bluuebunny/crossref_metadata_2025 huggingface dataset:
PHOTOTHROMBOSIS USING TWO DIFFERENT PHTHALOCYANINE ADMINISTRATION ROUTES: CONTINUOUS I.V. INFUSIO versus BOLUS I.V. INJECTION*
Authors: Tsilimbaris Miltiadis K. , Pallikaris Ioannis G. , Lydataki Stella
Published: January 2008

Abstract—

Chloraluminum sulfonated phthalocyanine was administered in 22 albino rabbits either by means of bolus i.v. (intravenous) injection (6 mg/kg) or as continuous i.v. infusion. Eight animals were used for the comparison of plasma phthalocyanine change as a factor of time, using the two administration methods. Photothrombosis of corneal neovascularization was carried out in the remaining 14 animals either after bolus phthalocyanine injection or after continuous i.v. infusion. Irradiation of the right eye's vessels started 10 min after the injection in the bolus group and 35 min after initiation of infusion in the continuous infusion group. The vessels of each animal's left eye were irradiated 1 h after the irradiation of the right eye. The initial peak of phthalocyanine concentration after bolus injection was followed by a rapid decrease of plasma levels. In the continuous infusion group,30–40 min after the initiation of infusion, plasma phthalocyanine concentration reached a plateau that remained stable for the rest of the80–90 min of infusion. Using bolus injection, a significant decrease of phthalocyanine concentration was accompanied by a significant increase of irradiation time necessary for neovascularization thrombosis in the two eyes. Irradiation time as well as phthalocyanine concentration did not differ between the two eyes using continuous infusion. Vascular photosensitivity seemed to be higher using continuous i.v. infusion of phthalocyanine. Continuous i.v. infusion represents an interesting alternative to bolus injection for phthalocyanine‐mediated corneal neovascularization photothrombosis.
EFFICACY OF PHOTODYNAMIC THERAPY ON ORIGINAL AND RECURRENT RAT MAMMARY TUMORS
Authors: Gibson Scott L. , Nguyen My Lien , Foster Thomas H. , White Geoffrey , Hilf Russell
Published: January 2008

Abstract— Photodynamic therapy has demonstrated efficacy toward primary, metastatic and recurrent human tumors. Here, we investigated the ability of photodynamic therapy, using Photofrinr̀, to inhibit growth of R3230AC mammary adenocarcinomas when tumors were treated as original implants and again as lesions recurring at the initial treatment site. The results demonstrate that both initial implants and lesions recurring after the first photodynamic treatment respond similarly to the same photodynamic therapy protocol, with mean tumor volume doubling times of ˜ 11 days in both cases. Cells cultured from original tumor implants or tumors that recurred after photodynamic treatment accumulate equivalent amounts of [14C]polyhematoporphyrin. Single cell suspensions prepared from either original or recurrent tumors from animals administered 5 mg/kg Photofrinr̀ and exposed to light in vitro displayed comparable phototoxicity. Additionally, examination of tumors by light microscopy revealed no morphological differences between the original tumor implants and the recurrent lesions. Taken together, these data indicate that lesions which recurred at the site of the initial photodynamic treatment were not resistant to a second identical course of photodynamic therapy.
RESOLUTION OF SPECTRAL AND LIFETIME HETEROGENEITY OF TRYPTOPHAN FLUORESCENCE IN BACTERIORHODOPSIN
Authors: Roy Mita , Periasamy N.
Published: January 2008

Abstract— The picosecond time‐resolved fluorescence decay of bacteriorhodopsin (BR) was analyzed by the maximum entropy method. Results showed five distributions of lifetimes indicating at least five decay components. A wavelength‐dependent study of emission decay of BR was carried out in the wavelength region from 310 to 390 nm. The decay at each wavelength was resolvable into four decay components by the discrete exponential analysis. The three short lifetime components (100 ± 20 ps, 400 ± 50 ps and 1.0 ± 0.1 ns) were independent of wavelength, whereas the longest lifetime component was wavelength dependent (varying from 4.1 ns at 310 nm to 5.7 ns at 390 nm). These results are inconsistent with the existing model of associating the fluorescence of bacteriorhodopsin with two or four lifetime components. An attempt is made to associate the five decay components with the emitting tryptophans of BR.
PHOTOSENSITIZED REDUCTION OF L‐BIOPTERIN IN THE ACTIVE TERNARY COMPLEX OF DIHYDROFOLATE REDUCTASE
Authors: Ledbetter John W. , Pfleiderer Wolfgang , Freisheim James H.
Published: January 2008

Abstract—

Photosensitized L‐biopterin induces the transfer of a hydrogen atom from the dihydronico‐tinamide moiety of NADPH to the biopterin ring. Sensitization occurs through the triplet state of both the lactim and lactam tautomers of L‐biopterin. Quenching kinetic analysis to measure the bimolecular rate constant demonstrated a greater reactivity for the lactim tautomer. Recombinant human dihydro‐folate reductase enzyme, for which these molecules are substrate and cofactor, enhanced the rate constant of the photosensitized H• transfer to the apparent lactim tautomer by eight times to 1.6 × 1010M‐1s‐1. When NADP+ replaced NADPH at the active site, no enhanced photoreduction of biopterin was observed, implying that the hydrogen atom comes from the reduced nicotinamide group and not as a result of protein conformational changes. This reduction at the active site represents a photoinitiated H• transfer in protein between substrate and cofactor.
SUBCELLULAR LOCALIZATION OF AND PHOTOSENSITIZATION BY PROTOPORPHYRIN IX IN HUMAN KERATINOCYTES AND FIBROBLASTS CULTIVATED WITH 5‐AMINOLEVULINIC ACID
Authors: Gaullier Jean‐Michel , Gèze Marc , Santus René , Melo Teresa Sa e. , Mazière Jean‐Claude , Bazin Marc , Morlière Patrice , Dubertret Louis
Published: January 2008

