bluuebunny/crossref_metadata_2025 huggingface dataset:
Solvent Polarity and pH Effects on the Spectroscopic Properties of Neutral Red: Application to Lysosomal Microenvironment Probing in Living Cells
Authors: Sousa Cristina , Sá e Melo Teresa , Gèze Marc , Gaullier Jean‐Michel , Mazière Jean Claude , Santus René
Published: January 2008

Neutral red is a lysosomal probe and a biological pH indicator. In aqueous solutions, the protonated (NRW) and neutral (NR) forms of monomeric neutral red exhibit distinct absorption maxima (535 and 450 nm, respectively) but have the same fluorescence with a maximum at 637 nm and a quantum yield of 0.02. The similarity of the fluorescence spectra at acidic and basic pH suggests deprotonation of cationic species in the first singlet excited state. The NR fluorescence strongly depends on the solvent polarity as shown by addition of increasing amounts of water to pure dioxane, which gradually shifts the fluorescence maximum from 540 nm in pure dioxane to 637 nm in water. The fluorescence quantum yield increases from 0.17 in dioxane to 0.3 upon addition of 7% water and then decreases, reaching 0.02 in pure water. Immediately after incubation of human skin fibroblasts with neutral red, excitation with 435 nm light produces a fluorescence whose maximum is recorded at 575 nm. This fluorescence is located in the perinuclear region and originates from large fluorescent intracytoplasmic spots, suggesting staining of the endoplasmic reticulum‐Golgi complex. At longer times, this fluorescence is shifted to 606 nm, suggesting slow diffusion of the lysosomotropic dye toward the more hydrated and acidic interior of ly‐sosomes. Addition of a lysosomotropic detergent to cells previously incubated with neutral red shifts the fluorescence to the blue. Thus, in complex biological systems, this probe cannot be a good pH indicator but is a very sensitive probe of lysosomal rnicroenvironrnents.
Fourier Transform Multipixel Spectroscopy and Spectral Imaging of Protoporphyrin in Single Melanoma Cells
Authors: Malik Zvi , Dishi Michal , Garini Yuval
Published: January 2008

Fourier transform multipixel spectroscopy was applied to subcellular localization of endogenous protoporphyrin (endo‐PP) in single living B16 melanoma cells during photosensitization. Continuous fluorescence spectra for each pixel were recorded using a Sagnac interferometer coupled to a charge‐coupled device camera. Multiple frames of data were acquired for each pixel composing the image, then they were stored as interferometric data and resolved as spectra for every pixel (103‐−4 × 103 point pixels in a single cell). The net result was the intensity I (x, y, λ), for each pixel of the image (x, y), at any wavelength (λ). The present study demonstrates the application of Fourier transformed multipixel spectroscopy for spectral imaging of melanoma cells incubated with 5‐aminolevulinic acid (ALA). The fluorescence image of ALA‐treated cells revealed endo‐PP all over the cytosol with a vesicular distribution, which represent mitochondria and endoplasmic reticulum compartments. Two main spectral fluorescence peaks were demonstrated at 630 and 670 nm, of monomeric and aggregated protoporphyrin, with intensities that differed from one sub‐cellular site to another. Photoirradiation of the cells induced point‐specific subcellular fluorescence spectrum changes and demonstrated photoproduct formation. Spectral‐image reconstruction revealed the subcellular distribution of porphyrin species in single photosensitized cells. Multipixel spectroscopy of exogenous protoporphyrin revealed an endosomal‐lysosomal compartment in aggregated states, whereas monomeric porphyrin species were localized mainly on the outer membrane. Photo‐products could be visualized at sites of formation in suhcellular compartments.
Erythemal Ultraviolet Insolation in New Zealand at Solar Zenith Angles of 30° and 45°
Authors: Ryan K. G. , Smith G. J. , Rhoades D. A. , Coppell R. B.
Published: January 2008

Solar UV radiometers with spectral responsivities that are close to the erythemaVcarcinogenic action spectrum of skin have been installed at several centers of population in New Zealand, including Auckland, 37°S, Wellington, 41°s and Christchurch, 43.5°S. The data set covers the period from the time the radiometry program commenced in 1988/1989 to the end of the southern summer, March 1995. The radiometers were recalibrated annually and the data were corrected for changes in the absolute responsivity of the radiometers. Erythemally effective UV irradiances at solar zenith angles of 30° and 45° were then extracted from the data set. No monotonic trend in these data is apparent, although there are statistically significant differences in mean irradiances from one year to the next. An example of this is the decrease observed in all sites following the Mt. Pinatubo eruption in June 1991. The maximum erythemally effective insolations at solar zenith angles of 30° and 45° were consistently lower in Christchurch than in the other two New Zealand sites. This could arise from higher levels of atmospheric turbidity andlor tropospheric ozone at this location. Also, a seasonal increase in erythemally effective UV insolation from spring to autumn was observed each year in all three New Zealand sites.
Quantitation of the Reactive Oxygen Species Generated by the UVA Irradiation of Ascorbic Acid‐Glycated Lens Proteins
Authors: Linetsky Mikhail , Ortwerth Beryl J.
Published: January 2008

The oxidation products of ascorbic acid rapidly glycate proteins and produce protein‐bound, advanced glycation endproducts. These endproducts can absorb UVA light and cause the photolytic oxidation of proteins (Ortwerth, Linetsky and Olesen, Photochem. Photobiol. 62, 454–463, 1995), which is mediated by the formation of reactive oxygen species. A dialyzed preparation of calf lens proteins, which had been incubated for 4 weeks with 20 mM ascorbic acid in air, was irradiated for 1 h with 200 mW/ cm2 of absorbed UVA light (λ > 338 nm), and the concentration of individual oxygen free radicals was measured. Superoxide anion attained a level of 76 μM as determined by the superoxide dismutase (SOD)‐depen‐dent increase in hydrogen peroxide formation and of 52 μM by the SOD‐inhibitable reduction of cytochrome c. Hydrogen peroxide formation increased linearly to 81 μM after 1 h. Neither superoxide anion nor hydrogen peroxide, however, could account for the UVA photolysis of Trp and His seen in this system.

