bluuebunny/crossref_metadata_2025 huggingface dataset:
UVB‐induced Cyclobutane Pyrimidine Dimer Frequency Correlates with Skin Cancer Mutational Hotspots in p53
Authors: Drouin Regen , Therrien Jean‐Philippe
Published: January 2008

Abstract— Ultraviolet light has been identified as the major carcinogen in skin cancer and the p53 tumor suppressor gene is a major target for UV‐induced mutations. The mutations are probably caused by unrepaired UV‐induced cyclobutane pyrimidine dimers (CPD) and possibly by the less frequent pyrimidine (6‐4) pyrimidone photoproducts. While hot spots for p53 mutations in human nonmela‐noma skin tumors correspond quite well to slow spots for CPD repair in cultured cells irradiated with the model mutagen 254 nm UVC (which is not present in terrestrial sunlight), they do not all coincide with sequences that are initially frequently damaged by 254 nm UVC. Using LMPCR (ligation‐mediated polymerase chain reaction), we show that environmentally relevant UVB light induces CPD at CC and PyrmC positions much more frequently than does UVC light, and that all eight skin cancer hot spots in p53 are also hot spots for UVB‐induced CPD. Our results show that methylation of dipyrimidine sites (PyrmCpG) is associated with an increase rate of CPD formation upon UVB irradiation. Consequently, DNA methylation may increase the mutagenic potential of UVB and explains that several p53 mutation hot spots are found at PyrmCpG. The distribution patterns of CPD formation and the photofootprint patterns found along exons 5 and 6 of p53 gene are suggestive of DNA folding into nucleosomes.
Molecular Dynamics Simulation of the Excited‐State Dynamics of Bacteriorhodopsin
Authors: Yamato Takahisa , Kakitani Toshiaki
Published: January 2008

Abstract— The excited‐state dynamics of bacteriorhodopsin was studied by molecular dynamics simulation. For the purpose of suppressing large displacement of amino acid residues on the surface of bacteriorhodopsin, positional restraints were imposed on these residues. A new method was developed to investigate the movement of amino acid residues upon photoexcitation and their role on the ultrafast photoisomerization of the chromophore. The structural change of bacteriorhodopsin was then traced up to 200 fs, i.e. until the formation of the intermediate I. We found that when all the conjugated bonds of the chromophore were allowed to twist freely in the excited state, many bonds including the C13=C14 bond twist in large scale within 100 fs. When only the C13=C14 bond and the single bonds were allowed to twist freely, the twisting took place at most 20° within 200 fs. From these results, it is claimed that a special potential surface is provided for the C13=C14 bond twisting by the protein environment in the course of the isomerization reaction, giving rise to the specific, ultrafast photoisomerization of bacteriorhodopsin. As a trace of such a mechanism, we observed that several functionally important residues incuding Asp85, Asp212 and Tyr185 responded quickly to the photoexcitation of the chromophore.
Nonlinear Dynamics of Intracellular Methylene Blue During Light Activation of Cell Cultures
Authors: Rück Angelika , Heckelsmiller Klaus , Akgün Nermin , Beck Gerd , Kunzi‐Rapp Karin , Schick Elisabeth , Steiner Rudolf
Published: January 2008

Abstract— Methylene blue (MB+) is a well‐known dye in medicine and has been discussed as an easily applicable drug for topical treatment in photodynamic therapy (PDT). Methylene blue can potentially be used as a redox indicator to detect the important redox reactions that are induced during PDT. The kinetics of this process was analyzed on a subcellular level with confocal laser scanning microscopy. BKEz‐7 endothelial cells were incubated 4 h with 1 μM MB+. The fluorescence dynamics of MB+ during irradiation with 633 nm light was observed with subcellular resolution. Images were acquired at 0.5 s intervals (frame rate 1 image/0.5 s). Fluorescence was observed in the red channel of the laser scanning microscope. Synchronously, the phase‐contrast image was visualized with the green channel. Morphological changes could therefore be correlated with the dynamics of MB+. In addition, the light‐dose‐dependent phototoxicity at 633 nm irradiation was determined by viable cell counting. After an induction period (phase I), fast fluorescent spikes could be observed in the whole cytoplasm, which decayed with a time constant of about 20 s (phase II), followed by a period of nearly constant fluorescence intensity (phase III) and exponential photobleaching (phase IV). Phase II exhibits highly nonlinear kinetics, which is hypothesized to correlate probably with a nonlinear quantal production of reactive oxygen species (ROS). Morphological cell changes were not observed during phase II. During phase III, a pycnotic cell nucleus developed. From the determination of viable cells we can conclude that a light dose applied within phase II was only sublethal in correlation with morphological observations. Overproduction of ROS leading finally to cell killing during phases III and IV is discussed.
Photodynamic Efficiency of Protoporphyrin IX: Comparison of Endogenous Protoporphyrin IX Induced by 5‐Aminolevulinic Acid and Exogenous Porphyrin IX
Authors: Tabata Kenji , Ogura Shun‐ichiro , Okura Ichiro
Published: January 2008

Abstract— Photodynamic efficiency of protoporphyrin IX (PP) accumulated in HeLa cells by the incubation of PP with HeLa cells was compared with that of accumulated PP formed from 5‐aminolevulinic acid (ALA) as a precursor. The ALA‐induced PP was photodynamically more efficient than exogenous PP. The difference is caused by monomelic PP concentration and PP localization site in HeLa cells. Exogenous PP was accumulated mainly in plasma membrane, and the membrane was strongly damaged by irradiation. The ALA‐induced PP was selectively accumulated in mitochondria and inactivated the mitochondrial function by irradiation.
Photomodulation of Oxidative Metabolism and Electron Chain Enzymes in Rat Liver Mitochondria
Authors: Yu Wei , Naim John O. , McGowan Margit , Ippolito Kim , Lanzafame Raymond J.
Published: January 2008