Abstract— The subcellular localization of protoporphyrin (PP) has been studied by microspectrofluo‐rometric techniques in NCTC 2544 keratinocytes incubated with 5‐aminolevulinic acid (ALA) for times up to 42 h. Whereas the plasma membrane shows strong staining, fluorescent spots are observed within the cytoplasm especially in the perinuclear region. Although the topographic pattern of the PP distribution does not change much with the incubation time with ALA, the fluorescence spectra suggest that the PP microenvironments are quite different at short and long incubation times. Addition of 18 uJW desferoxamine almost doubles the ALA‐induced PP concentration. Colocalization experiments with rhodamine 123, a mitochondrial probe, and lucifer yellow (LY) or neutral red (NR), two lysosome probes, demonstrate that at least some of these spots are of lysosomal origin. Study of the time evolution of the NR fluorescence under irradiation with visible light in the presence and absence of ALA demonstrates that lysosomes are damaged in cells that have synthesized PP. No PP fluorescence can be detected in mitochondria after incubation with ALA. However, photosensitization of mitochondria occurs under irradiation with visible light. Very little formation of lipofuscins by photosensitization with exogenous PP or ALA‐induced PP is observed with the NCTC 2544 keratinocytes, as compared to normal human fibroblasts.
THE ROLE OF UROCANIC ACID IN UV‐INDUCED IMMUNOSUPPRESSION: RECENT ADVANCES (1992–1994)
Authors: Norval Mary , Gibbs Neil K. , Gilmour Jill
Published: January 2008

Abstract— Cis ‐ urocanic acid (UCA), formed in the epidermis by UV irradiation of trans‐UCA, has been implicated as a mediator of the immunosuppression induced by UV exposure of the skin. This review covers recent work in which the wavelength dependence of cis‐UCA formation, the interaction of UCA isomers with DNA, the effects of UCA isomers on the immune system and their interaction with histamine are examined. Results are frequently conflicting, particularly when considering the possible mode of action of cis‐UCA but, overall, a multifaceted role for UCA in immunomodulation by UV radiation is substantiated.
INVESTIGATION OF LIGHT‐INDUCED CONFORMATION CHANGES IN SPIROPYRAN‐MODIFIED SUCCINYLATED POLY(l‐LYSINE)
Authors: Cooper Thomas M. , Stone Morley O. , Natarajan Lalgudi V. , Crane Robert L.
Published: January 2008

Abstract— To determine the maximum range of coupling between side‐chain photochromism and polypeptide conformation change, we modified the carboxylate side chains of succinylated poly(l‐lysine) with a spiropyran to form polypeptide I. The extent of modification was determined to be 35.5%. The spacer group length between the polypeptide a‐carbon and the dye was 12 atoms, providing minimum polypeptide‐dye interaction. Conformation changes were monitored by circular dichroism as a function of light adaptation and solvent composition (hexafluoroisopropanol [HFIP] vs trifluoroethanol [TFE]). Under all solvent compositions, the dark‐adapted dye was in the merocyanine form. Light adaptation by visible light converted the dye to the spiropyran form. When dissolved in TFE, I adopted a helical conformation insensitive to light adaptation. With increasing percentage HFIP, a solvent‐induced helix‐to‐coil transition was observed around 80% (vol/vol) HFIP. At 100% HFIP, both light‐ and dark‐adapted forms of I were in the coil state. Near the midpoint of the solvent‐induced helix‐to‐coil transition, light adaptation caused conformation changes. Applying helix‐to‐coil transition theory, we measured a statistically significant difference in coil segment‐HFIP binding constant for light‐ v.v dark‐adapted solutions (6.38 ± 0.03 M‐1vs 6.56 ± 0.03 M‐1), but not for the nucleation parameter σ (1.2 ± 0.4 10‐3 v.v 1.3 ± 0.3 × 10‐3). The small binding constant difference translated to a light‐induced binding energy difference of 17 cal/mol/monomer. Near the midpoint of the helix‐to‐coil transition, collective interactions between monomer units made possible the translation of a small energy difference (less than RT) into large macromolecular conformation changes. This work parallels similar behavior observed in poly(isocyanate) (Green, M. M. et al. J. Am. Chem. Soc.115, 4941–4942, 1993). The subtle differences in dye‐backbone interaction in I suggested a maximum coupling distance (12 atoms) beyond which polypeptide conformation and dye state are uncoupled.
INACTIVATION OF THE ACCEPTOR SIDE AND DEGRADATION OF THE D1 PROTEIN OF PHOTOSYSTEM II BY SINGLET OXYGEN PHOTOGENERATED FROM THE OUTSIDE
Authors: Chung Soon Kyu , Jung Jin
Published: January 2008