Singlet oxygen levels approached 1.0 mM as measured by the oxidation of histidine, which was consistent with singlet oxygen measurements by the bleaching of N,N‐dimethyl‐4‐nitrosoaniline. High concentrations of sodium azide, a known singlet oxygen quencher, inhibited the photolytic destruction of both His and Trp. Little or no protein damage could be ascribed to hydroxyl radical based upon quenching experiments with added mannitol. Therefore, superoxide anion and H2O2 were generated by the UVA irradiation of ascorbate advanced glycation endproducts, however, the major reactive oxygen species formed was singlet oxygen.
Photothermal Beam Deflection Calorimetry in Solution Photochemistry: Recent Progress and Future Prospects
Authors: Falvey Daniel E.
Published: January 2008

Abstract— Photothermal beam deflection (PBD) calorimetry is a technique that measures changes in the solvent's refractive index that accompanies photothermal heating. This method is capable of extracting both kinetic and thermodynamic information from photochemical reactions. A qualitative description of physical basis of time‐resolved PBD is presented. Several recent examples of its application to photochemical and photobiological problems are discussed. Finally, the advantages and limitations of PBD are described.
Equilibrium, Kinetics, Diffusion and Self‐Association of Proteins at Membrane Surfaces: Measurement by Total Internal Reflection Fluorescence Microscopy
Authors: Thompson Nancy L. , Drake Andrew W. , Chen Lixin , Broek Willem Vanden
Published: January 2008

Abstract— The equilibrium, kinetics, diffusion and self‐association of proteins at membrane/solution interfaces may deviate substantially from these processes in bulk solution. A set of methods for examining these phenomena combines substrate‐supported planar model membranes and the use of evanescent illumination with laser‐based, quantitative fluorescence microscopy. Measurement of the steady‐state, surface‐associated fluorescence can be used to examine the thermodynamic properties of proteins at membranes. When combined with fluorescence photobleaching recovery, this technique provides information about membrane‐binding kinetics; and when combined with fluorescence pattern photobleaching recovery, measurement of the translational diffusion coefficients of proteins weakly bound to membranes is possible. The use of polarized evanescent illumination can provide information about the orientation distributions of adsorbed fluorophores. Fluorescence correlation spectroscopy provides information about the self‐association (e.g. dimerization) of membrane‐associated proteins.
Comparative Photophysical Study of Disulfonated Aluminum Phthalocyanine in Unilamellar Vesicles and Leukemic K562 Cells
Authors: Dhami Suman , Rumbles Garry , MacRobert Alexander J. , Phillips David
Published: January 2008

Abstract— The photophysical properties of cis‐disulfonated aluminum phthalocyanine (AlPcS2) in unilamellar vesicles (liposomes) of DL‐a‐dipalmitoyl‐phosphatidylcholine have been measured. Both the fluorescence and triplet quantum yields decreased with increasing sensitizer concentration. The time‐resolved fluorescence decays, analyzed by both the sum of exponentials and decay time distribution analyses, are compared with those reported for AlPcS2 in leukemic K562 cells. Information on the pho‐todynamic transport and localization mechanism has been obtained by drawing correlations between the two systems, indicating active transport of the phthalocyanine into tumor cells involving lysosomal accumulation.
Solar Radiation Is Shown to Select for Pigmented Bacteria in the Ambient Outdoor Atmosphere
Authors: Tong Yongyi , Lighthart Bruce
Published: January 2008

Abstract— The percentage of pigmented to total bacteria in the outdoor atmospheric population was studied in the field and in controlled laboratory experiments to evaluate the effects of solar radiation (SR) on bacterial survival. The field experiments showed that the percentage of pigmented bacteria positively correlated with SR activity during clear summer days. The percentage was lowest during darkness before dawn and around midnight (ca 33%) and as the SR increased during the day, gradually increased to a maximum of ca 50–60% at noontime to early afternoon and decreased thereafter. In the laboratory the ambient outdoor atmospheric bacteria impacted on culture plates were exposed to simulated SR and a germicidal light. With increased exposure, more nonpig‐mented bacteria were killed and the percentage of pigmented bacteria gradually increased. These observations suggest an inverse relationship between the atmospheric bacterial survival and the percentage of pigmented bacteria contained therein, thus supporting the notion that pigmentation might provide protection for outdoor atmospheric bacteria from sunlight damage. As a consequence, viable pigmented bacteria (and other UV‐resistant forms) in the atmosphere could be enriched under areas of stratospheric ozone depletion.
Induction of 8‐Oxo‐7,8‐Dihydro‐2'‐Deoxyguanosine by Ultraviolet Radiation in Calf Thymus DNA and HeLa Cells
Authors: Zhang Xueshu , Rosenstein Barry S. , Wang Yan , Lebwohl Mark , Mitchell David M. , Wei Huachen
Published: January 2008

