bluuebunny/crossref_metadata_2025 huggingface dataset:
INVESTIGATIONS ON THE LIGHT‐EMITTING SPECIES IN THE REACTION OF METMYOGLOBIN AND METHEMOGLOBIN WITH HYDROGEN PEROXIDE
Authors: Stolze Klaus , Liu Yang , Nohl Hans
Published: January 2008

Abstract The formation of a compound I type ferryl complex in the reaction of methemoglobin (MetHb) and metmyoglobin (MetMyo) with hydrogen peroxide is accompanied by strong chemiluminescence. An approach to identify the nature of the light‐emitting species was made by the use of quenchers and sensitizers reacting with singlet oxygen and compounds interfering in the formation and reactivity of other reactive oxygen species.

Singlet oxygen is not the source of light emission. This could be concluded from the results obtained using the specific singlet oxygen trap 9,10‐anthracenedipropionic acid (ADPA) in combination with high‐performance liquid chromatography (HPLC) analysis. The singlet oxygen adduct of ADPA was not formed in the incubation systems (MetHb or MetMyo/H2O2). Instead, ADPA was oxidized by the ferryl ion to a different oxidation product, which was characterized by HPLC and IR spectroscopy. In the case of MetHb‐related chemiluminescence, light emission does not result from a single source. Both, SH‐groups and O2.¯ radicals are involved because blocking of thiol‐groups with N‐ethylmaleimide (NEM) and scavenging of O2.¯(by superoxide dismutase) suppressed chemiluminescence by 50% and 30%, respectively. Development of MetMyo‐related chemiluminescence is not dependent on thiol groups (which are not present in the globin moiety) and also 02.¯is not involved. Although generation of chemiluminescence in MetHb and MetMyo seems to follow different mechanisms, both types of light‐emitting species are sensitive to antioxidants, such as uric acid and ascorbate. The detection of the respective free radicals by means of ESR demonstrates that both MetHb‐ and MetMyo‐mediated chemiluminescence is associated with a strong one‐electron oxidizing species, which seems to be identical with the light‐emitting source itself. Also desferal, which was originally used to exclude the involvement of a Fenton‐type reaction, was readily oxidized to the nitroxide free radical associated with a strong decrease of chemiluminescence. This quenching effect was not dependent on iron complexation because the addition of iron was ineffective. In summary, chemiluminescence is not restricted to a single chemical process but is related to different one‐electron transfer reactions from globin residues to the oxo‐heme center.
DNA PHOTOLYASE FROM THE FUNGUS NEUROSPORA CRASSA. PURIFICATION, CHARACTERIZATION AND COMPARISON WITH OTHER PHOTOLYASES
Authors: Eker A. P. M. , Yajima H. , Yasui A.
Published: January 2008

Abstract A phr‐gene from the filamentous fungus Neurosporu crassa was overexpressed in Escherichia coli cells, yielding a biologically active photolyase. After purification till apparent homogeneity, the 66 kDa protein was found to contain equimolar amounts of 5,1O‐methenyltetrahydrofolic acid (MTHF) and FAD, classifying it as an MTHF‐type photolyase. Compared to other MTHF photolyases the absorption maximum of Neurosporu photolyase is shifted from ca 380 nm to 391 nm (t = 34 800), while an additional shoulder is present at 465 nm. In dark‐adapted enzyme the FAD chromophore is predominantly present in the oxidized form, in contrast with E. coli and Saccharomyces cerevisiue photolyase, which contain mainly semiquinone or fully reduced FAD, respectively. Preillumination or dithionite treatment converted oxidized FAD in Neurospora photolyase into the fully reduced form, with a concomitant shift of the absorption maximum from 391 to 396 nm and disappearance of the 465 nm shoulder. The action spectrum of photoreactivation coincides with the absorption spectrum of preilluminated (reduced) photolyase, extending the spectral region of MTHF‐type photolyases from 380 till 396 nm. A quantum yield of 0.57 was obtained for the overall repair reaction. Comparison of spectral properties of FAD in Neurospora photolyase and the model compound lumiflavin points to an apolar microenvironment of photolyase‐bound FAD. Neurosporu photolyase has distinct advantages over E. coli photolyase as it is more stable and contains a full complement of chromophores.
DISTANCE‐DEPENDENT FLUORESCENCE QUENCHING OF TRYPTOPHAN BY ACRYLAMIDE
Authors: Lakowicz Joseph R. , Zelent Bogumil , Gryczynski Ignacy , Kuba Józef , Johnson Michael L.
Published: January 2008