Abstract— The possible association of photodynamic sensitization with photoinhibition damage to the photosystem II complex (PS II) has been investigated using isolated intact thylakoids from pea leaves. For this study singlet oxygen (1O2), photoproduced by endogenous chromophores that are independent of the function of PS II, was assumed to be the major reactive intermediate involved in the photoinhibition process. When thylakoid samples preincubated with rose bengal were subjected to exposure to relatively weak green light (500–600 nm) under aerobic conditions, PS II was severely damaged. The pattern of the rose bengal‐sensitized inhibition of PS II was similar to that of high light‐induced damage to PS II: (1) the secondary quinone (QB)‐dependent electron transfer through PS II is inactivated much faster than the QB‐independent electron flow, (2) PS II activity is lost prior to degradation of the D1 protein, (3) diuron, an herbicide that binds to the QB domain on the D1 protein, prevents D1 degradation, and (4) PS II is damaged to a greater extent by the deuteration of thylakoid suspensions but to a lesser extent by the presence of histidine. Furthermore, it was observed that destroying thylakoid Fe‐S centers resulted in a marked reduction of high light‐induced PS II damage. These results may suggest that the primary processes of photoinhibition are mediated by 1O2 and that Fe‐S centers, which are located in some membrane components, but not in PS II, play an important role in photogenerating the activated oxygen immediately responsible for the initiation of photodamage to PS II.
CHANGES IN THE PATTERNS OF BIOSYNTHESIS and COMPOSITION OF AMINO ACIDS IN A MARINE PHYTOPLANKTER EXPOSED TO ULTRAVIOLET‐B RADIATION: NITROGEN LIMITATION IMPLICATED
Authors: GOES Joaquim I. , Handa Nobuhiko , Taguchi Satoru , Hama Takeo
Published: January 2008

Abstract—The impact of ultraviolet‐B radiation (UVBR) on the biosynthesis and the composition of intracellular dissolved free and combined amino acids was examined in nitrate‐replete and nitrate‐deficient cultures ofTetraselmissp. Several similarities were observed in the response ofTetraselmissp. to nitrogen deficiency and exposure to UVBR, in support of a view that UVBR affects amino acid synthesis in phytoplanktonviaits inhibitory effects on nitrogen assimilation into the cells. At levels that still permitted the uptake of carbon into the cells, both nitrogen deficiency and UVBR exposure resulted in a reduction in the overall rates of carbon incorporated into amino acids, an increase in the absolute concentrations of amino acids within the intracellular dissolved free amino acid (INDFAA) pool and a decrease in the total cellular amino acid (TCAA) pool. An examination of the patterns of carbon assimilation into individual amino acids in cells exposed to UVBR revealed similarities with the patterns in cells subject to nitrogen deficiency. The most conspicuous changes from the controls included an increased incorporation of13C into glutamic' acid (glutamic acid + glutamine) and aspartic acid and a marked reduction into alanine and valine. Changes in the concentrations of amino acids within the INDFAA and TCAA pools were also similar in nitrate‐deficient and UVBR‐exposed cells and resembled the carbon assimilation patterns. These results strongly suggest that UVBR‐induced changes in the biosynthesis and composition of amino acids are probablyviaits suppression of nitrogen assimilation into the cells.
POTENTIATION OF MEROCYANINE 540‐MEDIATED PHOTODYNAMIC THERAPY BY SALICYLATE and RELATED DRUGS
Authors: Traul Donald L. , Anderson Gregory S. , Bilitz James M. , Krieg Marianne , Sieber Fritz
Published: January 2008

Abstract— Simultaneous exposure to merocyanine 540 (MC540) and light of a suitable wavelength kills leukemia, lymphoma and neuroblastoma cells but is relatively well tolerated by normal pluripotent hematopoietic stem cells. This differential phototoxic effect has been exploited in preclinical models and a phase I clinical trial for the extracorporeal purging of autologous bone marrow grafts. Salicylate is known to potentiate the MC540‐mediated photokilling of tumor cells. Assuming that salicylate induces a change in the plasma membrane of tumor cells (but not normal hematopoietic stem cells) that enhances the binding of dye molecules it has been suggested that salicylate may provide a simple and effective means of improving the therapeutic index of MC540‐mediated photodynamic therapy. We report here on a direct test of this hypothesis in a murine model of bone marrow transplantation as well as in clonal cultures of normal murine hematopoietic progenitor cells. In both systems, salicylate enhanced the MC540‐sensitized photoinactivation of leukemia cells and normal bone marrow cells to a similar extent and thus failed to improve the therapeutic index of MC540 significantly. On the basis of a series of dye‐binding studies, we offer an alternative explanation for the potentiating effect of salicylate. Rather than invoking a salicylate‐induced change in the plasma membrane of tumor cells, we propose that salicylate displaces dye molecules from serum albumin, thereby enhancing the concentration of free (active) dye available for binding to tumor as well as normal hematopoietic stem cells.
Model Studies of DNA Photorepair: Enthalpy of Cleavage of a Pyrimidine Dimer Measured by Photothermal Beam Deflection Calorimetry
Authors: Scannell Michael P. , Yeh Syun‐Ru , Falvey Daniel E.
Published: January 2008

Abstract— The enzyme DNA photolyase mediates the repair of pyrimidine dimers. This repair step, a net retro [2+2] reaction, proceeds through either the cation or anion radical of the pyrimidine dimer. In order to understand how electron transfer makes the repair process possible, its energetics have been examined by photothermal beam deflection calorimetry, fluorescence quenching and quantum yield studies. The enthalpy for the cleavage reaction of cis‐syn 1,3‐dimethylthymine dimer itself was found to be ‐19 kcal/mol. In addition, from the redox potentials, the enthalpies for the cleavage reactions of the dimer cation radical and the anion radical were determined to be ‐19 kcal/mol and ‐28 kcal/mol, respectively.
Red Light‐Induced Appearance of Phosphotyrosine‐like Epitopes on Nuclear Proteins from Pea (Pisum sativum L.) Plumules
Authors: Tong Chii‐Gong , Kendrick Richard E. , Roux Stanley J.
Published: January 2008