Abstract— The levels of 8‐oxo‐7,8‐dihydro‐2'‐deoxyguanosine (8‐oxodGuo) in purified calf thymus DNA and HeLa cells were measured following exposure to either UVC, UVB or UVA wavelengths. This DNA damage was quantitated using HPLC coupled with an electrochemical detector. The 8‐oxodGuo was induced in purified DNA in a linear dose‐dependent fashion by each portion of the UV spectrum at yields of 100, 0.46 and 0.16 8‐oxodGuo per 105 2'‐deoxyguanosine (dGuo) per kJ/m2 for UVC, UVB and UVA, respectively. However, the amount of 8‐oxodGuo in HeLa cells irradiated with these UV sources decreased to approximately 2.0, 0.013 and 0.0034 8‐oxodGuo per 105 dGuo per kJ/m2, respectively. In contrast, the levels of cyclobutyl pyrimidine dimers were similar in both irradiated DNA and cells. Therefore, 8‐oxodGuo is induced in cells exposed to wavelengths throughout the UV spectrum although it appears that protective precesses exist within cells that reduce the UV‐induced formation of this oxidative DNA damage. Cell survival was also measured and the number of dimers or 8‐oxodGuo per genome per lethal event determined. These calculations are consistent with the conclusion that dimers play a major role in cell lethality for UVC‐ or UVB‐irradiated cells but only a minor role in cells exposed to UVA wavelengths. In addition, it was found that the relative yield of 8‐oxodGuo to dimers increased nearly 1000‐fold in both UVA‐irra‐diated cells and DNA compared with cells subjected to either UVC or UVB. These results are supportive of the hypothesis that 8‐oxodGuo, and possible other forms of oxidative damage, play an important role in the induction of biological effects caused by wavelengths in the UVA portion of the solar spectrum.
Extracorporeal Photochemotherapy (Photopheresis) Induces Apoptosis in Lymphocytes: A Possible Mechanism of Action of PUVA Therapy
Authors: Enomoto Dory N. H. , Schellekens Peter T. A. , Yong Si‐La , ten Berge Ineke J. M. , Mekkes Jan R. , Bos Jan D.
Published: January 2008

Abstract— The mechanism of action of psoralen plus UVA (PUVA) and photopheresis is not entirely understood. These therapies are assumed to be immunomodulating partly by gradually decreasing leukocyte viability. We investigated whether this delayed form of cell death was due to apoptosis. Untreated and treated (PUVA exposed) leukocytes obtained from six patients with systemic sclerosis and (untreated) leukocytes from healthy control individuals were studied. Qualitative gel electrophoresis and quantitative in situ nick translation analysis of DNA fragmentation was performed. Apoptosis of the treated cells did occur (gel electrophoresis) after 24 h. At t = 0 h, immediately after exposure to PUVA, there was no evidence of DNA fragmentation in the treated cells. The percentage of treated cells undergoing apoptosis was 20–55% at t = 24 h (in situ nick translation). The untreated leukocytes of the patients and the healthy individuals showed no distinctive rise in apoptotic cells. Apoptosis of the leukocytes after PUVA or photopheresis treatment might be a mechanism of action and might explain the therapeutic response.
The Relative UV Sensitizer Activity of Purified Advanced Glycation Endproducts
Authors: Ortwerth B. J. , Prabhakaram Malladi , Nagaraj R. H. , Linetsky Mikhail
Published: January 2008

Abstract— The oxidation products of ascorbic acid react with lens proteins to form advanced glycation endproducts (AGE) that are capable of generating reactive oxygen species when irradiated with UVA light. L‐Threose, the most active of these oxidation products, was reacted with N‐acetyl lysine and six AGE peaks were isolated by RP‐HPLC. Each peak exhibited fluorescence and generated superoxide anion and singlet oxygen in response to UV light. Solutions of these AGE peaks (50 μg/mL) generated5–10 nmol/mL of superoxide anion during a 30 min irradiation. This activity was 100‐fold less than the superoxide anion generated by kynurenic acid and 400‐fold less than riboflavin.

Ultraviolet irradiation generated from 1.2 to 2.7 μmol/mL of singlet oxygen with the purified threose AGE compounds. This activity was similar to that seen with other purified AGE compounds (pentosidine, LM‐1 and Ac‐FTP) and with kynurenine and 3‐OH kynurenine. This considerable singlet oxygen formation, however, was still 40‐fold less than that obtained with kynurenic acid and 100‐fold less than riboflavin under the same irradiation conditions. In spite of this lower sensitizer efficiency, the purified AGE generated20–60‐fold more singlet oxygen on a weight basis than either crude ascorbic acid glycated proteins or a preparation of water‐insoluble proteins from aged normal human lenses. On a molar basis, therefore, AGE could account for the sensitizer activity in these protein preparations if they represented less than 1% of the total amino acids.
Electron Transfer Quenching of the Rose Bengal Triplet State
Authors: Lambert Christopher R. , Kochevar Irene E.
Published: January 2008

Abstract— The potential for electron transfer quenching of rose bengal triplet (3RB2‐) to compete with energy transfer quenching by oxygen was evaluated. Rate constants for oxidative and reductive quenching were measured in buffered aqueous solution, acetonitrile and in small unilamellar liposomes using laser flash photolysis. Biologically relevant quenchers were used that varied widely in structure, reduction potential and charge. Radical ion yields (Øi) were measured by monitoring the absorption of the rose bengal semireduced (RB*3‐) and semioxidized (RB*‐) radicals. The results in solution were analyzed as a function of the free energy for electron transfer (δG) calculated using the Weller equation including electrostatic terms. Exothermic oxidative quenching was about 10‐fold faster than exothermic reductive quenching in aqueous solution. The quenching rate constants decreased as δG approached zero in both aqueous and acetonitrile solution. Exceptions to these generalizations were observed that could be rationalized by specific steric or electrostatic effects or by a change in mechanism. The results suggest that electron transfer reactions with some potential quenchers in cells could compete with formation of singlet oxygen [O2(1δg)]. Values of Øi were generally greater for reductive quenching and, for oxidative quenching, greater in acetonitrile than in buffer. Electron transfer quenching of 3RB2‐ in liposomes, below the phase transition temperature was slower than in solution for both lipid‐soluble and water‐soluble quenchers indicating that these reactions may not compete with formation of O2(1δg) during cell photosensitization.
Photochemical Reactions and Phototoxicity of Sterols: Novel Self‐perpetuating Mechanism for Lipid Photooxidation
Authors: Albro Phillip W. , Bilski Piotr , Corbett Jean T. , Schroeder Joanna L. , Chignell Colin F.
Published: January 2008

