in vivo using two laser excitations: 355 nm (Nd:YAG + tripling) for bluish autofluorescence and 610 nm (rhodamine 6G dye) for reddish autofluorescence and dye emission. Images were recorded through bandpass filters at 470 and 640 nm (autofluorescence) and at 680 nm (dye + autofluorescence) with an intensified charged‐coupled device camera. Autofluorescence as Ph‐a fluorescence images did not allow accurate LIF diagnosis of pancreatic carcinoma. An image processing, including for each pixel a computed division of Ph‐a fluorescence (after subtraction of reddish autofluorescence) by bluish autofluorescence intensity generated poorly contrasted tumor images in five of six and false tumor localization in one of three of the tumor‐bearing pancreas. A fitting of the digital 640 nm autofluorescence up to the mean 680 nm fluorescence intensity in pancreas prior to subtraction allowed a safe diagnosis to be made with well‐contrasted tumor images. To assess automation ability of the processing, a same fitting coefficient (mean of individual values) was applied. In this way, false‐negative (one of six) and false‐positive (two of six) images were present in tumor‐bearing animals as false‐positive in one‐half of the controls. A successful standardized procedure was then applied with a normalization of 640 and 680 nm pancreas intensities to a same set threshold prior processing. In opposition to thin‐layered hollow organs, such as bronchial tube or digestive tract, LIF imaging of carcinoma inserted in a compact organ is exhausting. The use of a dye excitable in the red wavelength range (610 nm for Ph‐a) may partly solve this problem, rendering LIF imaging more accurate and potentially automated.
Photochemical Labeling: Can Photoaffinity Labeling be Differentiated from Site‐Directed Photochemical Coupling?
Authors: Bouchet Marie‐Jeanne , Goeldner Maurice
Published: January 2008

Site‐directed photochemical labeling is a methodology designed to irreversibly and specifically label, through the action of light, a ligand binding site of a biological mac‐romolecule. Photoaffinity labeling, a widely used site‐directed labeling methodology, uses photosensitive ligand analogs generally obtained after chemical modification of the ligand by introducing an appropriate photoactivata‐ble moiety. This methodology can be applied to natural ligands showing inherent photosensitivity, without any additional modification, and which can be linked efficiently to their receptor binding site by direct photoac‐tivation. The emergence of an alternative methodology that links nonphotosensitive ligands to their receptors has raised the question of their potential use and their mechanisms of photocoupling. This article presents a series of examples that are meant to compare the general characteristics of the different site‐directed labeling reactions and proposes distinct photochemical activation processes between photoaffinity labeling and site‐directed photochemical coupling reactions. We suggest in particular that the former is necessarily a ligand‐mediated activation process while the latter might involve a receptor‐mediated mechanism.
Mechanistic Studies Relevant to Bromouridine‐Enhanced Nucleoprotein Photocrosslinking: Possible Involvement of an Excited Tyrosine Residue of the Protein
Authors: Norris Christopher L. , Meisenheimer Kristen M. , Koch Tad H.
Published: January 2008

The results of mechanistic studies on formation of uridine (U) and N‐acetyl‐in‐(5‐uridinyl)tyrosine N‐ethylam‐ide (2) from irradiation of aqueous, pH 7 solutions of bromouridine (BrU) and N‐acetyltyrosine JV‐ethylamide (1) are reported. Solutions were irradiated with monochromatic laser emission at 266, 308 and 325 nm. Quantum yield measurements as a function of excitation wavelength suggest that both products result from excitation of the tyrosine derivative followed by electron transfer to BrU, possibly with intermediacy of the hydrated electron. The BrU radical anion ejects bromide to form the uri‐dinyl radical, which then abstracts a hydrogen atom from 1 or adds to the aromatic ring of 1. Formation of adduct 2 is a model for photocrosslinking of nucleic acids bearing the bromouracil chromophore to adjacent tyrosine residues of proteins in nucleoprotein complexes. The value of 325 nm excitation in photocrosslinking, where the tyrosine chromophore is more competitive for photons, was demonstrated with an RNA bound to the MS2 bacteriophage coat protein; more than a 60% increase in the yield of photocrosslinking relative to that obtained with 308 nm excitation was achieved.
Interactions between Vitamin E Homologues and Ascorbate Free Radicals in Murine Skin Homogenates Irradiated with Ultraviolet Light
Authors: Kitazawa Manabu , Podda Maurizio , Thiele Jens , Traber Maret G. , Iwasaki Keiji , Sakamoto Kazutami , Packer Lester
Published: January 2008