Abstract We used GHz frequency‐domain fluorometry to investigate the time‐dependent intensity decays of N‐acetyl ‐L‐trytophanamide (NATA) when collisionally quenched by acrylamide in propylene glycol at 20°C. The intensity decays of NATA became increasingly heterogeneous in the presence of acrylamide. The NATA intensity decays were not consistent with the Collins‐Kimball radiation boundary condition (RBC) model for quenching. The steady‐state Stern‐Volmer plots show significant upward curvature. At low temperature in vitrified propylene glycol (‐60%), where translational diffusion cannot occur during the lifetime of the excited state, quenching of NATA by acrylamide was observed. The Smoluchowski and RBC quenching models do not predict any quenching in the absence of translational diffusion. Hence, these frequency‐domain and steady‐state data indicate a through‐space quenching interaction between NATA and acrylamide. The rate for quenching of NAT A by acrylamide appears to depend exponentially on the fluorophore‐quencher separation distance. Comparison of the time‐resolved and steady‐state data provides a sensitive method to determine the distance dependence of the fluorophore‐quencher interaction. The distance‐dependent rate of quenching also explains the upward curvature of the Stern‐Volmer plot, which is often observed for quenching by acrylamide. These results suggest that the distance‐dependent quenching rates need to be considered in the interpretation of quenching data of proteins by acrylamide.
THE PHOTOCHEMICAL PROPERTIES OF FLUOROALUMINUM PHTHALOCYANINE
Authors: Rosenthal I. , Shafirovich V. Y. , Geacintov N. E. , Ben‐Hur E. , Horowitz B.
Published: January 2008

Abstract Fluoride is known to inhibit the photodynamic activity of aluminum phthalocyanine in a variety of biological systems. In order to gain insight into this phenomenon, the effect of fluoride on the photophysical properties of free and albumin‐bound chloroaluminum phthalocyanine sulfonate (AlPcSn.) were studied. The association constant of NaF with AlPcSn, in aqueous solution was measured as 500 ± 20 M−1. This binding affects the photophysical properties of the dye: the absorption bands in the visible range are blue‐shifted by 6–8 nm, and this effect is mirrored in the fluorescence emission spectrum. Human serum albumin significantly quenched the dye fluorescence independent of the presence of fluoride ion. The transient absorption spectrum of the excited dye triplet is unchanged by NaF, but the quantum yield for its generation is increased by 50%, with no decrease in its lifetime. Formation of fluoroaluminum phthalocyanine complexes was also observed in tetrabutylammonium fluoride‐assisted solutions in wet acetonitrile. The fluoro‐AlPcSn, complex is a better photosensitizer for generation of singlet oxygen than the original dye‐hydroxyl ion complex, as confirmed using the imidazole‐N,N‐dimethyl‐4‐nitrosoaniline method. On the other band, the fluoro‐AlPcSn. complex exhibits an intense inhibitory effect on photohemolysis of red blood cells (RBC) even after the cells are washed to remove free dye and fluoride prior to irradiation, indicating that once the dye is attached to the cellular site, the fluoride ligand is no longer prone to displacement (by hydroxyl ion, for example). Nonetheless, it is clear from the spectroscopic data that the new fluoro complex is an efficient sensitizer for photo‐oxidation. Therefore, the reduced photodynamic action of the fluoro‐AlPcSn. complex on RBC (Ben‐Hur et al., Photochem. Photobiol. 58, 351–355, 1993) may result from a lowering of the efficiency of interaction of the fluorodye complex with sensitive cell target moieties.
EXCISION OF CYCLOBUTANE DIMERS IN GENOMIC AND EPISOMAL DNA IN HUMAN CELLS
Authors: Mitchell David L. , Cleaver James E. , Lutze Louise H.
Published: January 2008

Abstract Direct determination has been made of cyclobutyl pyrimidine dimer induction and excision repair in an episomal SV40 DNA population in vivo. Maintaining SV40‐transformed human (GM637) cells in confluent culture results in amplification of a mutant SV40 episome to high copy number. T4 endonuclease V was used to quantify the induction and repair of cyclobutane dimers in the SV40 episome and genomic DNA of the same cells. Differences in both parameters were observed cyclobutane dimers were induced at 1.5–2‐fold greater frequency in episomal DNA and excised at a reduced rate compared to genomic DNA in the host cells.
COMPARATIVE POTENCY OF DIFFERENT UV SOURCES IN REDUCING THE DENSITY AND ANTIGEN‐PRESENTING CAPACITY OF LANGERHANS CELLS IN C3H MICE
Authors: El‐Ghorr Ali A. , Pierik Frank , Norval Mary
Published: January 2008