Abstract— Sterols are important lipid components that may contribute to phototoxicity. We have found that phototoxic response in earthworms is related to sterols extractable with lipophilic solvents. The photochemically active compounds in worm lipids are 5,7,9(11),22‐ergostatetraen‐3bT‐ol (9‐DHE) and 5,7,9(11)‐cholestatrien‐3bT‐ol (9‐DDHC), respectively. Human skin lipids are known to contain 9‐DHE. We have also found 9‐DDHC in human skin, which is reported here for the first time. In the presence of an excess of the corresponding 5,7‐dienes (ergosterol or 7‐dehydrocholesterol), these photoactive sterols constitute a self‐regenerating source of singlet molecular oxygen (1O2) during irradiation in vivo or in vitro with UVA bT15‐400 nm). The quantum yield for photosensitization of 1O2 by 9‐DHE was estimated to be 0.09. The 1O2 is scavenged by the dienes and the rate constant for 1O2 quenching by ergosterol was found to be 1.2 times 107M‐1 s‐1 in methyl t‐butyl ether (MTBE). This scavenging ultimately leads to the production of 5,8‐endo‐peroxide and hydrogen peroxide. Photochemically induced superoxide radical was also produced on irradiation of sterol 5,7,9‐trienes and trapped with the spin trap 5,5‐dimeth‐yl‐1‐pyrroline W‐oxide (DMPO). The production of singlet oxygen, peroxides and radicals by the sterols may be significant in the cell damaging and tumor promoting action of UVA light on skin.
Initiation of Apoptosis versus Necrosis by Photodynamic Therapy with Chloroaluminum Phthalocyanine
Authors: Luo Yu , Kessel David
Published: January 2008

Abstract— While chloroaluminum phthalocyanine is a highly effective photosensitizer of murine leukemia P388 or L1210 cells, the mode of cell death varies as a function of the PDT dose. When cells were incubated with 0.3 mUM of the sensitizer, a light dose of 45 mJ cm‐2 (670 5 nm) yielded a 90% apoptotic cell population within 60 min. The sensitizer localized throughout the cytoplasm and catalyzed both lysosomal and mitochondrial photodamage at this light dose. Higher light doses yielded progressively more membrane photodamage and inhibited the apoptotic response as determined by the examination of Hochst dye HO 33342‐IabeIed nuclei, DNA fragmentation on gels and a poly(adenosylribose) polymerase (PARP)‐cleavage assay. Pulse‐field gel electrophoresis revealed nonspecific DNA degradation to particles 50 kbp at the higher PDT doses but neither PARP cleavage nor apoptotic nuclei
Photodynamic Therapy with Topical dL‐aminolevulinic Acid Delays UV Photocarcinogenesis in Hairless Mice
Authors: Stender I.‐M. , Bech‐Thomsen N. , Poulsen T. , Wulf H. C.
Published: January 2008

Abstract— Photodynamic therapy (PDT) with topical application of 8‐aminolevulinic acid (ALA) followed by irradiation with visible light (ALA‐PDT) is a relatively new and promising experimental treatment of superficial premalignant and malignant skin neoplasms. The purpose of this study was to determine whether ALA‐PDT can prevent photocarcinogenesis in hairless mice exposed to solar UV. A total of 140 mice was divided into seven groups of 20 mice each. Group 1: solar‐UV exposure. Group 2: solar UV and a cream base + visible light once a week. Group 3: solar UV and ALA‐PDT once a week. Group 4: solar UV and ALA‐PDT once every second week. Group 5: solar UV and ALA‐PDT every fourth week. Group 6: ALA‐PDT once a week. Group 7: no treatment. The time to first and to second tumor 1 mm was registered. Predefined endpoints, such as one tumor a 4 mm or an area of small confluent tumors on the back of the mice were criteria for withdrawal from the experiment. The time to first and to second tumor was significantly longer in the ALA‐PDT‐treated mice than in mice only exposed to solar UV and solar‐UV/cream base‐visible light (P < 0.005). However, we observed an increased death and accident rate in the ALA‐PDT‐treated groups compared to the groups not treated with ALA‐PDT (chi‐square test,P= 0.0250). Significantly more ALA‐PDT‐treated mice were withdrawn because of a tumor 4 mm (P= 0.0005). The UV unexposed mice developed no tumors. Repetitive treatments with ALA‐PDT delay photoinduced carcinogenesis in mice.
Oxyradical‐mediated Clastogenic Plasma Factors in Psoriasis: Increase in Clastogenic Activity after PUVA
Authors: Filipe P. , Emerit I. , Youssefi A. Alaoui , Levy A. , Cernjavski L. , Freitas J. , de Castro J. L. Cirne
Published: January 2008

Abstract— Psoriasis is a common skin disorder characterized by hyperproliferation and incomplete differentiation of epidermal keratinocytes. Psoralen plus UVA (PUVA) is one of the treatments proposed for this disease. We had reported previously that exposure of regular blood cultures from healthy donors to PUVA leads to chromosomal breakage via the formation of transferable clastogenic materials, a phenomenon inhibitable by superoxide dismutase. In the present paper we show that these clastogenic factors (CF) are also formed in vivo. The CF were found in about 50% of the psoriasis patients studied (14 out of 31). In PUVA‐treated psoriasis patients, the clastogenic activity of the plasma increased significantly between the first and the last (16th) exposure to PUVA. We hypothesize that CF formation in psoriasis is similar to that in other diseases accompanied by oxidative stress, in particular chronic inflammatory diseases with autoimmune reactions such as lupus erythematosus, progressive systemic sclerosis, rheumatoid arthritis and others. Increased superoxide production by phagocytes, formation of lipid peroxidation products and release of cytokines are considered to be responsible for the superoxide‐stimulating and chromosome‐damaging properties of patients' plasma. During PUVA therapy, superoxide generated via the interaction of psoralen with UVA may contribute to CF formation in addition to superoxide from inflammatory cells. An increased risk of cancer and leukemia is observed in diseases accompanied by CF formation. Therefore CF may contribute to the well‐known risk of photocarcinogenesis by PUVA therapy. This additional risk may be preventable by antioxidants and superoxide scavengers.
Generation and Characterization of Psoralen Radical Cations
Authors: Wood Paul D. , Johnston Linda J.
Published: January 2008