The mechanism of oxidation of ascorbic acid in mouse skin homogenates by UV light was investigated by measuring ascorbate free radical formation using electron spin resonance signal formation. Addition of vitamin E (α‐tocopherol or α‐tocotrienol) had no effect, whereas short‐chain homologues (2,5,7,8‐tetramethyl‐6‐hydroxy‐chroman‐2‐carboxylic acid [Trolox] and 2,2,5,7,8‐penta‐methyl‐6‐hydroxychromane [PMC]) accelerated ascorbate oxidation. The similar hydrophilicity of ascorbate, Trolox and PMC increased their interaction, thus rapidly depleting ascorbate. When dihydrolipoic acid was added simultaneously with the vitamin E homologues, the accelerated ascorbate oxidation was prevented. This was due to the regeneration of ascorbate and PMC from their free radicals by a recycling mechanism between ascorbate, vitamin E homologues and dihydrolipoic acid. Potentiation of antioxidant recycling may be protective against UV irradiation‐induced damage. The rate of ascorbate oxidation in the presence of vitamin E homologues was enhanced by a photosensitizer (riboflavin) but was not influenced by reactive oxygen radical quenchers, superoxide dismutase or 5,5‐dimethyl‐l‐pyrroline‐iV‐ox‐ide. These experimental results suggest that the UV irradiation‐induced ascorbate oxidation in murine skin homogenates is caused by photoactivated reactions rather than reactive oxygen radical reactions.
Simultaneous Two‐Photon Activation of Type‐I Photodynamic Therapy Agents
Authors: Fisher W. G. , Partridge W. P. , Dees C. , Wachter E. A.
Published: January 2008

The excitation and emission properties of several psoralen derivatives are compared using conventional single‐photon excitation and simultaneous two‐photon excitation (TPE). Two‐photon excitation is effected using the output of a mode‐locked titanium: sapphire laser, the near infrared output of which is used to promote non‐resonant TPE directly. Specifically, the excitation spectra and excited‐state properties of 8‐methoxypsoralen and 4′‐aminomethyl‐4,5,8‐trimethylpsoralen are shown to be equivalent using both modes of excitation. Further, in vitro feasibility of two‐photon photodynamic therapy (PDT) is demonstrated using Salmonella typhimurium. Two‐photon excitation may be beneficial in the practice of PDT because it would allow replacement of visible or UV excitation light with highly penetrating, nondamag‐ing near infrared light and could provide a means for improving localization of therapy. Comparison of possible laser excitation sources for PDT reveals the titanium: sapphire laser to be exceptionally well suited for nonlinear excitation of PDT agents in biological systems due to its extremely short pulse width and high repetition rate that together provide efficient PDT activation and greatly reduced potential for biological damage
Potential Involvement of Both Type I and Type II Mechanisms in M13 Virus Inactivation by Methylene Blue Photosensitization
Authors: Abe H. , Ikebuchi K. , Wagner S. J. , Kuwabara M. , Kamo N. , Sekiguchi S.
Published: January 2008

We have investigated the mechanism of virus photoinactivation with methylene blue (MB) by conducting deuterium oxide (D2O), azide ion (N3‐) and oxygen‐dependent, studies. Inactivation of M13 bacteriophage and singlet oxygen (1O2) generation by MB photosensitization were irradiation dose dependent. Inactivation of M13 was enhanced by D2O and inhibited by N3‐, suggesting that 1O2 participates in M13 inactivation by MB photosensitization. However, N3‐ did not inhibit M13 inactivation completely. On the other hand, deoxygenating the reaction solution still caused 52‐67% of M13 inactivation observed in the presence of oxygen. These results suggest that 102‐mediated (Type II) and sensitizer‐mediated (Type I) reactions may both play roles in M13 inactivation by MB photosensitization.
Structure‐Activity and Mechanism Studies on Silicon Phthalocyanines with Plasmodium falciparum in the Dark and Under Red Light
Authors: Zhao Xiao‐Juan , Lustigman Sara , Kenney Malcolm E. , Ben‐Hur Ehud
Published: January 2008