Abstract— As assayed by western blot analysis, red light induces the appearance of epitopes recognized by anti‐phosphotyro‐sine antibodies in several pea nuclear proteins. The im‐munostaining is blocked by preadsorbing the antibodies with phosphotyrosine but not by preadsorbing them with phosphoserine or phosphothreonine. This light response is observed whether the red light irradiation is given to pea plumules or nuclei isolated from the plumules. The red‐light‐induced response seen in plumules is reversible by a subsequent far‐red‐light irradiation, indicating that the likely photoreceptor for this response may be phy‐tochrome. By immunoblot analysis pea phytochrome A, but not phytochrome B, can be detected in proteins extracted from pea nuclear chromatin‐matrix preparations. Phytochrome A and the protein bands immuno‐stained by anti‐phosphotyrosine antibodies can be solu‐bilized from unirradiated pea chromatin by 0.3 M NaCl, but irradiating this preparation with red light does not induce the appearance of phosphotyrosine‐like epitopes in any nuclear proteins. These results suggest that the association of phytochrome with purified pea nuclei is such that its conversion to the far‐red light‐absorbing form can induce a post‐translational epitope change in nuclear proteins in vivo.
Damage Threshold of Normal Rat Brain in Photodynamic Therapy
Authors: Chen Q. , Chopp M. , Madigan L. , Dereski M. O. , Hetzel F. W.
Published: January 2008

Normal brain tissue response to photodynamic therapy (PDT) must be quantified in order to implement PDT as a treatment of brain neoplasm. We therefore calculated the threshold for PDT‐induced tissue necrosis in normal brain using Photofrin (porfimer sodium, Quadralogic Technologies Inc., Vancouver, BC) as the photosensitizer. The absolute light fluence‐rate distribution for superficial irradiation and effective attenuation depth were measured in vivo using an invasive optical probe. Photosensitizer uptake in cerebral cortex was measured with chemical extraction and fluorometric analysis. Photodynamic therapy‐induced lesion depths at various drug dose levels were measured as a biological end point. The PDT threshold for normal brain necrosis was calculated as in the magnitude of 1016 photons/cm3. Thus normal rat brain is extremely vulnerable to PDT damage. This suggests that extra precautions must be exercised when PDT is used in brain.
Laser Flash Photolysis of 2,2′‐Dithiobls(pyridine N‐oxide): Reactivity of N‐Oxypyridyl‐2‐thio Radical
Authors: Alam Maksudul M. , Watanabe Akira , Ito Osamu
Published: January 2008

Abstract— Reaction kinetics of radicals produced by the nanosecond laser flash photolysis of 2,2′‐dithiobis(pyridine N‐oxide) and related compounds have been studied. The transient absorption band at 360 nm was attributed to the radical in which the unpaired electron mainly localizes on the S atom (N‐oxypyridyl‐2‐thio radical). The reactivities of the radical for conjugated dienes are lower than those of the pyridyl‐2‐thio radical, suggesting that a considerable unpaired electron density on the S atom delocalizes onto the N‐oxypyridine moiety. The addition reaction rate of the radical to the conjugating diene was accelerated with hydrogen‐bonding solvents and with addition of the cation, which may stabilize the N+‐O‐ canonical structure, increasing the unpaired electron density on the S atom. By the photolysis of N‐hydroxypyridine‐2‐thione, the N‐O bond was predominantly dissociated producing a pyr‐idyl‐2‐thio radical. By the photolysis of its anion, photoejection took place followed by the N‐O bond fission, yielding pyridine‐2‐thione.
Probing Bile Salt Aggregates by Fluorescence Quenching
Authors: Ju C. , Bohne C.
Published: January 2008

Abstract— The combination of steady‐state and time‐resolved quenching experiments was employed to study the aggregation behavior of sodium cholate at concentrations below 50 mAf. Naphthalene, anthracene and pyrene were used as fluorescent probe molecules, and protection by the aggregates from aqueous quenchers, as well as the onset of aggregation at low sodium cholate concentrations, was dependent on the shape of the probes. Protection from aqueous quenchers was inferred by comparing the efficiency for dynamic quenching in the absence and presence of sodium cholate and was best for naphthalene followed by pyrene and anthracene. Static quenching was observed, suggesting that probe molecules are located in an aggregate environment that also contains iodide. The incorporation of pyrene at low sodium cholate concentrations, as well as the small degree of static quenching observed, suggest that the shape complementarity, i.e. hydrophobic surface and packing, between pyrene and sodium cholate is optimum for aggregate formation.
Reversible Photobleaching of Fluorescein Conjugates in Air‐Saturated Viscous Solutions: Singlet and Triplet State Quenching by Tryptophan
Authors: Periasamy N. , Bicknese S. , Verkman A. S.
Published: January 2008

Abstract— Fluorescence recovery after photobleaching (FRAP) measurements on air‐saturated aqueous solutions of fluorescein made viscous with glycerol or sucrose revealed a rapid component of fluorescence recovery with exponential time constants of 30‐120 μs at viscosities of 15‐300 cP. The rapid recovery process was not related to fluorophore translational diffusion and was insensitive to fluorophore concentration and the additive used to increase solution viscosity. At constant viscosity, the rate of reversible photobleaching recovery increased 2.5‐fold in an O2‐ vs N2‐saturated solution. The relative efficiency of reversible‐to‐irreversible photobleaching decreased with increasing photobleaching time and/or beam intensity. Reversible photobleaching was also detected for conjugates of fluorescein with dextrans and proteins in viscous media. In screening triplet state quenchers that might influence the reversible recovery, it was found that tryptophan enhanced the rate of reversible photobleaching recovery (two‐fold increase at 8 mM) and quenched the fluorescein singlet state (Stern‐Volmer constant, 12 M−1). Analysis of fluorescein lifetimes and photobleaching parameters for a series of fluorescein‐labeled proteins with different numbers of tryptophans were also carried out. The results provide evidence for an oxygen‐dependent, reversible photobleaching mechanism for the fluorescein chromophore involving triplet state relaxation. The identification of reversible fluorescein photobleaching has important implications for FRAP measurements of rapid solute diffusion in biological systems.
An Improved Algorithm of Resolution of Fluorescence Spectra into Quencher Accessibility‐Associated Components
Authors: Burstein E. A.
Published: January 2008