Abstract—Low‐level laser irradiation has been applied in a variety of laboratory studies and clinical trials for photobiostimulation over the last three decades. Considerable skepticism exists regarding the concept of photostimulation within the medical community. One of the major difficulties with photoirradiation research is that it lacks experimentally supportable mechanisms for the alleged photobiostimulatory effects. This study was undertaken to determine whether oxidative metabolism and electron chain enzymes in rat liver mitochondria can be modulated by photoirradiation. Oxygen consumption, phosphate potential, and energy charge of rat liver mitochondria were determined following photoirradiation. Activities of mitochondrial enzymes were analyzed to assess the specific enzymes that are directly involved with the photostimulatory process. An argon‐dye laser at a wavelength of 660 nm and at a power density of 10 mW/cm2was used as a photon source. Photoirradiation significantly increased oxygen consumption (0.6 J/cm2and 1.2 J/cm2,P< 0.05), phosphate potential, and the energy charge (1.8 J/cm2and 2.4 J/cm2,P< 0.05) of rat liver mitochondria and enhanced the activities of NADH: ubiquinone oxidoreductase, ubiquinol: ferricytochrome C oxidoreductase and ferrocytochrome C: oxygen oxidoreductase (0.6 J/cm2, 1.2 J/cm2, 2.4 J/cm2and 4.8 J/cm2,P< 0.05). The activities of succinate ubiquinone oxidoreductase, ATPase, and lactate dehydrogenase were not affected by photoirradiation.
Protein‐A‐mediated Targeting of Bacteriochlorophyll‐IgG to Staphylococcus aureus: A Model for Enhanced Site‐Specific Photocytotoxicity
Authors: Gross Shimon , Brandis Alexander , Chen Louise , Rosenbach‐Belkin Varda , Roehrs Susanne , Scherz Avigdor , Salomon Yoram
Published: January 2008

Abstract— A model for studying the efficiency of photodynamic action with a photosensitizer placed exclusively on the bacterial cell wall has been used. Bacteriochlorophyllide molecules, conjugated to rabbit immunoglobulin G (IgG), were synthesized. The conjugated pigment bacteriochlo‐rophyll (Bchl)‐IgG bound with high specificity to protein‐A residues naturally exposed on the cell wall of the bacterium Staphylococcus aureus Cowan I. In bacterial suspensions the phototoxicity of the targeted conjugates (0.5‐2.5 pigment per IgG molecule) was dose dependent (LD50= 1.7 μM) in the presence of light (Λ > 550 nm) and inhibited by native IgG but not by ovalbumin, suggesting selective interaction with protein‐A on the bacterial cell wall. No dark toxicity was noticed even with the highest conjugate concentration tested. In contrast, the photocytotoxicity of bacteriochlorophyll‐serine (Bchl‐Ser, LD50= 0.07 μM) used as a nontargeted control was not inhibited by IgG. In spite of its lower apparent potency, Bchl‐IgG was found to be 30 times more efficacious than Bchl‐Ser: At LD50, only 66000 Bchl‐IgG molecules were bound per bacterium compared to 1900 000 molecules of Bchl‐Ser. The higher efficacy of Bchl‐IgG is explained by its exclusive position on the bacterial cell wall. Consequently, photogeneration of oxidative species is confined to the cell wall and its vicinity, a seemingly highly susceptible domain for photodynamic action. In considering the design of cell‐specific sensitizers for bacterial and cancer therapies, it would be beneficial to identify the more discretely sensitive subcellular domains as targets.
Isolation and Partial Characterization of a Novel Psoralen‐Tyrosine Photoconjugate from a Photoreaction of Psoralen with a Natural Protein
Authors: Sastry Srinivas S.
Published: January 2008

The photoreaction of psoralens with DNA is a well‐characterized reaction. However, the photoreactions of psoralens with proteins is not very well understood. Our objective was to isolate an amino acid‐psoralen photochemical adduct. We photoreacted 8‐methoxypsoralen (8‐MOP) with T7 RNA polymerase, a protein that carries out the fundamental biological process of transcription. Amino acid composition analysis of the photoreacted polymerase revealed that tyrosines quantitatively reacted with 8‐MOP. From the acid hydrolysates of the photo‐conjugated T7 RNA polymerase, an 8‐MOP‐tyr adduct was partially purified by HPLC. The purified 8‐MOP‐tyr adduct and related parent compounds were analyzed by UV‐visible absorption, fluorescence and mass spectroscopy. Excitation/absorption spectra suggested that the pyrone of the original 8‐MOP was modified in the isolated photoadduct, and that the adduct probably contained a benzofuran. Chemical ionization mass spectrometry was consistent with the photoaddition of tyr to 8‐MOP with a conservation of the overall mass (±1 atomic mass units). As far as we know, this work represents the first instance of isolation and partial characterization of an amino acid‐psoralen photoadduct.
Recent Trends in the Evaluation of Photochemical Insertion Characteristics of Heterobifunctional Perfluoroaryl Azide Chelating Agents: Biochemical Implications in Nuclear Medicine
Authors: Pandurangi Raghoottama S. , Karra Srinivasa R. , Kuntz Robert R. , Volkert Wynn A.
Published: January 2008

Achieving high efficiency covalent attachment of molecular probes to biomolecules by photolabeling techniques remains an elusive goal of chemists and biologists. Developments in the understanding of the fundamental photochemistry of perfluoroaryl azides suggest a new means of attaching diagnostic and therapeutic radionuclei to proteins and antibodies through heterobifunctional chelating agents containing a photoactive terminus. Such an approach requires establishment of the coordination chemistry of ligating systems, appropriate molecular design of the photolabel, analytical method development for critical analysis of photolabeled products and retention of immunoreactivity of antibodies in the postlabeling stage. In this review, we summarize recent developments in photolabeling technology and discuss new challenges for application of the technology in the field of nuclear medicine.
Formation and Processing of UV Photoproducts: Effects of DNA Sequence and Chromatin Environment
Authors: Pfeifer Gerd P.
Published: January 2008