Abstract Although broadband UV‐B irradiation has been shown to induce selective immunosuppression in a variety of experimental systems, the wavelength dependence of the immunomodulation and the initial events in the skin remain unclear. In the present study three UV lamps were used at suberythermal doses on C3H mice: a conventional broadband UV‐B source (270–350 nm), a narrowband UV‐B source (311–312 nm) and a UV‐A source (320400 nm). Their effects on the photoisomerization of the naturally occumng trans‐isomer of urocanic acid (UCA) to cis‐UCA, on the density of Langerhans cells and on the ability of epidermal cells to stimulate allogeneic lymphocytes in the mixed skin lymphocyte reaction (MSLR) were ascertained. Broadband UV‐B irradiation was more efficient than narrowband UV‐B at reducing the density and function of Langerhans cells, while UV‐A irradiation was least effective. These changes were most pronounced immediately following irradiation, were dose dependent and were only detected in UV‐exposed areas of skin. There was a close correlation between the UV‐induced reduction in Langerhans cell density and the formation of cis‐UCA in the epidermis. This correlation was not detected between the reduction in the MSLR response following UV irradiation in vivo and cis‐UCA formation.
PHOTOSENSITIZED FORMATION OF 8‐HYDROXY‐2′‐DEOXYGUANOSINE AND DNA STRAND BREAKAGE BY A CATIONIC meso‐SUBSTITUTED PORPHYRIN
Authors: Nicotera Thomas M. , Munson Benjamin R. , Fiel Robert J.
Published: January 2008

Abstract Cationic porphyrins, known to have a high affinity for DNA, are useful tools with which to probe a variety of interactions with DNA. In this study we have examined both DNA strand scission and oxidative DNA base damage, measured by 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) formation, using a photoactivated cis‐dicationic por‐phyrin. The data demonstrated a dose‐dependent formation for each type of DNA damage. Inhibition of strand scission and 8‐OHdG formation with the singlet oxygen scavenger 1,3‐diphenylisobenzofuran and with MgCl2 and no apparent effect by D2O suggests that a singlet oxygen mechanism generated in close proximity to the DNA may be responsible for the damage. However, a nearly complete inhibition of 8‐hydroxy‐2′‐deoxyguanosine formation in 75% D2O and the substantial enhancement of 8‐hydroxy‐2′‐deoxyguanosine formation in a helium atmosphere by photoactivated porphyrin rules out singlet oxygen as a primary mechanism for this process. These data indicate that distinct mechanisms lead to 8‐OHdG formation and strand scission activity.
DOUBLY ALLYLIC HYDROPEROXIDE FORMED IN THE REACTION BETWEEN STEROL 5,7‐DIENES AND SINGLET OXYGEN
Authors: Albro Phillip W. , Corbett Jean T. , Schroeder Joanna L.
Published: January 2008

Abstract Ergosterol and 7‐dehydrocholesterol, common 5,7‐conjugated diene sterols, react with photochemically produced singlet oxygen very efficiently to yield, in parallel pathways, the corresponding 5,8‐endoperoxides and the 7β‐hydroperoxy‐5,8(9),22‐trienol or ‐5, 8(9)‐dienol, respectively. The hydroperoxides decompose in an acid‐catalyzed reaction to generate hydrogen peroxide and the 5, 7, 9(1 1), 22‐tetraenol or 5, 7, 9(11) trienol, respectively, with 1:l stochiometry. The molar ratio of endoperoxide to hydroperoxide was constant (16:5) with two different reaction solvents, two different photosensitizers, and at all time points between 5 min and 3 h from the start of irradiation. Ergosterol did not react with either hydrogen peroxide or superoxide ion under our reaction conditions. Inhibition studies with nitrogen, 2,5‐dimethylfuran, βcarotene, and tert‐butanol confirmed the involvement of singlet oxygen in these reactions. The unstable hydroperoxide would be expected to have undesirable biological consequences if formed in vivo.
ASSESSMENT OF DIELECTRIC ENRICHMENT AROUND TWO FLUOROPHORES IN BINARY SOLVENTS
Authors: Zurawsky Walter , Scarlata Suzanne
Published: January 2008