Abstract—Radical cations of psoralen, 8‐methoxypsoralen(8‐MOP) and 5‐methoxypsoralen have been generated by photosensitized electron transfer in acetonitrile and aqueous buffer/acetonitrile (1:1) and have absorption maxima at 600, 650 and 550 nm, respectively. The radical cations have lifetimes of 5 p.s under these conditions, are unreactive toward oxygen and show behavior typical of ar‐ylalkene radical cations in their reactivity toward nucle‐ophiles and the precursor psoralens. Direct 355 nm excitation of 8‐MOP in aqueous buffer at physiological pH results in monophotonic photoionization to give 8‐MOP*+with a quantum yield of 0.015.The 8‐MOP*+reacts with both guanosine and adenosine mononucleotides (k= 2.5 times 109and 3.4 times 107M‐1s1, respectively)viaelectron transfer to give the purine radical cations, but does not react with pyrimidine mononucleotides. These results suggest that reactions of psoralen radical cations generated by electron transfer or photoionization may be involved in psoralen/UVA therapy.
Photoinactivation of Virus Infectivity by Hypocrellin A
Authors: Hirayama Junichi , Ikebuchi Kenji , Abe Hideki , Kwon Kil‐Won , Ohnishi Yoshiko , Horiuchi Motohiro , Shinagawa Morikazu , Ikuta Kazuyoshi , Kamo Naoki , Sekiguchi Sadayoshi
Published: January 2008

Abstract— We investigated the photoinactivation of virus infectivity by hypocrellin A and its mechanism. The titers of vesicular stomatitis virus (VSV) and human immunodeficiency virus type 1 (HIV‐1), both of which are enveloped viruses, were reduced upon illumination with hypocrellin A in a concentration‐dependent manner, whereas canine parvovirus, a nonenveloped virus, was not killed. The removal of oxygen or addition of sodium azide or bT‐carotene both inhibited VSV inactivation. Mannitol and superoxide dismutase had no effect on VSV inactivation. These results indicate that singlet oxygen was involved in the process of VSV inactivation. Of the three major VSV membrane proteins, peripheral membrane protein M was most damaged by the hypocrellin A phototreatment.
Nano‐ and Microsecond Time‐Resolved FTIR Spectroscopy of the Halorhodopsin Photocycle
Authors: Dioumaev Andrei K. , Braiman Mark S.
Published: January 2008

Abstract— Step‐scan Fourier transform infrared spectroscopy with 50 ns time resolution was applied to the early stages of the photocycle of halorhodopsin (hR) for the temperature range 3‐42° C. Kinetic data analysis with global fitting revealed two distinct kinetic processes associated with relaxations of the early red‐shifted photoproduct hK; these processes have time constants T1⋍ 280 ns and T2⋍ 360 μs at 20°C. Spectral features demonstrate that the T1 process corresponds to a transition between two distinct bathointermediates, hKE and hKL. The vibrational difference bands associated with both T1 and T2 transitions are spread throughout the whole 1800‐900 cm−1 range. However, the largest bands correspond to ethylenic C=C stretches, fingerprint C‐C stretches and hydrogen out‐of‐plane (HOOP) wags of the retinal chromophore. The time evolution of these difference bands indicate that both the T1 and T2 decay processes involve principally a relaxation of the chromophore and its immediate environment. The decay of the intense HOOP vibrations is nearly equally divided between the T1 and T2 processes, indicating a complex chromophore relaxation from a twisted nonrelaxed conformation in the primary (hKE) bathointermediate, to a less‐twisted structure in hKL, and finally to a roughly planar structure in the hypsochromically shifted hL intermediate. This conclusion is also supported by the unexpectedly large positive entropy of activation observed for the T1 process. The two relaxations from hKE to hL are largely analogous to corresponding relaxations (KE→ KL→ L) in the bacterior‐hodopsin photocycle, except that the second step is slowed down by over 200‐fold in hR.
Characterization of Lamprey Rhodopsin by Isolation from Lamprey Retina and Expression in Mammalian Cells
Authors: Hisatomi Osamu , Ishikawa Makoto , Tonosaki Akira , Tokunaga Fumio
Published: January 2008

Abstract— A visual pigment was extracted from lamprey retina and was expressed in cultured mammalian cells (293S) using a cDNA fragment isolated from lamprey retina. The extracted pigment, a putative lamprey rhodopsin, had an absorption maximum at 503 nm. The recombinant lamprey rhodopsin, reconstituted with 11‐cis‐retinal, showed an absorption maximum at about 500 nm. Both pigments reacted with an anti‐bovine rhodopsin antibody (Rh29), which recognizes the short photoreceptor cells in lamprey retina. Unlike rhodopsins of higher vertebrates, the lamprey rhodopsin bleached gradually in the presence of 100 mM hydroxylamine even in the dark. Our results suggest that, despite its high similarities with other vertebrate rhodopsins, lamprey rhodopsin has a character different from those of higher vertebrates.
UV Light‐induced Crosslinking of the Complementary Strands of Plasmid pUC19 DNA Restriction Fragments
Authors: Pospíšilová Šáka , Kypr Jaroslav
Published: January 2008