Cyclobutane pyrimidine dimers and (6–4) photoproducts are the two major classes of lesions produced in DNA by UVB and UVC irradiation. Their distribution along genes is nucleotide sequence‐dependent. In vivo, the frequency of these lesions at specific sites is modulated by nucleosomes and other DNA binding proteins. Repair of UV photoproducts is dependent on the transcriptional status of the sequences to be repaired and on the chromatin environment. The formation of DNA photolesions by UV light is responsible for the induction of mutations and the development of skin cancer. To understand the mechanisms of UV mutagenesis, it is important to know how these lesions are formed, by which cellular pathways they are repaired and how they are dealt with by DNA polymerases.
Reactivity of Singlet Oxygen Toward Proteins: The Effect of Structure in Basic Pancreatic Trypsin Inhibitor and in Ribonuclease A
Authors: Michaeli Albert , Feitelson Jehuda
Published: January 2008

The reactions of singlet oxygen, 1O2, with large peptides have been described previously. It was found that even in these systems, which in their native form are generally not supposed to possess a stable structure in solution, the polypeptide does impede the access of 1O2 to the amino acids that react readily with 1O2. Here we describe the 102 reaction with two proteins of well‐defined structure. The quenching of 1O2 by bovine pancreatic trypsin inhibitor (BPTI) and by ribonuclease A (RNase A) was compared to that of a solution at the same concentration as those of its constituent amino acids that react readily with 1O2. The proteins were studied in their native form, when partly denatured by splitting their S‐S bonds and when fully denatured. It was found that while in the native form the quenching rate constant was seven times lower in BPTI (2.2 vs 15.2 times 107WM‐1 s‐1) and three times lower in RNase A (11.0 vs 32 times 107M‐l s‐1) than in a mixture of its constituent amino acid residues, it increased upon denaturation reaching in the fully denatured state the value of the corresponding amino acid mixture. More striking is the effect of the protein structure when comparing the fraction of the encounters between 1O2 and protein, which cause damage to the protein, as reflected in the decrease of its biological activity. This decrease is assumed to be due to the chemical (oxidative) reactions of 1O2 in the protein. In the exceptionally stable BPTI the fraction of such encounters was 0.05 and in RNase A it was 0.2, whereas for the amino acid tryptophan in solution, 0.7 of the collisions with 1O2 led to a chemical reaction.
Time and Dose Dependence of Acceptance of UV‐lnduced Syngeneic Tumor Implants in Chronically UV‐Exposed Hairless Mice
Authors: Sontag Yvonne , Steerenberg Peter , Garssen Johan , van Loveren Henk , van der Leun Jan C. , van Vloten Willem A. , de Gruijl Frank R.
Published: January 2008

The kinetics of skin cancer induction by UV radiation has been extensively studied in hairless mice and described by Weibull statistics in which the time till 63% of the mice bear tumors is a primary parameter. However, the kinetics of the associated immunosuppression remained to be determined. To this end, we implanted a syngeneic UV‐induced skin carcinoma cell line (T51/6.53) in the ventral skin of HRA/SKH hairless mice after various periods of daily dorsal UV exposure, either 150 or 75 mj/cm2 per day UV from F40 sunlamps (regimens that when continued yield 63% of the mice with 1 mm tumors in 11.5 or 16.2 weeks, respectively). Both exposure regimens achieved a 100% acceptance (after 7 and 16 weeks, respectively). The implants failed to grow in all unirradiated control mice, but the percentage of mice in which the implants grew increased with the UV treatment time and dose. The estimated times to 63% implant acceptance were 4.3 ± 0.8 and 8.2 ± 0.8 weeks for the high and low daily doses, respectively. As reported earlier for shaved haired mice, there appears to be a straight reciprocity between daily UV dose and the time to tumor acceptance, i.e. the latter fully depends on the cumulative UV dose, whereas the tumor induction does not. The latter probably also depends on a pure elapse of time, i.e. U V‐independent processes. A further analysis of the Weibull description indicates that immunosuppression toward the tumor requires fewer UV‐driven steps than tumor induction.
Photoreaction of New Psoralen Analogs with DNA: Sequence and Mutation Specificity in the Escherichia coli lacZ Gene
Authors: Collet Marianne , Sage Evelyne , Piette Jacques
Published: January 2008

New thio‐ and seleno‐analogs of psoralen were synthesized and analyzed for their photoreactivity toward DNA. Using oligonucleotides of denned sequence, we first showed that these derivatives predominantly generated interstrand crosslinks at 5′‐TpA sites. We also observed a surprisingly high reactivity of 7H‐thiopyrano[3,2‐f][l]benzofuran‐7‐one (PSO[0‐S]) with the BamHl and PstI oligomers, giving rise to the formation of crosslinks at 5′‐ApT sites and of the thymidine‐psoralen‐cytosine type. Next, the sequence specificity in the photochemical binding of all the compounds was investigated in two DNA fragments encompassing the lacZ gene of Escherichia coli, using the T4 DNA polymerase sequencing methodology. Resulting maps demonstrated that thio‐and seleno‐analogs of psoralen preferentially photoreact‐ed with thymine and cytosine residues. The AT‐rich sequences proved to be particularly reactive sites as did adjacent thymines, especially at C‐surrounding residues. Likewise, photoaddition at cytosines in CA/AC context was observed. It was highly significant that all of the derivatives exhibited similar sequence specificities with only minor differences. However, PSO(O‐S) differed from the other heteropsoralens. Photoadducts occurred with a higher frequency at AC and CA dinucleotides, and new sites were detected. A comparison with 8‐methoxypsor‐alen photobinding is also reported. Finally, the mutagenic consequences of photoadducts induced in M13mp19 DNA by PSO(O‐S) were determined in a forward system that detects all classes of mutagenic events. The high phototoxicity exhibited by PSO(O‐S) could be attributed to crosslinks, and the comparison of the observed mutational specificity with the photoadduct distribution within the same gene showed that mutations were targeted at potential monoadduct sites where photolesions were detected in our footprinting experiments
An Assay for the Quantitation of Photofrin in Tissues and Fluids
Authors: Bellnier David A. , Greco William R. , Parsons John C. , Oseroff Allan R. , Kuebler Alex , Dougherty Thomas J.
Published: January 2008