Syntheses for the new photosensitizers HOSiPc‐OSi(CH3)2(CH2)3N(CH2)1 or 3(CH3)2, Pc 34 and Pc 25, have been developed and the order of activity of these photosensitizers and the previously reported photosensi‐tizer Pc 4, HOSiPcOSi(CH3)2(CH2)3N(CH3)2, in the dark and with broad‐band red light toward Plasmodium falciparum in red blood cell (RBC) suspensions has been studied. The order of activity has been found to be Pc 4 Pc 34 Pc 25. Thus, the activity of the photosensitizers under both sets of conditions is inversely proportional to the length of their terminal amino alkyl chains. The 50% inhibition dye concentration (IC50) in the dark for the parasites in RBC suspension with Pc 4 is 24 nM and the dye concentration and light fluence that yield:3 log10 of parasite inactivation with Pc 4 are 2 mM and 3 J/cm2, respectively. The synthesis of DNA and proteins by the parasites in culture was strongly inhibited by Pc 4 in the dark while parasite lactate dehydrogenase (pLDH) activity was unaffected. With Pc 4 and light, DNA and protein synthesis of the parasites in culture was strongly inhibited, pLDH activity of the parasites was moderately inhibited and ribosome density of the parasite cells was reduced. Gel electrophoresis studies showed that synthesis of all parasite proteins was inhibited to a similar extent. These results suggest that Pc 4 both in the dark and with light inactivates the cells by disturbing their machinery for the synthesis of not just one but a whole series of proteins. It is concluded that Pc 4 and light may be able to serve as a practical sterilization combination not only for HIV and other viruses but also for malaria parasites in RBC concentrates, and that Pc 4 by itself may have potential as a chemotherapeutic agent toward malaria.
Intercomparison of Effective Erythemal Irradiance Measurements from Two Types of Broad‐Band Instruments during June 1995
Authors: Wengraitis Stephen , Benedetta Danielle , Sliney David H.
Published: January 2008

The biologically effective global solar ultraviolet radiation (UVR) measurements from a multiband UVR monitor and a conventional broadband UVR monitor are compared. The measurements were performed during the varied weather conditions of June 1995. We compared the daily total exposures measured by both instruments, as well as the ratio of the measured doses throughout the course of each day. The daily total exposures agreed within approximately 11% throughout the month. The ratio between the measured doses held at 1.12 between 0900 and 1700 h (solar zenith angles ≅16‐52°). The ratio decreased from 1.12 to 0.90 during the next 90 min outside that period (solar zenith angles ≅52‐72°) and decreased further beyond that point. Spectral response and cosine response mismatch between the instruments are discussed as the possible cause of discrepancies between the measured doses. Implications for erythemal irradiance monitoring and suggestions for further study are discussed.
Laser Line‐Scanning Confocal Fluorescence Imaging of the Photodynamic Action of Aluminum and Zinc Phthalocyanines in V79–4Chinese Hamster Fibroblasts
Authors: Scully A. D. , Ostler R. B. , MacRobert A. J. , Parker A. W. , Lara C. de , O'Neill P. , Phillips D.
Published: January 2008

Confocal fluorescence microscopy, using a newly constructed laser line‐scanning confocal microscope, was applied to an investigation of the early stages of photoinduced destruction of V79–4Chinese hamster fibroblasts using aluminum and zinc phthalocyanines as photosensitize. Results obtained in this work show that aluminum and zinc phthalocyanines, once internalized, localize in perinuclear sites that are disrupted upon light exposure resulting in fluorescence redistribution. The combination of laser‐line scanning with charge‐coupled device detection used in the confocal microscope developed in this work can enable rapid high‐resolution sequential imaging, which is ideal for studying photoinduced intracellular fluorescence dynamics.
Delayed Hyperresistance of Endothelial Cells to Photodynamic Inactivation After Contact with Hemin
Authors: Lin Fubao , Bertling Chad J. , Geiger Peter G. , Girotti Albert W.
Published: January 2008