Restriction fragments of pUC19 DNA were irradiated by various doses of UV light and analyzed by denaturing (alkaline) agarose gel electrophoresis. The irradiation generated retarded species whose mobility indicated two crosslinked DNA strands. Quantitative analysis of the experimental data provided an empirical equation relating the fraction of crosslinked DNA molecules to their length and to the dose of their irradiation by UV light. This equation can be used to predict the crosslinking behavior of pUC19‐like DNA molecules whose primary structures do not much differ from a random nucleotide sequence. The amount of interstrand crosslinks increased with the (A+T) content of the pUC19 DNA fragments but the dependence was not clear‐cut to indicate that oligonucleotide composition of DNA played a significant role as well.
Modification of Global Erythemally Effective Irradiance by Clouds
Authors: Thiel Stephan , Steiner Karl , Seidlitz Harald K.
Published: January 2008

The role clouds play in the modification of global radiation is still a major uncertainty in the risk assessment of UV effects on ecological systems and human health. This study presents cloud transmission data obtained from measurements with Robertson‐Berger meters and simultaneous cloud observations. The global transmission of erythemally weighted irradiance depends strongly on cloud amount and can be described by a cubic function. The comparison with results derived from long‐term records of total global irradiance indicates no statistically significant difference between the attenuation of ery‐themal and total global radiation. The large variance of data results from lumping together data from different cloud types. Classification of data according to cloud forms yields a more statisfactory fit. The coefficient of the cubic term characterizes the ability of various cloud forms to attenuate UV radiation. It varies between 0.4 for high clouds and approximately 1.0 for cumulonimbus. This attenuation parameter allows a quantitative description of the cloud influence on irradiance and therefore a more accurate risk assessment.
Comparative Action Spectrum for Ultraviolet Light Killing of Mouse Melanocytes from Different Genetic Coat Color Backgrounds
Authors: Hill Helene Z. , Hill George J. , Cieszka Krystyna , Plonka Przemyslaw M. , Mitchell David L. , Meyenhofer Markus F. , Xin Patrick , Boissy Raymond E.
Published: January 2008

The photobiology of mouse melanocyte lines with different pigment genotypes was studied by measuring colony‐forming ability after irradiation. The cell lines were wild‐type black (melan‐a) and the mutants brown (melan‐b) and albino (melan‐c). Four lamps emitting various UV wavelengths were used. These were germicidal (UVC, 200–280 Dm), 82.3% output at 254 nm, TL01 (UVB, 280–320 nm), 64.2% at 310–311 nm, FS20, broadband with peak output at 312 nm and Alisun‐S (UVA, 320–400 nm), broadband with peak output at 350–354 nm. Appropriate filtration reduced the contaminating UVC to nonlethal levels for the longer waverange lamps. Wild‐type melan‐a was resistant to UVC and UVA compared to the other two cell lines, but the differences were small. The melan‐c cell line was more resistant to UVB and markedly more resistant to FS20 than the pigmented lines. With the exception of FS20 responses, melan‐b was more sensitive than melan‐a to killing by the various UV lamps. There were more pyrimidine dimers (cyclobutane dimers and 6–4 photoproducts) produced in melan‐a than in melan‐c cells by UVC, UVB and FS20 lamps. Unlike melan‐c, melan‐a and melan‐b showed a strong free radical signal of melanin character with a detectable contribution of pheomelanin‐like centers. The contribution of pheome‐lanin was higher in melan‐b than in melan‐a, while the total melanin content in these two cell lines was comparable. The abundant melanin granules of wild‐type melan‐a melanocytes were well melanized and ellipsoidal, whereas those of melan‐b melanocytes tended to be spherical. In the albino line (melan‐c) the melanocytes contained only early‐stage melanosomes, all of which were devoid of melanin. The results indicate that pigment does not protect against direct effect DNA damage in the form of pyrimidine dimers nor does it necessarily protect against cell death. High pigment content is not very protective against killing by UVC and UVA, and it may photosensitize in UVB the very wavelength range that is of greatest concern with respect to the rising incidence in skin cancer, especially melanoma. It is clear from these studies that, in pigment cells, monochromatic results cannot predict polychromatic responses and that cell death from solar irradiations is a complex phenomenon that depends on more than DNA damage.
Benzophenone Photoprobes for Phosphoinositides, Peptides and Drugs
Authors: Prestwich Glenn D. , Dormán György , Elliott John T. , Marecak Dale M. , Chaudhary Anu
Published: January 2008

Benzophenones (BP) and related aryl ketone photophores have become established as the photoactivatable group of choice for high‐efficiency covalent modification of hydrophobic regions of binding proteins, including enzymes and receptors that recognize peptide hormones, (oligonucleotides and nucleosides, phosphoinositides, inositol polyphosphates and a wide variety of therapeutic molecules. This review presents the advantages of BP as pho‐toafnnity labels and provides specific examples from the last 3 years of applications of BP‐containing ligands used in biochemistry.
Peroxidase Activity May Play a Role in the Cytotoxic Effect of Indole Acetic Acid*
Authors: de Melo Mariza Pires , Curi Tania Cristina Pithon , Curl Rui , Mascio Paolo Di , Cilento Giuseppe
Published: January 2008