A method for determining the concentration of Photofrin in tissues and biological fluids was developed. The procedure is based on the dissolution of biological material with Solvable a commercially available tissue so‐lubilizer, followed by porphyrin‐specific fluorescence detection and measurement. It was found necessary to use a quadratic standard curve for the estimation of unknown Photofrin concentrations. While this method is limited to compounds that are stable in strong base, it has the advantages of being sensitive, rapid and low cost.
Chronically Ultraviolet‐exposed Human Skin Shows a Higher Mutation Frequency of Mitochondrial DNA as Compared to Unexposed Skin and the Hematopoietic System
Authors: Berneburg Mark , Gattermann Norbert , Stege Helger , Grewe Markus , Vogelsang Kathrin , Ruzicka Thomas , Krutmann Jean
Published: January 2008

Normal ageing processes are associated with an accumulation of mutations within the mitochondrial (mt) DNA. The most frequent mutation is a 4977 base pair (bp) deletion known as common deletion. In order to test the hypothesis that chronically sun‐exposed skin is characterized by an increased mutation frequency of mtDNA, the mutation frequency of the common deletion between skin and another replicating tissue (the hematopoietic system) and chronically sun‐exposed versus sun‐protected skin was compared in the same individuals. This was done by comparing the amount of mutated mtDNA molecules with the whole mitochondrial genome in the same specimen with a semiquantitative polymerase chain reation method, thus allowing direct comparison of different tissues. In all skin specimens the common deletion could be observed. In contrast only 3 of 10 blood samples revealed detectable amounts of the common deletion. Comparison of sun‐exposed versus sun‐protected skin exhibited a higher content of the common deletion in sun‐exposed skin in 7 of 10 individuals. Additionally, a hitherto undescribed mtDNA mutation was detected exclusively in human skin. These studies indicate that exposure of human skin to solar radiation leads to an accumulation of mtDNA mutations, possibly via oxidative damage, which may play an important role in photoageing.
An Unexpected Spectrum of p53 Mutations from Squamous Cell Carcinomas in Psoriasis Patients Treated with PUVA
Authors: Wang Xiao Mei , McNiff Jennifer Madison , Klump Vincent , Asgari Maryam , Gasparro Francis P.
Published: January 2008

Photochemotherapy employing 8‐methoxypsoralen and long‐wavelength ultraviolet radiation (UVA, 320‐400 nm) is widely used in the treatment of psoriasis. The pho‐toactivation of psoralens in skin cells leads to formation of DNA photoadducts which may be responsible, at least in part, for the efficacy of these photochemotherapies. However, mutations arising from these adducts may also lead to the well‐characterized increased incidence of squamous cell carcinoma. Mutations in the p53 tumor suppressor gene have been detected in many human cancers. To determine whether p53 mutations occur in squamous cell carcinomas in PUVA patients, PCR was used to amplify the exons (5‐9) in which other studies have found a high frequency of point mutations. Gel electrophoresis was used to detect single‐strand conformational polymorphisms. Aberrantly migrating bands were excised, reamplined and sequenced. Thirty‐four specimens from 10 patients were examined. Specimens from one patient who had received no phototherapy as well as from normal controls were also analyzed. Five of the 10 patients showed at least one p53 mutation. In contrast to previously reported psoralen‐induced p53 mutations in mice, the expected psoralen type mutations at alternating AT sites were not detected. All but two of the altered sequences occurred at dipyrimidine sites which is typical of solar type mutations. Two C→T mutations and two dipyrimidine mutations (CC→TT) were found. Other mutations included: C→G, G→T, C→A and an 18 bp deletion. A review of therapeutic history of these patients showed that some had also received UVB phototherapy. Furthermore, because sunlight is thought to be beneficial for psoriasis, nontherapeutic, casual UVB exposure cannot be excluded. Our observations suggest that the SCC may have arisen from the solar mutations and that PUVA may enhance tumor progression or immune suppression
Photobiological Properties of 1′‐Thieno‐4,6,4′‐trimethylangelicin
Authors: Bordin F. , Marzano C. , Baccichetti F. , Carlassare F. , Vedaldi D. , Falcomer S. , Lora S. , Rodighiero P.
Published: January 2008