Hemin (ferriprotoporphyrin IX), the oxidized prosthetic group of hemoglobin, is a potential source of prooxidant iron in heavily vascularized tumors. We have evaluated hemin's effects on photodynamic inactivation of bovine artery endothelial cells, using a partially purified oligo‐meric fraction of hematoporphyrin derivative (HPD‐A) as the sensitizing agent. Confluent cells in 5% serum/ RPMI medium showed a progressive loss of thiazolyl blue (MTT)‐detectable viability when irradiated with broadband visible light in the presence of HPD‐A. Cells pretreated with desferrioxamine (DFO) were substantially less sensitive to photokilling, implying that non‐heme iron plays a role in cytotoxic activity. Hemin (10–20μM) had remarkably different effects on photokilling, depending on the time interval between adding it to cells and exposing them to photodynamic action. For example, cells were more sensitive when photostressed immediately after 1 h hemin treatment and washing but much more resistant when photostressed 23 h later. Similar responses were observed when cells were challenged with glucose oxidase. Immunoblot analysis following hemin treatment revealed a progressive induction of the heavy (H) subunit of ferritin that paralleled the development of hyperresistance. After incubation with saturating levels of the synthetic iron donor [55Fe]ferric‐8‐hydroxyquino‐line, hemin‐stimulated cells contained about four times more immunoprecipitable ferritin 55Fe than controls. This is consistent with the notion that sequestration of toxic iron as a result of induction of H‐chain‐enriched ferritin is a key factor in hyperresistance. Inflammatory injury in tumor vasculatures could expose endothelial and neoplastic cells to chronic hemoglobin‐derived iron. Consequent upregulation of ferritin could impact negatively on the efficacy of photodynamic therapy and other oxidant‐based cancer therapies.
Modulation of Porphyrin Derivatives Accumulation in C6 Glioma Cells by Drugs Acting on β‐Adrenergic Receptors. A Spectrofluorometric Study
Authors: Croce A. C. , Mares V. , Lisa V. , Krajci D. , Bottiroll G.
Published: January 2008

The pharmacological modulation of the uptake of porphyrin derivatives in cultured C6 glioma cells was investigated by means of spectrofluorometric analysis both in single cells and in cell homogenates. The influence of drugs acting as β‐receptor agonists or antagonists was studied in cells grown to semiconfluency. Isoproterenol (ISO), a β‐receptor agonist, enhanced the intracellular fluorescence intensity of both Photofrin and protoporphyrin IX (PpIX). A treatment with a β‐receptor antagonist I‐propranolol (PRO), simultaneous with ISO, resulted in an intracellular Photofrin fluorescence signal comparable to that of the control cells, indicating the specificity of the pharmacological action. The pharmacological treatment seemed particularly effective with the aggregated species. This is suggested by the relative increase of the band at 670 nm, being greater than that in the 630 nm band in the emission spectra of Photofrin and PpIX, and by the comparison of the fluorescence intensity on cell homogenates measured both in the absence and in the presence of cetyltrimethyl‐ammonium bromide as a detergent.
Sensitized Photooxidation of Di‐ and Tripeptides of Tyrosine*
Authors: Criado Susana , Soltermann Arnaldo T. , Marioli Juan M. , Garcia Norman A.
Published: January 2008

This paper studies the dye‐sensitized photooxidation of tyrosine (tyr) and tyr di‐ and tripeptides (tyr‐tyr and tyr‐tyr‐tyr) mediated by singlet molecular oxygen (O,[l]) in alkaline media. Photooxidation quantum efficiencies (φr) were obtained by determining the overall and reactive rate constants of interaction with the oxidative species, employing the time‐resolved O,(l) phosphorescence detection method and static‐photolysis actinometric method, respectively. The interaction of O,(l)‐tyr derivatives occurs through an intermediate encounter complex with polar character. Ionization of the phenolic OH group of tyr derivatives and the polarity of the solvent favors the overall interaction. Nevertheless, & values decrease when changing from water to MeCN‐water medium. This indicates that the reactive deactivation of the encounter complex, probably an entropy‐controlled step, may be affected by solvent polarity in the same way as those processes in which charg‐ es are neutralized along the reaction pathway. Photooxidation quantum efficiencies indicate that the contribution to O,(′) physical quenching (a second alternative deactivation route for the encountered complex [O,(′)‐tyr derivatives]) increases with the complexity of the peptide. As a result, the selfprotection of the peptidic entity against physical quenching also increases. The information obtained from the fractional consumption mol OJmol tyr derivative (in tyr, the di‐ and tripeptides and the respective methyl ester of tyr and the tripeptide), together with the evolution (either consumption and/or generation) of primary amino groups upon photosensitized irradiation of the same compounds clearly indicates that the photooxidation of di‐ and tri‐tyr peptides proceeds with the breakage of peptidic bonds. As a consequence, in the final balance each tyr unity behaves as an independent photooxidizable target.
Kinetic Spectroscopy of Erythrosin Phosphorescence and Delayed Fluorescence in Aqueous Solution at Room Temperature*
Authors: Duchowicz Ricardo , Ferrer María Luisa , Acuñ A. Ulises
Published: January 2008