Abstract We investigated dielectric enrichment, or the preferential interaction between a probe and a polar solvent, using two different fluorophores in binary solvents that will not form strong hydrogen bonds with the probe. The first probe, 6‐propionyl(N,N‐dimethylamino)naphthalene (PRODAN), undergoes a large change in dipole moment in the excited state. We found that its emission energy varies linearly with the dielectric parameter f(D) [f(D) = 2(D ‐ 1)/(2D + 1)] in acetone/benzene and acetonekyclohexane mixtures. The emission of the second probe, N‐acetyl tryptophanamide (NATA), also varies linearly with f (D) in ethylether/acetonitrile solvents. This lack of preferential solvation was further investigated by studies of NATA in acetonitrile/methanol mixtures where hydrogen bonding is possible. We found that NATA‐methanol interactions are not energetic enough to give rise to significant preferential solvation. Since preferential solvation does not occur even for weakly hydrogen bonding solvents it is thus highly unlikely that dielectric enrichment, which takes place in the absence of specific interactions, occurs with any frequency. We postulate that previous reports of dielectric enrichment were due to a lack of consideration of changes in f (D) of the solvent.
ALFALFA SEEDLINGS GROWN OUTDOORS ARE MORE RESISTANT TO UV‐INDUCED DNA DAMAGE THAN PLANTS GROWN IN A UV‐FREE ENVIRONMENTAL CHAMBER
Authors: Takayanagi Shinnosuke , Trunk John G. , Sutherland John C. , Sutherland Betsy M.
Published: January 2008

Abstract The relative UV sensitivities of alfalfa seedlings grown outdoors versus plants grown in a growth chamber under UV‐filtered cool white fluorescent bulbs have been determined using three criteria: (1) level of endogenous DNA damage as sites for the UV endonuclease from Micrococcus luteus. (2) susceptibility to pyrimidine dimer induction by a UV challenge exposure and (3) ability to repair UV‐induced damage. We find that outdoor‐grown plants contain approximately equal frequencies of endogenous DNA damages, are less susceptible to dimer induction by a challenge exposure of broad‐spectrum UV and photorepair dimers more rapidly than plants grown in an environmental chamber under cool white fluorescent lamps plus a filter that removes most UV radiation. These data suggest that plants grown in a natural environment would be less sensitive to UVB‐induced damage than would be predicted on the basis of studies on plants grown under minimum UV.
OBSERVED AND PREDICTED MINIMAL ERYTHEMA DOSES: A COMPARATIVE STUDY
Authors: Diffey B. L.
Published: January 2008

Abstract This study compared how well minimal erythema doses predicted using the reference action spectrum for UV erythema proposed by the International Commission on Illumination (CIE) in 1987 agreed with those observed in phototesting a large number of subjects with normal responses to sunlight to six different wavelengths of UV radiation (UVR) between 300 and 400 nm. It was found that, within the limits of experimental error, the hypothesis that the CIE reference action spectrum is a valid predictor of the erythemal effectiveness of different wavelengths of UVR could not be dismissed. There is no strong reason, therefore, why the CIE action spectrum should not continue to be used as a reference to compare the erythemal effectivenesses of different broadband sources. However, close examination of the residuals from the regression analysis suggested that the erythemal sensitivity of skin at longer UV wavelengths (>350 nm) in the population studied here is greater than predicted from the CIE reference action spectrum.
AN 125I‐LABELED N6‐SUBSTITUTED AZIDO ANALOG OF NAD+ FOR THE PHOTOAFFINITY LABELING OF NAD+‐LINKED ENZYMES
Authors: Chen Pei‐Lin , Ensor Charles Mark , Tai Hsin‐Hsiung
Published: January 2008