Some photobiological properties of l'‐thieno‐4,6,4′‐tri‐methylangelicin (TTMA), a new isoster of 4,6,4′‐trimeth‐ylangelicin (TMA) were studied in comparison with the parent compound. The TTMA absorbs UVA light and photobinds in vitro to DNA more efficiently than TMA; however, in Ehrlich cells in vivo TTMA linked to DNA to a lesser extent than the parent compound. In general, the formation of damage into DNA is in line with this last result: In fact, TTMA and TMA form equivalent amounts of interstrand cross‐links (ISC) both in vitro in linearized PM2 DNA and in vivo in HeLa cells. In this system TTMA induces DNA‐protein cross‐links (DPC) more efficiently than TMA; on the contrary, no significant amounts of single‐strand breaks were detected with both compounds. The antiproliferative activity of TTMA is consistent with these results, being only slightly more pronounced than that of TMA. Experiments carried out using double irradiation demonstrated that these drugs are capable of inducing antiproliferative effects by bi‐photonic reactions, including the formation of both ISC and DPC. Thus, replacement of the oxygen atom by a sulfur increases the UV absorption of the drug and its capacity to photobind to DNA in vitro but does not yield a comparable enhancement of its photosensitizing properties in vivo; this might be due to various reasons, for instance to an increase in the lipophilic character that could modify the behavior in vivo.
Structural Volume Changes upon Photoisomerization of the Bilirubin‐Albumin Complex: A Laser‐Induced Optoacoustic Study‡
Authors: Williams Rene M. , McDonagh Antony F. , Braslavsky Silvia E.
Published: January 2008

The Z → E photoisomerization of the 1:1 complexes of human serum albumin (HSA) and several bilirubins (BR‐IXα, ‐IIIα, meso‐IXα and a mixture of ‐IXα, ‐IIIα and XHIα) affords in every case an almost negligible structural volume change (δVR = 0) within detection limits (i e. less than2–4 cm3/mol for this isomerization with very low quantum yield) as determined by laser‐induced optoacoustic spectroscopy. Based on previous model studies of photoisomerizations in aqueous environment, this negligible small change is interpreted as indicating that the part of the molecule undergoing photoisomerization is not exposed to water but is located in a hydrophobic protein cavity that shields the molecule from the aqueous medium. The BR‐protein interaction within this cavity seems to be very loose in view of the small structural volume change observed. The energy difference between the Z and E isomers of the BR‐HSA complexes was estimated to be less than 4 kJ/mol, probably close to zero (ΔH = 0).
Carotenohematoporphyrins as Tumor‐Imaging Dyes. Synthesis and In Vitro Photophysical Characterization*
Authors: Tatman Dereck , Liddell Paul A. , Moore Thomas A. , Gust Devens , Moore Ana L.
Published: January 2008

Multichromophoric dyes for use in tumor imaging have been synthesized and photophysically characterized. Structurally, these dyes are dyads and triads that consist of one or two carotenoid polyenes covalently attached to hematoporphyrin (HP) or hematoporphyrin dimethyl ester (HPDME) moieties via ester linkages. The ground‐state absorption of each compound shows that the electronic interaction between the chromophores is small. The fluorescence quantum yield for the dyad monocar‐oteno‐ HPDME is 0.033 and the dicaroteno‐HPDME triads have yields between 0.016 and 0.007, all of which are reduced with respect to the parent compound HPDME (0.09). Global analysis of the transient fluorescence decays of the dyads and triads requires two exponential components (˜5–6ns and ˜1–2ns) to fit the data, while a single exponential component with a lifetime of 9.3 ns describes the decay data of the parent HPDME. Possible mechanisms for the observed porphyrin fluorescence quenching by the nearby carotenoid are discussed. Nanosecond transient absorption reveals a carotene triplet with maximum absorption at 560 nm and a 5.0 μs lifetime. No transient was detected at 450 nm, indicating rapid (10 ns) triplet energy transfer from the hematoporphyrin to the carotenoid moieties in fluid as well as in rigid media. The yield of triplet energy transfer from the porphyrin to the carotenoid moiety is unity. Singlet oxygen, O2(1δg), studies support the transient absorption data, as none of these compounds is capable of sensitizing O2(1δg). Liposome vesicles were used to study the photophysical characteristics of the dyes in phospholipid membranes. Singlet oxygen was not sensitized by the dyads and triads in liposomes. Transient absorption measurements suggest that the triads are substantially aggregated within the phospholipid bilayer, whereas aggregation in the dyads is less severe.
Time‐Resolved Study of the Sensitized Formation and Decay of 1O2(1δg) in the Presence of Cyclodextrins*
Authors: Sanramé Carlos N. , Rossi Rita H. de , Argüello Gustavo A.
Published: January 2008

The effect of β‐cyclodextrin and β‐hydroxypropyl‐cyclodextrin on some properties of the aromatic ketone 1‐H‐phenalen‐1‐one and its sulfonate derivative 1‐H‐phen‐alen‐1‐one‐2‐sulfonic acid was measured in aqueous solution. From the changes in the UV‐visible range of the absorption spectra, the association equilibrium constants for the formation of inclusion complexes were determined. Because these ketones are very efficient sensitizers for the generation of singlet oxygen, time‐resolved infrared luminescence was used to measure the lifetime of singlet oxygen in D2O. Cyclodextrins are weak deactivating agents of singlet oxygen; the upper limits for the bimolecular deactivation constants are 2 times 105M−1s−1 and 1 times 105M−1s−1 for β‐cyclodextrin and β‐hydroxypropyl‐cyclodextrin, respectively. Besides, they do not affect noticeably the extent of formation of singlet oxygen; this result is explained in terms of relocation of the sensitizer (exit from the cyclodextrin cavity) in the triplet excited state.
Photoaddition to DNA by Nonintercalated Chlorpromazine Molecules
Authors: Kochevar Irene E. , Garcia Carmelo , Geacintov Nicholas E.
Published: January 2008