The photophysics and polarization of the phosphorescence and delayed fluorescence of erythrosin in conditions compatible with the current biological applications of the dye (aqueous buffers at pH 7.4 at ambient temperatures) and in ethanol have been studied as a function of dye concentration (10 −7‐10−5M) and temperature (245–333K). The emission decay is strictly single exponential and the detailed kinetic analysis of all the rate processes connected with the emitting T1 state showed that (1) the lowering of the emission lifetime at the higher temperatures is due to a very efficient self‐quenching process, (2) the back intersystem crossing rate Tx S1 is temperature dependent (δETS7 kcal mol−1) but the T1S0 is not (Ea0.1 kcal mol−1) and (3) both intersystem crossing processes are very sensitive to solvent polarity, which accounts for the solvent dependence of the phosphorescence yield and lifetime. The high value of the phosphorescence anisotropy (r0= 0.25 lt 0.006) is independent of the excitation and emission wavelengths, and its evolution in time accurately reflects the rotational restrictions in solid solutions. The relevance of these findings to studies with protein‐dye conjugates is also outlined to facilitate the design and interpretation of phosphorescence depolarization experiments that probe the (μs‐ms dynamics of biomolecules and supramolecular systems.
pH‐Dependent Extraction of Ca 2+ from Photosystem II Membranes and Thylakoid Membranes: Indication of a Ca 2+‐Sensitive Site on the Acceptor Side of Photosystem II
Authors: Semin Boris K. , Ivanov Llja I. , Rubin Andrei B. , Carpentier Robert
Published: January 2008

The action of low pH treatment (pH 3.6) known to release Ca2+ from the oxygen‐evolving complex in photosystem II (PSII) membranes and to induce Ca2+‐revers‐ible inhibition of electron transport at the acceptor side of PSII in thylakoid membranes (TM) was compared in PSII membranes and TM. The rate of the inactivation of electron transport by low pH was four times higher in TM than in PSII membranes. Ferricyanide accelerated the inactivation of PSII membranes but decreased it in the case of TM. Low pH treatment also greatly modified the fluorescence induction kinetics in both preparations, but significant differences have been found in the fluorescence induction kinetics of treated TM and PSII membranes. Calcium restored the electron transport activity and fluorescence induction kinetics in PSII membranes and TM, whereas diphenylcarbazide restored these functions only in PSII membranes. The reactivation of Ca‐depleted PSII membranes was more effective in the dark, whereas the reactivation of TM required weak light. In the case of PSII membranes subjected to low pH citrate buffer, maximal reactivation was observed at 60 mM Ca2+ but for TM about 10 mM Ca2+ was required and 60 mM fully inhibited electron transport in TM during reactivation. These results indicate that the Ca‐dependent inactivation of the acceptor side of PSII in TM after low pH treatment cannot be explained by the extraction of Ca2+ from the oxygen‐evolving complex. It is rather suggested that the Ca2+ involved in this inhibition is bound to the acceptor side of the photosystem near to the QA‐non‐heme iron binding site and may participate in the binding of a polypeptide of the PSII light antenna complex to the PSII reaction center.
In Situ Molecular Dosimetry and Tumor Risk: UV‐lnduced DNA Damage and Tumor Latency Time‡
Authors: Gruijl Frank R. de , Berg Rob J. W.
Published: January 2008

In UV carcinogenesis there is a fundamental chain of causal events from UV‐induced DNA damage through mutations up to tumor formation: each of the early events should be predictive of the ultimate tumor risk. Instead of the UV surface exposure, the in situ load of DNA damage should be a more direct measure of the carcinogenicity. To explore this further we measured cy‐clobutane thymine dimer loads of epidermal cell suspensions from chronically UV‐exposed hairless SKH‐1 mice; skin samples were taken after various time periods under different daily exposures. Although the average load per cell decreased in the course of time due to dilution of damage in an increasing epidermal hyperplasia, the amount of thymine dimers in a column of epidermis (i.e. per mm2 of skin area) became stationary, and this amount increased with higher daily exposure. The median tumor latency time, tso, is inversely related to this stationary load. Extrapolation of a fitted relationship would imply a t50 between 450 and 1430 days for spontaneous skin carcinomas. The present data suggest that the skin strives to maintain a maximum level of tolerable DNA damage by lowering the average genotoxic load in vital cells in a hyperplastic reaction: pseudo‐repair by dilution. This would also explain the strong hyperplastic reactions in DNA repair‐deficient mouse strains. An understanding of these short‐term adaptive reactions can refine our assessments of skin cancer risks in humans.