Abstract 125I‐N6‐(N‐[6‐N‐{5‐iodo‐4‐azidosalicyl}‐aminohexyl]‐aminocarbamoylmethyl)‐nicotinamide adenine dinucleotide (125I‐N6‐I‐ASA‐AH‐NAD+) was synthesized by coupling N6‐([6‐aminohexyl]‐carbamoylmethyl)‐NAD+ with 4‐azidosalicylic acid N‐hydroxysuccinimide ester followed by radioiodination. The utility of 125I‐N6‐I‐ASA‐AH‐NAD+ as an effective site‐directed photoprobe was demonstrated by the photolabeling of both glutamate dehydro‐genase and 15‐hydroxyprostaglandin dehydrogenase. Both enzymes can be saturated with labeled probe with apparent dissociation constants comparable to those reported for NAD+. Photoincorporation of the probe into both enzymes was found to be protected specifically by NAD+. These results indicate that 125I‐N6‐I‐ASA‐AH‐NAD+ can be a specific photoprobe for NAD+‐linked enzymes.
A COMPARISON OF DIRECI AND LIPOSOMAL ANTIBODY CONJUGATES OF SULFONATED ALUMINUM PHTHALOCYANINES FOR SELECTIVE PHOTOIMMUNOTHERAPY OF HUMAN BLADDER CARCINOMA
Authors: Morgan Janet , Lottman Henri , Abbou Claude C. , Chopin Dominique K.
Published: January 2008

Abstract There is a need to improve the selectivity of photodynamic therapy and for better targeting of tumor cells within specific tumor compartments. Selective in vitro phototoxicity of a human bladder carcinoma cell line 647V has been achieved by targeting sulfonated aluminum phthalocyanines (AlSPc) with monoclonal antibodies. Aluminum tetra‐3 sulfonyl chloride phthalocyanine (PC) or rhodamine sulfonyl chloride were directly coupled to antibodies by a sulfonamide linkage and AlSPc or carboxyfluorescein were encapsulated in liposomes of the small unilamellar vesicle type (SUV) bearing antibody. Antibody E7 (IgM subclass), which recognized an antigenic determinant expressed on 647V but was absent on T24 a control human bladder carcinoma cell line, and a control IgM antibody were used. The effects of the two types of conjugate were compared. Immunofluorescence studies on living cells demonstrated specific cell surface localization of conjugates at 4°C and internalization at 37°C. Phototoxicity was measured by 3‐(4,5‐dimethylthiazol‐2–5‐diphenyltetrazolium) bromide assay after exposing A1SPc‐sensitized cells to red light. Significant AlSPc dose‐dependent phototoxicity of the order 4°C < 4°C plus 37°C < 37°C was observed with E7‐SUV and E7‐PC in the range 1–8 μM AlSPc. At equimolar AlSPc doses absolute toxicity was similar for the two conjugate types, but at equimolar antibody doses, the liposomal conjugate was more effective by up to 13‐fold. Addition of urine during illumination decreased toxicity, which was attributed to the presence of protective elements. The results suggest that photosensitizers such as AlSPc could be used for antibody‐directed therapy and in particular for selectively damaging tumor cells of the epithelial cell compartment in bladder carcinoma by intrabladder administration. The therapeutic ratio, which takes into account both specific and nonspecific toxicity, was greater for the liposome conjugate than for the direct conjugate indicating their greater suitability for in vivo instillation.
LIGHT QUENCHING OF FLUORESCENCE: A NEW METHOD TO CONTROL THE EXCITED STATE LIFETIME AND ORIENTATION OF FLUOROPHORES
Authors: Lakowicz Joseph R. , Gryczyski Ignacy , Kuba Józef , Bogdanov Valery
Published: January 2008

Abstract Experimental studies have recently demonstrated that fluorescence emission can be quenched by laser light pulses from modem high‐repetition rate lasers, a phenomenon we call “light quenching.” In this overview article, we describe the possible effects of light quenching on the steady‐state and time‐resolved intensity and anisotropy of fluorophores. One can imagine two classes of experiments. Light quenching can occur within the single excitation pulse, or light quenching can be accomplished with a second time‐delayed quenching pulse. The extent of light quenching depends on the amplitude of the emission spectrum at the quenching wavelength. Different effects are expected for light quenching by a single laser beam (within a single laser pulse) or for a time‐delayed quenching pulse. Depending upon the polarization of the light quenching beam, light quenching can decrease or increase the anisotropy. Remarkably, the light quenching can break the usual z‐axis symmetry of the excited state population, and the measured anisotropy (or polarization) depends upon whether the observation axis is parallel or perpendicular to the propagation direction of the light quenching beam. The polarization can increase to unity under selected conditions. Quenching with time‐delayed light pulses can result in step changes in the intensity or anisotropy, which is predicted to result in oscillations in the frequency‐domain intensity and anisotropy decays. These predicted effects of light quenching, including oscillations in the frequency‐domain data, were demonstrated to occur using selected fluorophores. The increasing availability and use of pulsed laser sources requires consideration of the possible effects of light quenching and offers the opportunity for a new class of two‐pulse or multiple‐pulse time‐resolved experiments where the sample is prepared by the excitation pulse and subsequent quenching pulses to modify the excited state population, followed by time‐ or frequency‐domain measurement of the optically prepared excited fluorophores.
ABSORPTION SPECTRUM OF HEMATOPORPHYRIN DERIVATIVE in vivo IN A MURINE TUMOR MODEL
Authors: Cubeddu Rinaldo , Canti Gianfranco , Musolino Mario , Pifferi Antonio , Taroni Paola , Valentini Gianluca
Published: January 2008