Chlorpromazine (CPZ) forms photoadducts with DNA and photosensitizes DNA strand breaks. These reactions may be responsible for the reported photomutagenicity of CPZ and for the well‐known cutaneous and ocular phototoxicity associated with this drug. We have investigated whether CPZ molecules that are intercalated between base pairs in double‐stranded (ds) DNA are the absorbing species for the photoaddition reaction. Quenching of CPZ fluorescence by ds‐DNA gave nonlinear Stern‐Volmer plots, indicating that more than one type of complex is formed. Linear dichroism spectra of CPZ in the presence of ds‐DNA showed a minimum at 345 nm, indicating that the absorption maxima of intercalation complex(es) are red‐shifted compared to the absorption maximum of free CPZ at 307 nm. The sum of the absorption of all CPZ complexes with ds‐DNA, obtained from dialysis experiments, was broadened and maximized at about 315 nm, indicating that complexes not involving intercalation dominate the absorption spectrum at X < 350 nm. The wavelength dependence for covalent binding of CPZ to DNA was determined by irradiating 3H‐CPZ in the presence of ds‐DNA at 310, 322, 334, 346, 358 and 370 nm. The resulting spectrum correlated closely with the absorption spectrum of nonintercalated CPZ rather than with the spectrum of intercalated CPZ, indicating that the latter species is not the chromophore for the photoaddition reaction.
Generation of High‐Oxidation States of Myoglobin in the Nanosecond Time‐Scale by Laser Photoionization
Authors: Candeias Luis P. , Steenken Steen
Published: January 2008

Abstract— The 248 nm laser flash photolysis of myoglobin in various redox states (oxy, met and ferryl) in neutral aqueous solution yielded hydrated electrons with concurrent changes in the visible absorption spectrum of the heme. The results could be ascribed to the photoionization of both the peptide and the heme group, in approximately equal yields. The ionization of met‐ and ferrylmyoglobin was biphotonic, but that of oxymyoglobin was a mixture of mono‐ and biphotonic processes. Using appropriate electron and radical scavengers, the changes in the heme absorption could be investigated at times ≥100 ns and were shown to be associated with a +1 increase of the formal oxidation state of the heme. Using this method, the formal iron(V) state of native myoglobin could be spectroscopically characterized for the first time. Its absorption, blue‐shifted and less intense relative to the ferryl state, is reminiscent of that of the compound I of peroxidases, which contains a ferryl‐oxo (iron[IV]) group and a porphyrin radical cation. On this basis, the same structure is proposed for the formal iron(V) state of native myoglobin. The transition from oxy‐ to metmyoglo‐bin took ‐5 μs, which may reflect the kinetics of exchange of oxygen with water as ligand. The transitions from the met to the ferryl state, and from ferryl to iron(V) states were faster (?250 ns), consistent with processes that involve proton or electron movements but no changes in the iron coordination state.
Near‐Infrared Raman Spectroscopy for In Vitro Detection of Cervical Precancers
Authors: Mahadevan‐Jansen Anita , Mitchell Michele Follen , Ramanujamf Nirmala , Malpica Anais , Thomsen Sharon , Utzinger Urs , Richards‐Kortumt Rebecca
Published: January 2008

Abstract— In this study, we investigate the potential of near‐infrared Raman spectroscopy to differentiate cervical precancers from normal tissues, inflammation and metaplasia and to differentially diagnose low‐grade and high‐grade precancers. Near infrared Raman spectra were measured from 36 biopsies from 18 patients in vitro. Detection algorithms were developed and evaluated relative to histopathologic examination. Algorithms based on empirically selected peak intensities, ratios of peak intensities and a combination of principal component analysis for data reduction and Fisher discriminant analysis for classification were investigated. Spectral peaks were tentatively identified from measured spectra of potential chromophores. Empirically selected normalized intensities can differentiate precancers from other tissues with an average sensitivity and specificity of 88 ± 4% and 92 ± 4%. Ratios of un‐normalized intensities can differentiate precancers from other tissues with a sensitivity and specificity of 82% and 88% and high‐grade from low‐grade lesions with a sensitivity and specificity of 100%. Using multivariate methods, intensities at eight frequencies can be used to differentiate precancers from all other tissues with a sensitivity and specificity of 82% and 92% in an unbiased test. Raman algorithms can potentially separate benign abnormalities such as inflammation and metaplasia from precancers. Comparison of tissue spectra to published and measured chromophore spectra indicate that the most likely primary contributors to the tissue spectra are collagen, nucleic acids, phospholipids and glucose 1‐phos‐phate. These results suggest that near‐infrared Raman spectroscopy can be used for cervical precancer diagnosis and may be able to accurately separate samples with inflammation and metaplasia from precancer.
Identification and Quantification of Intrinsic Optical Contrast for Near‐infrared Mammography
Authors: Quaresima Valentina , Matcher Stephen J. , Ferrari Marco
Published: January 2008

Near‐infrared spectroscopy has been used to quantify the composition of healthy female breast tissue in vivo. By collecting transillumination spectra in the wavelength range 680–1100 nm at 7–9 positions on the breasts of five female volunteers, an attempt was made to quantify the intra‐ and intersubject variability of breast composition. The dominant absorbers are water, lipids and hemoglobin. Hemoglobin concentration in the breast is substantially lower than that in the brain or muscle (less than 10 μM). The measured deoxyhemoglobin concentration can vary by up to 100% between different positions on the same breast. Water and lipid concentrations can show similar variability. Phantom and simulation studies demonstrate that this variability is not due to the effects of tissue boundaries on the measurements. The low hemoglobin concentration implies that optical breast imaging should be performed at wavelengths below about 850 nm to ensure that the image contrast comes predominantly from hemoglobin. Intrasubject variability could have implications for the ability of optical imaging to discern tumors from background contrast variations.
Phantoms for Noninvasive Blood Glucose Sensing with Near Infrared Transmission Spectroscopy
Authors: Burmeister Jason J. , Chung Hoeil , Arnold Mark A.
Published: January 2008