Peroxidase activity in neutrophils is higher than in thioglycollate macrophages, while in lymphocytes this enzyme activity is very low. Indole‐3‐acetic acid is oxidized by peroxidase and the role of this enzyme in the cytotoxic effect of the compound was evaluated by measuring oxygen consumption, light emission and cell death in neutrophils, macrophages and lymphocytes. The increase in light emission, oxygen consumption and rate of cell death in cells cultured in the presence of indole‐3‐acetic acid presented a direct correlation with the peroxidase activity of the cells as follows: neutrophils > thioglycollate macrophages > resident macrophages > lymphocytes. Indeed, in lymphocytes that possess very low peroxidase activity, indole‐3‐acetic acid did not result in an increase in light emission or oxygen consumption and it was not cytotoxic.
Antiviral Activities of Photoactive Perylenequinones
Authors: Hudson J. B. , Imperial V. , Haugland R. P. , Diwu Z.
Published: January 2008

Nine perylenequinones (PQ), including some familiar naturally occurring pigments, were compared for their light‐mediated antiviral efficacies. Calphostin C was the most active compound against the two target viruses, herpes simplex virus type 1 and Sindbis virus. Hypo‐crellins A and B were also very active. However, three cercosporin‐like PQ were substantially less active in spite of their high quantum yields of singlet oxygen, whereas phleichrome, another efficient singlet oxygen producer, showed no detectable antiviral activity. One other PQ, which was a very weak singlet oxygen producer, also showed no antiviral activity. None of the active compounds showed significant antiviral activity in the dark. Thus, for some groups of PQ there was correlation between quantum yield of singlet oxygen C02) and antiviral efficacy, but there are evidently other structural features of PQ that influence activity.
Changes in Optical Properties of Human Whole Blood in vitro Due to Slow Heating
Authors: Nilsson Annika M. K. , Lucassen Gerald W. , Verkruysse Wim , Andersson‐Engels Stefan , van Gemert Martin J. C.
Published: January 2008

Optical properties of human whole blood were investigated in vitro at 633 nm using a double integrating sphere set‐up. The blood flow was maintained at a constant rate through a flow cell while continuously heating the blood at 0.2–1.lC/min from approximately 25 to 55°C in a heat exchanger. A small, but rather abrupt decrease in the scattering asymmetry factor (g‐factor) of 1.7 ± 0.6% and a similar increase in the scattering coefficient of 2.9 ± 0.6% were observed at approximately 45–46°C yielding an increase in the reduced scattering coefficient of 40 ± 10%. Furthermore, a continuous, manifest increase in the absorption coefficient was seen with increasing temperature, on average 80 ± 70% from 25 to 50°C. The effect of the heating on the blood cells was also studied under a white‐light transmission microscope. A sudden change in the shape of the red blood cells, from discshaped to spherical, was observed at approximately the same temperature at which the distinct changes in g‐factor and scattering coefficient were observed, i.e. at 45–46°C. The results indicate that this shape transformation could explain the sudden change in scattering properties.
The Synthesis, Photophysical and Photobiological Properties and in vitro Structure‐Activity Relationships of a Set of Silicon Phthalocyanine PDT Photosensitizers
Authors: Li Ying‐Syi , Zaidi S. I. A. , Rodgers Michael A. J. , Mukhtar Hasan , Kenney Malcolm E. , Oleinick Nancy L. , He Jin , Larkin Hedy E. , Rihter Boris D.
Published: January 2008

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Fluorescence Lifetime Imaging of Experimental Tumors in Hematoporphyrin Derivative‐Sensitized Mice
Authors: Cubeddu Rinaldo , Canti Gianfranco , Pifferi Antonio , Taroni Paola , Valentini Gianluca
Published: January 2008

Tumor detection has been carried out in mice sensitized with hematoporphyrin derivative (HpD) by measuring the spatial distribution of the fluorescence lifetime of the exogenous compound. This result has been achieved using a time‐gated video camera and a suitable mathematical processing that led to the so‐called “lifetime images.” Extensive experimental tests have been performed on mice bearing the MS‐2 fibrosarcoma or the L1210 leukemia. Lifetime images of mice show that the fluorescence decay of HpD is appreciably slower in the tumor than in healthy tissues nearby, allowing a reliable detection of the neoplasia. The lengthening of the lifetime in tumors depends little on the drug dose, which in our experiments could be lowered down to 0.1 mg/kg body weight, still allowing a definite tumor detection. In order to ascertain the results achieved with the imaging apparatus, high‐resolution spectroscopy, based on a time‐correlated single photon counting system, has also been performed to measure the fluorescence lifetime of the drug inside the tumor and outside. The outcomes obtained with two techniques are in good agreement.
A Role for Neuropeptides in UVB‐Induced Systemic Immunosuppression
Authors: Garssen Johan , Buckley Theresa L. , Loveren Henk Van
Published: January 2008

The aim of this study was to investigate the possible role of sensory nerves in UV light‐induced systemic immunomodulation. Contact hypersensitivity to the low molecular weight compound picrylchloride was used as a model for cellular immunity that can be suppressed by low (i.e. suberythemal) doses of UV light even after exposure at a distant locus (i.e. systemic immunosuppression). In sensory nerve‐depleted mice, achieved by two subcutaneous injections with the neurotoxin capsaicin before the age of 4 weeks, UV light exposure failed to inhibit contact hypersensitivity responses to picrylchloride. This indicates that sensory nerves are at least partially involved in the induction of systemic immunosuppression by UV light. In order to analyze whether sensory neuropeptides, such as calcitonin gene‐related peptide (CGRP) and tachykinins, are involved in UV light‐induced systemic immunosuppression, mice were pretreated with selective antagonists prior to each UV light exposure. These experiments indicated that CGRP but not the tachykinins plays a crucial role in the UV light‐induced systemic immunosuppression.
Characterization and Purification of the Mammalian COP9 Complex, a Conserved Nuclear Regulator Initially Identified as a Repressor of Photomorphogenesis in Higher Plants
Authors: Wei Ning , Deng Xing‐Wang
Published: January 2008