Abstract‐Time‐resolved reflectance was used to measure the absorption spectrum of hematoporphyrin derivative (HpD) in vivo in a murine tumor model. Reflectance measurements were performed in the 600–640 nm range on mice bearing the L1210 leukemia. Then the animals were administered 25 mg/kg body weight of HpD intraperito‐neally. One hour later the reflectance measurements were repeated. Fitting of the data using the diffusion theory allowed assessment of the absorption coefficient before and after the administration. As a difference between the latter and the former data, the in vivo absorption spectrum of HpD was evaluated. Maximum absorption was measured at 620–625 nm. Similar spectral behavior was obtained for HpD in solution in the presence of low‐density lipoproteins.
RESOLUTION OF THE INDIVIDUAL TRYPTOPHANYL CONTRIBUTIONS TO THE NEAR‐ULTRAVIOLET DICHROIC ACTIVITY OF APOMYOGLOBIN
Authors: Sirangelo Ivana , Irace Gaetano , Bismuto Ettore
Published: January 2008

Abstract –The individual tryptophanyl contributions to the near‐ultraviolet dichroic activity of apomyoglobin in its native conformation have been resolved. This was accomplished by comparing the spectra of two classes of apomyoglobin with different aromatic residue contents and observing the effect of a specific modification of indole residues. The circular dichroism (CD) spectra of apomyoglobins containing two tryptophanyl residues, i.e. Trp A‐5 and A‐12, show the presence of a positive peak centered at 292 nm, attributable to indolic chromophore, which is missing in the CD spectrum of tuna apomyoglobin possessing only Trp A‐12. Moreover, the specific modification of Trp A‐5 by 2‐hydroxy‐5‐nitrobenzyl bromide is shown by the lack of the 292 nm peak and the appearance of a positive band at longer wavelength. The pH dependence of the position of this band suggests that it arises from the 2‐hydroxy‐5‐nitrobenzyl moiety. The results suggest that Trp A‐12 does not substantially contribute to the optical activity in the near ultraviolet.
ULTRAVIOLET‐INDUCED CELL DEATH IS INDEPENDENT OF DNA REPLICATION IN RAT KANGAROO CELLS
Authors: Miyaji Eliane N. , Menck Carlos F. M.
Published: January 2008

Rat kangaroo (Potorous tridactylus) cells have an efficient repair system for photoreactivation of lethal lesions induced by 254 nm UV. However, this ability is lost with increasing time after UV, being completely ineffective after 24 h. Critical events leading to UV‐induced cell death must occur within this period of time. DNA synthesis was inhibited by the DNA polymerase inhibitor aphidicolin and the loss of the capability to photorepair lethal lesions was maintained as for replicating cells. Similar data were obtained in synchronized cells UV irradiated immediately before S phase. Under the same conditions, the ability to remove cyclobutane pyrimidine dimers by photoreactivation in these cells remained unchanged 24 h after irradiation. These data indicate that the critical events responsible for UV‐induced cell death occur in the absence of DNA replication.
TARGETING OF PHOTOTOXIC DRUGS TO ANTIGEN‐SPECIFIC T LYMPHOCYTES in vitro USING ANTIGEN‐PRESENTING CELL MEMBRANES
Authors: Cchang Nan‐Hua , Rayner David C. , Boggs Joan M.
Published: January 2008