In vivo spectra from human subjects can be simulated with a phantom composed of different layers of water, fat and muscle tissue. All three components are necessary to simulate in vivo spectra collected over the combination spectral region (5000–4000 cm−1). Muscle tissue is not required, however, to accurately simulate overtone spectra (6600–5400 cm−1). The near‐IR spectral characteristics of fat and muscle tissue from several animal sources are essentially identical to those found for human tissue, hence, the animal source for these phantom components is not critical. Thickness of each tissue layer can be determined by a regression analysis where the in vivo spectrum of interest is regressed against standard absorbance spectra of the necessary model components (water, fat and muscle). In general, in vivo overtone spectra collected across human webbing tissue with a thickness of 6.7 mm can be simulated with water layer thicknesses ranging from 5.0 to 6.4 mm combined with fat layer thicknesses from 1.4 to 4.2 mm.
Photoinduced Electron Transfer in Dry Gelatin Containing Bound Methylene Blue
Authors: Smith Gerald J. , Harriman Anthony , Woolhouse Anthony D. , Haskell Timothy G. , Barnes Thomas H.
Published: January 2008

Excited states of methylene blue bound to dry gelatin undergo electron‐transfer quenching reactions with neighboring amino acid residues to give reduced, methylene blue‐free radicals. At the low loadings of methylene blue bound to gelatin used in this work, the absorption spectra indicated that the methylene blue existed principally in its monomeric form. The rates of methylene blue fluorescence quenching depended on the temperature at which the gelatin was dried. There was no detectable fluorescence characteristic of the localized excited singlet state of methylene blue when the dye is bound to gelatin dried at 15°C, where a significant proportion of the protein exists in its a‐helical form. Instead, weak emission with a maximum at 645 nm is observed. It is inferred from this that the rate of quenching of any localized excited singlet states of the dye bound to a‐helical gelatin is very fast thereby competing efficiently with radiative relaxation. The weak emission at 645 nm is ascribed to a fluorescent exciplex formed between bound methylene blue and a neighboring electron/proton donor residue of the gelatin. While fluorescence characteristic of the localized methylene blue singlet state was observed from dyed gelatin dried at 50°C, i.e. in its random coil form, the triplet state is subject to rapid quenching by electron/proton transfer with protein moieties.
Furocoumarin‐induced Epidermal Melanogenesis Does Not Protect Against Skin Photocarcinogenesis in Hairless Mice
Authors: Kipp Christian , Lewis Emma J. , Young Antony R.
Published: January 2008

Topical 6,4,4′‐trimethylangelicin (TMA) plus UVA was used to induce intense epidermal pigmentation in inbred HRA.HRII‐c/+/Skh hairless pigmented mice over a 13 day period. Subsequent UVB/UVA exposure was used to assess the photoprotective properties of this tan using skin tumors as an endpoint. Comparisons were always made with sibling albino mice. The TMA/UVA treatment was shown to be not carcinogenic when treated mice were compared with untreated control mice over 25 weeks. The tan faded despite daily exposure to UVB/UVA and did not afford any protection when TMA/UVA‐treated mice with subsequent UVB/UVA were compared with pigmented mice treated with UVB/UVA only. In one group, the TMA‐induced tan was maintained by application of TMA three times a week prior to UVB/UVA for the duration of the experiment. This treatment was associated with a significant increase in tumor risk in both pigmented and albino mice compared to groups treated with UVB/UVA alone. Although pigmented mice had a significant photoprotective advantage, it was shown to be outweighed by the carcinogenic risks of the TMA maintenance treatment when they were compared with mice that did not have this treatment. Nonpretanned pigmented mice developed mild pigmentation during UVB/UVA treatment that was shown to have no protective effect when those mice were compared with albinos. We conclude that induced epidermal tanning with or without furocoumarin enhancement is not an effective way to prevent skin cancer in the HRA.HRII‐c/+/Skh mouse model.
GRP78 Induction by Calcium lonophore Potentiates Photodynamic Therapy Using the Mitochondrial Targeting Dye Victoria Blue BO
Authors: Morgan Janet , Whitaker James E. , Oseroff Allan R.
Published: January 2008

The cationic photosensitizing triaryl methane dye Victoria Blue BO (VBBO) localizes in mitochondria and causes oxidative damage to this organelle during photodynamic therapy (PDT). Oxidative stresses from other photosensitizers induce a variety of stress proteins. The endoplasmic reticulum (ER)‐based, calcium‐binding stress protein GRP78 is a putative protective factor for photo‐sensitizers such as Photofrin® that damage multiple intracellular sites and for several cytotoxic agents. In the current study VBBO‐PDT was found to induce glucose‐regulated protein (GRP)78. However, in contrast to other drugs, rather than being protected, human squamous carcinoma cells (FaDu) induced to express GRP78 by calcium ionophore A23187 became more sensitive to PDT. A line of Chinese hamster ovary cells (C.‐1) constitutively overexpressing GRP78 also were more sensitive. Cytotoxicity of the A23187 treatment and VBBO was synergistic, with more than 11‐fold potentiation with light irradiation, but was only additive in the dark. The in‐creased cell killing was not due to differences in VBBO uptake or to changes in the intracellular localization of VBBO caused by calcium ionophore or GRP78. Thus, GRP78 appears to enhance rather than protect against VBBO‐induced mitochondrial photodamage and contributes to cell death. This novel finding possibly may stem from the effects of GRP78, ER Ca2+ stores and ATP consumption on the Ca2+ and ATP‐dependent mitochondrial permeability transition that may be evoked by PDT damage to the mitochondrial respiratory chain. The work suggests interventions that may potentiate PDT with mitochondrial targeting sensitizers and potential enhancements in efficacy when GRP78 is upregulated biologically or pharmacologically.
Photodynamic Treatment with Benzoporphyrin Derivative Monoacid Ring A Produces Protein Tyrosine Phosphorylation Events and DNA Fragmentation in Murine P815 Cells
Authors: Granville David J. , Levy Julia G. , Hunt David W. C.
Published: January 2008