The COP9 complex has been identified as a repressor of photomorphogenesis in Arabidopsis. Here we demonstrate that the COP9 complex is also present in mammals. Specific antibodies were generated against human counterparts of the Arabidopsis COP9 and COP11, the two known subunits of plant COP9 complex. Using these antibodies, we showed that indeed mammalian COP9 and COP11, also known as GPS1, could be coimmuno‐precipitated using either of the two specific antibodies, definitively confirming that they are physically part of the same complex. Further, the mammalian COP9 and COP11/GPS1 were cofractionated in the same large molecular weight fractions of about 500 kDa and were absent from the monomeric fractions. The mammalian COP9 complex was present in all organs examined but abundances vary. Indirect immunofluorescence studies suggested that the mammalian COP9 complex is largely nuclear localized. Both conventional biochemical and affinity purifications of the COP9 complex from pig spleen indicated that the mammalian COP9 complex consists of eight distinct subunits. These findings indicate that mammals also have a COP9 complex with conserved molecular composition and biochemical and cellular properties similar to the higher plant counterpart.
Kinetics and Mechanism of the Photosensitized Oxidation of Furosemide*
Authors: Zanocco A. L. , S. G. Günther , M. E. Lemp , Fuente J. R. de la , U. N. Pizarro
Published: January 2008

Detection of O2(1δg) phosphorescence emission, γmax = 1270 nm, following laser excitation and steady‐state competitive methods was employed to measure total rate constants, kT, for the reactions of the diuretic furosemide, 2‐methylfurane and furfurylamine with singlet oxygen in several solvents. Correlation of kT values with solvent parameters and product identification shows that the reaction mechanism is strongly solvent dependent. In aliphatic alcohols, the dependence of kT on solvent parameters is similar to the one observed for triethylamine, suggesting a reaction mechanism involving partial charge transfer from the amino group to the singlet oxygen. In nonprotic solvents, the dependence of kT on solvent parameters resembles the behavior found for 2‐methylfur‐ane and furfurylamine, implying that mostly a 2 + 4 cy‐cloaddition mechanism of singlet oxygen to furane ring of furosemide occurs in these solvents. These mechanistic differences are explained in terms of hydrogen‐bonding interactions between the carboxylic group in the aromatic ring and the amino group of furosemide. Furthermore, direct generation of C2(1δg) by furosemide was detected. Quantum yields of 0.047 ± 0.003 and 0.078 ± 0.004 were determined in acetonitrile and benzene, respectively. This last result may be related, at least partially, to the photodynamic effects of this diuretic drug.
Tanning in Human Skin Types II and III Offers Modest Photoprotection Against Erythema
Authors: Sheehan John M. , Potten Christopher S. , Young Antony R.
Published: January 2008

We have investigated the photoprotective properties of tanning using erythema as an endpoint. Previously unexposed buttock skin sites of 16 young, healthy adults (8 skin type II, and 8 skin type III) were exposed daily (Mon‐Fri) for 2 weeks to 0.5 and 0.75 minimal erythema doses (MED) of solar‐simulated radiation (SSR). Erythema and melanin levels were assessed daily both visually and quantitatively using a reflectance device. One week after the last tanning treatment, MED reassessments were made on pretreated sites and on adjacent nontreat‐ed sites, including sites from which stratum corneum was removed by tape stripping. Compared to skin type II, similar daily SSR treatments produced less erythema and more evident tanning in skin types III. Independent of skin type, all volunteers showed an increased MED value when assessed on the 0.75 MED‐ and 0.5 MED‐treated sites compared to the MED value assessed on adjacent untreated sites. We express any increase in MED as an induced protection factor (IPF), i.e. (MED post‐tan/MED pre‐tan). Our data show mean IPF of 1.4 and of 2.1 in the 0.5 and 0.75 MED‐treated sites respectively, in skin types II. Similar values were obtained in skin types III with IPF of 1.5 and 2.3 for the 0.5 and 0.75 MED‐treated sites, respectively. In all cases, removal of the stratum corneum lowered the IPF by about 20%. Our results show that SSR‐induced melanogenesis, whether in skin type II or III, offers only moderate protection against erythema and suggest that SSR‐induced stratum corneum thickening affords less photoprotection than tanning.
Photoproduction and Direct Spectral Detection of Singlet Molecular Oxygen (102) in Keratinocytes Stained with Rose Bengal
Authors: Bilski Piotr , Kukielczak Barbara M. , Chignell Colin F.
Published: January 2008

In vivo, keratinocyte skin cells are exposed to photoox‐idative processes, some of which can be mediated by singlet molecular oxygen (1O2), a species that is very difficult to detect spectrally in cells. We photosensitized 1O2 in cultured HaCaT keratinocytes stained with rose bengal (RB) that localizes exclusively inside the keratinocyte hydrophobic regions, as evidenced by strongly red‐shifted absorbance and intense fluorescence. We used keratinocytes grown in a monolayer on a plastic coverslip and in suspension. The phosphorescence spectrum (1200–1350nm) from 1O2 was strongest when the coverslip containing RB‐stained keratinocytes was irradiated in air. The spectral intensity decreased when the coverslip was immersed in D2O during irradiation and was almost completely quenched when it was irradiated while immersed in water. Water not only shortens the 1O2 lifetime but also reabsorbs part of the 1O2 phosphorescence, processes that do not occur when 1O2 is produced in a keratinocyte layer exposed to air. Because the RB was inside keratinocytes, singlet oxygen must also be produced inside the keratinocytes. However, the sensitivity to the extracellular environment suggests that most of the detectable 1O2 phosphorescence originates from those 1O2molecules that escaped from the cell through its membrane into D2O or into the air, where 1O2 has longer lifetimes. Our results confirm directly that 1O2 is indeed photosensitized in living cells by RB. They also suggest that keratinocyte monolayers may be a good cell model to examine in vitro the production of 1O2 by other photo‐sensitizers of environmental and photomedical interest.