Abstract— A novel algorithm (QUENCH) is described that resolves fluorescence spectra into components differing in the accessibility (the Stern‐Volmer constants) for small quencher molecules. In contrast to the known algorithms, QUENCH first evaluates Stern‐Volmer constants of the components using the total set of intensities in spectra measured at several quencher concentrations and then calculates contributions of each component in the emission intensities at every wavelength value. The component spectra, revealed with QUENCH in the tryptophan fluorescence of ribosomal S7 protein, are very similar to the log‐normal components calculated using the SIMS program (Abornev, S. M. and E. A. Burstein Mol. Biol. [Engl, transl.] 26, 890‐897 1992). The QUENCH method is applicable for the component resolution of fluorescence spectra of any liquid system containing fluorophores of different accessibility to small quenchers.
UVB‐lnduced Decreased Resistance to Trichinella spiralis in the Rat Is Related to Impaired Cellular Immunity
Authors: Goettsch W. , De Gruijl F. R. , Van Loveren H. , Garssen J.
Published: January 2008

Abstract— Our laboratory has demonstrated in preliminary experiments that UVB exposure using the Kromayer lamp can induce increased numbers of Trichinella spiralis larvae in carcasses of infected Wistar rats, without affecting specific antibody titers to this parasite. In this study, orally T. spiralis‐infected Wistar rats were exposed to subery‐themal doses of UVB radiation using FS40 lamps during different time periods before or after infection. A significant increase in the number of T. spiralis larvae was found in the carcasses of rats that were UVB irradiated daily for 7 consecutive days in the second week after infection. Additionally, increased numbers of larvae were also detected histologically in the tongue of rats that were exposed the first and the second week after infection. Lymphocyte stimulation assays using mesenteral lymph node cells indicated that UVB exposure also impaired the specific lymphocyte response to T. spiralis. Moreover, DTH responses to T. spiralis were severely impaired in rats that were UVB irradiated daily for 7 consecutive days in the second week after infection. Thus, these data combined with the data of the Kromayer study indicate that exposure of rats to FS40 irradiation following oral infection with T. spiralis leads to increased numbers of larvae in systemic sites and impaired T‐cell immunity to the parasite.
Inactivation of Photosensitizing Merocyanine Dyes by Plasma, Serum and Serum Components
Authors: Anderson Gregory S. , Günther Wolfgang H. H. , Searle Roger , Bilitz James M. , Krieg Marianne , Sieber Fritz
Published: January 2008

Abstract— Merocyanine dyes with an oxygen in the electron donor heterocycle were rapidly degraded by plasma, serum and serum components. Replacement of the oxygen by a sulfur or selenium atom rendered the dyes refractory to degradation. The degradation of labile merocyanine dyes was temperature dependent and oxygen independent. The plasma component that was responsible for the degradation of merocyanine dyes was sensitive to heat and detergent, suggesting an enzymatic process. The identification of the structural requirements for sensitivity/resistance to degradation provides the experimenter with a simple means to manipulate the stability of mer‐ocyanines in high serum or plasma environments and may expand the clinical utility of merocyanine photosen‐sitizers beyond their traditional role in the extracorporeal purging of bone marrow grafts.
PUVA Treatment Selectively Induces a Cell Cycle Block and Subsequent Apoptosis in Human T‐Lymphocytes
Authors: Johnson Ray , Staiano‐Coico Lisa , Austin Lisa , Cardinale Irma , Nabeya‐Tsukifuji Reiko , Krueger James G.
Published: January 2008

Psoralen plus UVA (320–400 nm radiation; PUVA) is a highly effective therapy for cutaneous diseases caused by skin infiltration with normal or neoplastic T‐lympho‐cytes. In comparing the effects of pharmacologically relevant, low‐dose PUVA treatment on growth of human keratinocytes, peripheral blood leukocytes (PBMC), and T‐lymphocyte cell lines, we determined that PBMC or T‐lymphocytes were >50‐fold more sensitive to cytotoxic effects of PUVA, while antiproliferative effects were produced by similar PUVA levels in all cell types. Low doses of PUVA (10 ng/mL 8‐methoxypsoralen and 1–2 J/cm2) were highly cytotoxic for phytohemagglutinin‐activated normal lymphocytes or transformed T‐lymphocytes as assessed by two viability assays and by flow cytofluo‐rometry. Altered lymphocyte morphology, nuclear fragmentation, TUNEL+ nuclei or nuclear fragments, and the appearance of a sub‐G, DNA peak indicated that cell death occurred by apoptosis, beginning about 1 day after PUVA treatment and continuing for several days thereafter. From assessment of cell cycle progression in mi‐mosine‐synchronized cells, PUVA treatment markedly slowed cell cycle progression, eventually producing cell cycle arrest and apoptotic entry. We propose that the probable basis for disease remissions (psoriasis, cutaneous T‐cell lymphoma) produced by PUVA treatment is through selective cytotoxic effects on clonal T‐lymphocyte populations that are concentrated in diseased skin.
The Effects of ex vivo Handling Procedures on the Near‐Infrared Raman Spectra of Normal Mammalian Tissues
Authors: Shim Martin G. , Wilson Brian C.
Published: January 2008