Treatment with benzoporphyrin derivative monoacid ring A (BPD‐MA, verteporfin) and broad‐spectrum fluorescent light rapidly produced apoptosis in murine P815 mastocytoma cells. Fragmentation of DNA, a fundamental characteristic of cells undergoing apoptosis, was evident within 3 h following the photodynamic treatment. Western immunoblot analysis using the specific antiphosphotyrosine monoclonal antibody 4G10 indicated that molecular species of >200 kDa were phosphorylated on tyrosine residues during or immediately following the irradiation of cells loaded with BPD‐MA. Increased tyrosine phosphorylation of a 15 kDa protein was evident by 15 min postirradiation. In the absence of light, BPD‐MA did not affect the status of cellular protein tyrosine phosphorylation or cause DNA fragmentation. The protein kinase inhibitor staurosporine prevented tyrosine phosphorylation of the >200 kDa species but did not affect tyrosine phosphorylation of the 15 kDa protein or the level of DNA fragmentation produced by the photo‐dynamic treatment. The protein tyrosine phosphorylation events observed for P815 cells treated with cytotoxic levels of BPD‐MA and light may not be directly related to the induction of the apoptotic cell death pathway.
Chemiluminescence of Horseradish Peroxidase and Acetaldehyde Related with Gallic Acid and Hydrogen Peroxide Interaction
Authors: Yoshiki Y. , Yuan H. , Lida T. , Kawane M. , Okubo K. , Ishizawa T. , Kawabata S.
Published: January 2008

Abstract— This study focuses on the fact that the chemiluminescence in the visible region is emitted from the H2O2/gallic acid/ horseradish peroxidase (HRP) and the H2O2/gallic acid acetaldehyde (MeCHO) systems. The concentration dependence of chemiluminescence intensity that led to the different response of HRP and MeCHO toward H2O2 indicates that the photon emission participates with peroxidase activity including an electron transfer reaction. From our experimental results, in this study, we postulated a reaction process for chemiluminescence based on a one‐electron redox shuttle from H2O2 by peroxidase. The photon intensity and spectra data from the H2O2/ HRP and the H2O2/MeCHO systems with various cate‐chins were not only affected by HRP and MeCHO but also corresponded with the chemical structure of cate‐chins. The energy calculated from the spectra is 47–64 kcal/mol. These results suggested that the chemiluminescence of both systems arose from excited carbonyl compounds produced by an intermediate of the alkyl radical and the metal‐bound hydroxyl (compound II species). Hydroxyl radical inhibition, showing a notable increase from the gallic acid addition, makes the decay of the hydroxyl form of heme iron the most likely candidate for the chemiluminescence.
Birefringence Measurement of Rapid Structural Changes during Collagen Denatu ration
Authors: Sankaran Vanitha , Walsh Joseph T.
Published: January 2008

Abstract— Linear birefringence, an optical property that results from a material's structure and composition, can be used to study dynamic changes in tissue structure. Single, 200 μs‐long pulses from a Ho:YAG laser emitting 2.1 μm radiation were used to induce changes in the linear birefringence of rat tail tendon. Such changes were measured on a millisecond timescale. The measured rate coefficients describing the denaturation are not predicted by previous studies of collagen denaturation induced by slower, lower‐temperature heating. Two types of laser‐induced collagen denaturation can be differentiated: thermal denaturation, which appears rate‐limited, and thermomechanical denaturation, which is observed at higher laser radiant exposures. Neither process is described by standard Arrhenius‐type kinetic models.
Electron Transfer Fluorescence Quenching of Blepharisma japonicum Photoreceptor Pigments
Authors: Angelini Nicola , Quaranta Annamaria , Checcucci Giovanni , Song Pill‐Soon , Lenci Francesco
Published: January 2008

Abstract— The hypericin analogs blepharismin (BP), oxyblepharismin (OxyBP) and stentorin (ST), the photosensing chromophores responsible for photomotile reactions in the ciliates Blepharisma japonicum (red and blue cells) and Stentor coeruleus, represent a new class of photoreceptor pigments whose chemical structures have recently been determined. In the case of ST it has been shown that the first excited singlet state can be deactivated by donation of an electron to an appropriate acceptor molecule (e.g. a quinone molecule). This charge transfer can be considered a possible mechanism for the primary photoprocess for the photomotile responses in S. coeruleus. To determine whether an electron transfer process also occurs in the deactivation of excited blepharismin, we studied the fluorescence quenching of OxyBP in dimethyl‐sulfoxide (DMSO) and in ethanol using electron acceptors with different reduction potentials. Under our experimental conditions ground state and excited state complexes (like fluorescent exciplexes) are not formed between the fluorophore and the quenchers. In DMSO the bimolecular quenching constant values (kq) calculated on the basis of the best fitting procedures clearly show that the quenching efficiency decreases with the quencher negative reduction potential, E0. The kq (M‐1 s‐1) and E0 (V) values are, respectively, 7.8 times 109 and ‐0.134 for 1,4‐benzoquinone, 8.9 times 109 and ‐0.309 for 1,4‐naphthoquinone, 2.4 times 109 and ‐0.8 for nitrobenzene, 0.009 times 109 and ‐1.022 for azobenzene and 0 and ‐1.448 for benzophenone. These findings point to the conclusion that upon formation of the encounter complex between OxyBP and the quencher, an electron is released from excited OxyBP to the quencher, similar to what happens in ST. It is suggested that in the pigment granules such a light‐induced charge transfer from excited blepharismin to a suitable electron acceptor triggers sensory transduction processes in B. japonicum.