Recent studies in the literature have investigated the feasibility of tissue diagnostics based on Raman spectroscopy. The majority of these compare the ex vivo spectra of normal and diseased tissue. Due to the time lapse between tissue excision and spectroscopic examination, samples must be frozen or otherwise preserved to maintain their native biochemical states. In order to establish optimum procedures for ex vivo Raman spectroscopy of tissue, the effects of tissue drying, formalin fixing, snap freezing, tissue freezing in optimal cutting temperature (OCT) medium and extended post‐thaw durations were studied to determine if any of these handling procedures introduced spectral artifacts. Experiments on representative tissues indicated that tissue heating due to the excitation light did not change the spectra significantly. With minor exceptions, OCT and formalin did not contaminate tissue spectra, so that samples stored for histological examination could also be studied with Raman spectroscopy. Tissue dehydration caused disruption of the protein vibrational modes, which caused spectral artifacts. It is concluded that ex vivo tissue samples should be frozen in OCT. Prior to spectral analysis, the tissue should then be acclimatized at room temperature in phosphate‐buffered saline (PBS) and immersed in PBS during spectroscopic examination.
Time‐Resolved (CW) Electron Paramagnetic Resonance Spectroscopy: An Overview of the Technique and Its Use in Organic Photochemistry
Authors: Forbes Malcolm D. E.
Published: January 2008

Abstract— Steady‐state and time‐resolved electron paramagnetic resonance (TREPR) experiments are described. Comparison of the TREPR continuous wave method to other time domain EPR techniques such as Fourier transform EPR (FT‐EPR) is made, and the advantages and disadvantages of each are presented. The role played by several mechanisms of chemically induced dynamic electron spin polarization (CIDEP) in the appearance of the spectra is explained. The advantages of using higher frequency spectrometers than the standard X‐band (9.5 GHz) are presented and discussed. Examples are presented that are relevant to organic photochemistry and electron donor‐acceptor chemistry. The use of TREPR to study polymer photodegradation, polymer chain dynamics, free radical initiator chemistry and biradical spin exchange interactions is described. Emphasis is placed on magnetic field effects studied by multiple frequency TREPR in these systems. Finally, several future directions in the field are discussed in terms of new developments in microwave and magnetic field technology.
Glutathione Response after UVA Irradiation in Mitotic and Postmitotic Human Skin Fibroblasts and Keratinocytes
Authors: Niggli Hugo J. , Applegate Lee A.
Published: January 2008

Abstract— Since Hayflick's pioneering work in the early sixties, human diploid fibroblasts have become a widely accepted in vitro model system. Recently, Bayreuther and co‐workers extended this experimental approach showing that fibroblasts in culture resemble, in their design, the hemopoietic stem‐cell differentiation system. They found that the chemical agent mitomycin C accelerates the differentiation pathway from mitotic to postmitotic fibroblasts. We measured the response of endogenous glutathione levels after UVA irradiation (320‐400 nm) in mitotic and mitomycin C‐induced postmitotic human skin fibroblasts and foreskin‐derived keratinocytes. The initial levels in mitotic foreskin derived human fibroblasts were 14.4 nmol glutathione per mg protein, whereas a 30% higher value was obtained in matching foreskin‐derived keratinocytes. Similiar elevated levels of this important intracellular free radical scavenging system were found in fibroblasts of a donor suffering from xeroderma pigmentosum. Furthermore, three to four times higher levels of glutathione in mitomycin C‐treated mitotic fibroblasts have been determined. In mitotic skin fibroblasts, UVA irradiation resulted in a depletion of glutathione up to 90% following a fluence of 1.0 MJ/m2UVA radiation. Higher initial glutathione levels were found in keratinocytes and mitomycin C‐treated skin fibroblasts. In these fibroblasts a 70% depletion was detected and a much lower depletion (10‐20%) was seen in some keratinocyte cell lines following fluences up to 1.0 MJ/m2. The depletion in skin fibroblasts was retained after 24 h following a fluence of 0.75 MJ/m2UVA light. In view of the fact that glutathione has been shown to be involved in a variety of metabolic processes and plays a role in cellular protection against UVA radiation, our results imply that the fibroblast differentiation system is a very useful tool to unravel the complex mechanism of UVA‐induced oxidative stress.
Fluorescence and Time‐Resolved Photoacoustics of Hypericin Inserted in Liposomes: Dependence on Pigment Concentration and Bilayer Phase
Authors: Losi Aba
Published: January 2008

Abstract— Solvatochromic effects on the energy of visible absor‐bance and fluorescence transitions in hypericin have been studied in organic solutions and in phosphatidylcholine liposomes, suggesting that the molecule in this last case is preferentially located in the polar aprotic zone close to the lipid‐water interface. Nonradiative and radiative decays of the pigment have also been studied, combining photocalorimetric and fluorescence techniques, as a function of the pigment concentration in the liposomal phase and of the gel‐to‐sol thermotropic transition of the phos‐pholipidic bilayer. The results show that hypericin can sense the phase transition by exhibiting a stepwise increasing of the fluorescence quantum yield; concomitantly photoacoustic data indicate that in the gel phase, above a certain concentration of hypericin, clustering of the pigment promotes the formation of nonradiative long‐lived species, whereas when the bilayer is in the sol phase ultrafast nonradiative pathways become the main deactivation channels upon increasing concentration. As a consequence, as shown by photoacoustic results, the photosensitized formation of singlet oxygen is prevented when the local concentration of hypericin in the lipidic phase is greater than about 30 mM.
Ex vivo Application of δ‐Aminolevulinic Acid Induces High and Specific Porphyrin Levels in Human Skin Tumors: Possible Basis for Selective Photodynamic Therapy
Authors: Fritsch Clemens , Batz Janine , Bolsen Klaus , Schulte Klaus‐Werner , Zumdick Matthias , Ruzicka Thomas , Goerz Giinter
Published: January 2008

Abstract— In photodynamic therapy with topically applied δ‐aminolevulinic acid porphyrins are acting as photosensitiz‐ers. The profile of porphyrin metabolites in normal or in neoplastic skin after administration of δ‐aminolevulinic acid has not been determined in detail yet. Thus, to study porphyrin biosynthesis in human skin an organ culture model was developed. Explant pieces of normal skin, ker‐atoacanthoma, and basal cell carcinoma were incubated with 1 niM δ‐aminolevulinic acid for 36 h. Levels of δ‐aminolevulinic acid, porphyrins and porphyrin metabolites were measured in tissues and supernatants. After incubation with δ‐aminolevulinic acid, higher porphyrin levels were demonstrated in tumors as compared to normal skin. In supernatants, most of formed porphyrins, preferentially highly carboxylated porphyrin metabolites, were measured. The pattern of synthesized porphyrins differed between normal and neoplastic skin explants. In tissues of basal cell carcinomas protoporphyrin was preferentially shown and tissues of keratoacanthomas were characterized by a predominance of coproporphyrin as compared to normal skin. The results show that explant cultures offer an easy approach to examine the porphyrin biosynthesis of various tissues. The tumor‐specific δ‐aminolevulinic acid metabolism indicates additional porphyrin metabolites such as coproporphyrin apart from protoporphyrin as effective photosensitizers and may offer a novel approach to tumor‐selective photodynamic damage.
Analysis of UVB‐modulated Gene Expression in Human Keratinocytes by mRNA Differential Display Polymerase Chain Reaction
Authors: Abts Harry F. , Breuhahn Kai , Michel Gunter , Kohrer Karl , Esser Peter , Ruzicka Thomas
Published: January 2008

Abstract— EUltraviolet (UV) light is the most important environmental insult to skin. Even a single exposure to UVB radiation can result in inflammation and may also lead to DNA damage and apoptosis in the acute response of the cutaneous tissue. To elucidate the complex alterations of gene expression in human keratinocytes underlying these UV responses we took advantage of differential display polymerase chain reaction (DD‐PCR) technology's ability to detect qualitative and quantitative changes in gene expression in more than two cell populations simultaneously. We demonstrate that low‐dose UVB (100 Jm‐2) leads to both induction and down regulation of different genes during the 24 h after irradiation in a time‐dependent manner. In addition to the identification of known genes as possible effectors or targets in the UV response of human keratinocytes, we here identify a new sequence that is negatively regulated by UVB irradiation and was termed HUR 7 (HaCaT UV repressed). In general our results showed that DD‐PCR is a useful tool in the analysis of quantitative changes of mRNA levels in human keratinocytes after UV irradiation. The identification of new UVB‐repressed genes offers the opportunity to identify unrecognized molecular mechanisms in the UV response of human cells.
Energetics of Photoinduced Electron Transfer in the Indole‐O2 Cluster in Gas Phase: Possible Consequences for Photoexcited Tryptophan in Solution
Authors: Weinkauf R. , Schiedt J.
Published: January 2008

Abstract— A direct process for an activationless electron transfer from photoexcited tryptophan to molecular oxygen is proposed. By photodetachment of mass‐selected indole‐O2‐ clusters in gas phase a neutral indole+ O2‐ charge‐separated exciplex state is found at 2.25 0.2 eV above the neutral ground state. By theory also, the existence of an excited charge separated state at 3.05 0.2 eV is postulated. In gas phase both charge‐separated cluster states are energetically below the first singlet states 1Lb and 1La and the lower even below the first triplet state T1 of indole. In gas‐phase clusters these energetics imply a very efficient quenching of photoexcited indole by fast electron transfer to oxygen. We discuss a similar mechanism for tryptophan‐O2 in aqueous environment and find it without activation barrier and presumably extremely fast. In the collisional tryptophan*‐O2 complex the efficiency and the time scale of the charge transfer process should be mostly solvent independent. In polar solvent a complete charge separation and free superoxide formation are expected. We correlate this model with previous fluorescence and phosphorescence quenching data of excited tryptophan by O2 and propose electron transfer to be the relevant process.
Femtosecond Spectroscopic Studies on the Red Light‐Absorbing Form of Oat Phytochrome and 2,3‐Dihydrobiliverdin
Authors: Rentsch Sabine , Hermann Gudrun , Bischoff Mark , Strehlow Dietmar , Rentsch Manfred
Published: January 2008

Abstract— The excited state behavior of the red light‐absorbing form of phytochrome (Pr) was studied on the femtosecond time scale. After excitation of Pr with 75 fs laser pulses at 616 nm the kinetics of the transient absorption changes was recorded at selected wavelengths probing mainly the bleaching of the Pr ground‐state absorption and the stimulated emission. The kinetic data obtained indicate the population of an excited state with a 3 ps lifetime immediately after excitation. This state precedes the formation of another excited state with a 32 ps lifetime. The decay of the latter state is followed by the appearance of a first product state that is assumed to represent lunii‐R. In addition, 2,3‐dihydrobiliverdin, which is considered to be an adequate model of the Pr chro‐mophore, was included in the femtosecond studies. The absorption difference spectra recorded at various delay times show an immediate bleaching of the ground‐state absorption. Simultaneously with bleaching a broad transient absorption appears between 410 and 525 nm. The data analysis yields similar kinetic components as they were observed in the decay of Pr. It is suggested from this finding that within the first tens of picoseconds after excitation the excited‐state properties of Pr are mainly determined by the properties of the chromophore itself.
Direct Measurements for Isomer Ratio of Retinal in Bacteriorhodopsin by an Electrochemical Method
Authors: Koyama Koichi
Published: January 2008

Abstract— The composition of retinal isomers in bacteriorhodopsin (bR) in purple membrane (PM) was determined by photoelectric response measurements using a sandwich‐type electrochemical cell. The measured amplitude of the photocurrent obtained from a dark‐adapted sample was 55% lower than that from a light‐adapted sample. This ratio, 55:45, would correspond to the 13‐cis/aU‐trans isomer ratio of retinal in the dark if the 13‐cis form of the pigment did not give a response. This amplitude change correlated with the visible spectral shift of bR. The isomer ratio in the dark depended only weakly on the temperature of the electrolyte, whereas the retinal isomerization rate strongly depended on the temperature and the pH of the electrolyte in the cell. Our results indicate that photoelectric response is elicited only by a species originating from bR containing all‐trans retinal and that the behavior of the response in the dark is associated with the pKa of the proton release kinetics of Asp‐85.
Subcellular Localization of Sulfonated Tetraphenyl Porphines in Colon Carcinoma Cells by Spectrally Resolved Imaging
Authors: Malik Zvi , Amit Ifat , Rothmann Chana
Published: January 2008

Subcellular localization of the dye, 5,10,15,20‐tetra(4‐sul‐fonatophenyl)porphine (TPPS4) and the more hydrophobic dye, 5,10,15,20‐tetra(1‐sulfonatophenyl)porphine (TPPS1), in murine colon carcinoma cells was studied by spectrally resolved imaging (SRI) combined with image processing techniques. Spectrally resolved imaging enabled the acquisition of multipixel fluorescence spectra (>104) from a single cell. Demarcation of specific localization sites and segregation of the irrelevant fluorescence were based on the pixel spectra and by operating the functions of spectral similarity mapping (SSM), principal component analysis (PCA) and spectral classification. The SRI revealed the fine details of the photochemical process that clarify some aspects of subcellular damage. The SRI depicted the differences between TPPS4 and TPPS, with respect to their initial localization and their fate at the end of the photochemical effect. The dye TPPS4 was localized initially in lysosomal vesicles, and upon irradiation fluorescence was seen in the nucleus as well as in vesicles. Some of the vesicles were closely related to the nucleus, as resolved by SSM, PCA and spectral classification. Additional light exposure stimulated relocalization of TPPS4 into the nucleus as well as into the nucleolus, which was clearly depicted by SSM and PCA. Spectral classification showed a third, weak residual cytoplasmic array around the nucleus. The dye TPPS, concentrated in a Golgi‐like complex and was resolved in the nuclear envelope and in small vesicles: it was not redistributed into other compartments upon photosensitization. Serum supplementation to the incubation media of colon carcinoma cells treated with TPPS4 or TPPS, did not change the localization patterns. Pixel spectra of the two dyes in the cells showed spectral shifts and expanded shoulders due to microenvironmental effects. Thus, the chemical nature of the sulfonated phenyl porphines, and not their interaction with serum proteins, was the main determinant of their binding to the lysosomes, nucleus, nucleolus, nuclear envelope or Golgi.
Free Radical Production upon Photoinduced Electron Transfer Reactions in the Presence of Vinyl Monomers. Effect of Water Addition*
Authors: Encinas M. V. , Lissi E. A. , Rufs A. M. , Altamirano M. , Cosa J. J.
Published: January 2008

Free radical fragments produced in the photoinduced electron transfer from triethylamine (TEA) to excited pyrenebutyltrimethylammonium (*PyBu+) lead to 1‐vi‐nyl‐2‐pyrrolidinone (VP) and 2‐hydroxyethyl methacry‐late (HEMA) polymerization. Experiments carried out in water/acetonitrile solvent mixtures showed that the polymerization rate of VP increases upon increasing the water content, whereas the polymerization rate of HEMA follows the opposite trend. These results are interpreted in terms of the strong dependence on the solvent properties of the photochemical behavior of PyBu+ in the presence of the amine or monomers. Thus, the *PyBu+ quenching by VP is almost negligible in both solvents (water and acetonitrile). Whereas, the *PyBu+ quenching rate constant by HEMA in water is 4 times 109M−l s−1 and decreases four orders of magnitude in acetonitrile. The quenching of *PyBu+ by TEA in aqueous solutions is controlled by hydrogen‐bonding interactions between water molecules and the amine. Quantum yields of the pyrene radical anion (φPy) also strongly depend on the water content, decreasing from 0.28 to 0.015 upon going from acetonitrile to water.
A Comparative Analysis of Silicon Phthalocyanine Photosensitizers for in vivo Photodynamic Therapy of RIF‐1 Tumors in C3H Mice
Authors: Anderson Cathy Y. , Freye Kristin , Tubesing Karen A. , Li Ying‐Syi , Kenney Malcolm E. , Mukhtar Hasan , Elmets Craig A.
Published: January 2008

Photofrin® photodynamic therapy (PDT) has recently received FDA approval for the palliative treatment of to‐tally and partially obstructing esophageal malignancies. However, there is a need for new PDT photosensitizers because Photofrin has a number of undesirable features. The purpose of this study was to evaluate the efficacy of four amine‐bearing silicon phthalocyanines—Pc4, Pc10, Pc12 and Pc18—as potential PDT photosensitizers. Equimolar concentrations of these Pc were found to be highly effective at causing the regression of RIF‐1 tumors trans‐planted to C3H/HeN mice. The amount of Pc4 necessary to cause an equivalent amount of tumor regression in this model system was substantially less than the amount of Photofrin. The cutaneous phototoxicity of the silicon Pc photosensitizer was assessed by the utilization of the murine ear‐swelling model. When C3H mice were exposed to 167 J/cm2 of polychromatic visible light from a UVB‐filtered solar simulator, which emitted UV radiation and visible light above 320 nm, the Pc produced little, if any, cutaneous photosensitivity. These results indicate that Pc4, Pc10, Pc12 and Pc18 are at least as effective as Photofrin in PDT protocols, while at the same time addressing many of the drawbacks of Photofrin.