We have used the complex of antigen with class II major histocompatibility proteins (la) in membrane‐bound form to target a phototoxic compound to antigen‐specific T cell hybridomas in vitro. The iodoacetamidyl ester of phototoxic pyrene was bound covalently to antigen‐presenting cells (APC), and protein antigens were added to the cells for processing, presentation and targeting of the drug to three different T hybridomas specific for myelin basic protein (MBP), ovalbumin (OVA) and keyhole limpet hemocyanin (KLH). The B hybridoma LS102.9 was used as APC to present MBP, KLH and either a tryptic digest of OVA or the synthetic peptide OVA323–339to these T cells. A transformed B lymphoma, which expresses trinitrophenol (TNP)‐specific surface IgM, A20‐HL, was used to present TNP conjugates of KLH and OVA to T cells. Either the antigen‐bearing intact APC or Ia+ membranes shed spontaneously from them were used as drug carriers to target pyrene to the T cells. In the dark, or in the absence of pyrene, both the intact APC or the shed membranes stimulated interleukin‐2 (IL‐2) production by the T cells in an antigen‐specific way. After UVA (320–400nm) irradiation, both forms of these drug carriers had an antigen‐specific toxic effect on the T hybridoma cells with receptors for the antigen that they carried. Both spontaneous T cell proliferation and antigen‐induced IL‐2 production were inhibited. The shed membranes had a more antigen‐specific toxic effect than the intact APC, which tend to settle out with the T cells in the microtiter plates, possibly causing nonspecific contact. These results indicate that the antigen‐la complex in membrane‐bound form can be used to antigen‐target cytotoxic drugs to antigen‐specific T cells. The Ia+ membranes shed from APC may be useful to target drugs to antigen‐specific clones of T cells in vivo.
BLUE LIGHT PERCEPTION BY ENDOGENOUS REDOX COMPONENTS OF THE PLANT PLASMA MEMBRANE
Authors: Asard Han , Horemans Nele , Briggs Winslow R. , Caubergs Roland J.
Published: January 2008

b‐Type cytochromes of the higher plant plasma membrane may be reduced by irradiation with actinic blue light (light‐induced absorbance change). Although this reaction has been reported to depend on the presence of an exogenous oxygen‐scavenging system, significant cytochrome reduction was obtained in bean hook (Phaseolus vulgaris L. cv. “Limburgse Vroege”) plasma membranes without any addition. An endogenous oxygen‐consuming reaction is apparently sufficient to achieve a proper redox balance. A blue light‐mediated absorbance change with absorbance minima at 450 and 475 nm precedes cytochrome b reduction and indicates the presence of a flavoprotein in the plasma membrane fraction.

Cytochrome b reduction by blue light in the absence of an oxygen scavenger is highly sensitive to flavin photosensitizers. Glucose oxidase, which has previously been used to lower the oxygen concentration in membrane samples, was demonstrated to have a photosensitizing effect. Inhibitors of flavin photochemical reactions (KI and phenylacetic acid) were highly effective in preventing cytochrome b reduction. These results indicate that the blue light‐mediated reaction probably involves an endogenous plasma membrane flavoprotein as the photoreceptor.

As plasma membrane NADH‐dependent oxidoreductases potentially are flavoproteins these experiments raise the question whether a plasma membrane cytochrome b and a flavin‐enzyme may cooperate in blue light reactions. Evidence is also discussed, suggesting the possible involvement of oxygen radicals in the blue light‐induced cytochrome b reduction.
INFLUENCE OF CHANGES IN THE PHOTON PROTECTIVE ENERGY DISSIPATION ON RED LIGHT‐INDUCED DETRAPPING OF THE THERMOLUMINESCENCE Z‐BAND
Authors: Hagen Christoph , Pascal Andrew A. , Horton Peter , Inoue Yorinao
Published: January 2008

Abstract—

‐Thermoluminescence emission at 110 K (Z‐band) was markedly diminished when thylakoid membranes were exposed to red light during or after Z‐band charging with blue light. Analysis of this phenomenon showed that deactivation of Z‐band‐emitting chlorophyll species occurred preferentially on the low temperature side of the glow curve, and red light of670–680 nm was most efficient in the deactivation. In order to test our hypothesis that this detrapping is related to local heating effects caused by dissipation of absorbed energy, we measured thermoluminescence glow curves and Z‐band emission spectra from spinach leaf discs and thylakoid membranes during induction of nonphotochemical chlorophyll fluorescence quenching. Pretreatment of the plant material was designed to achieve different levels of (1) de‐epoxidized xanthophylls in the photosynthetic apparatus and (2) the proton concentration in the thylakoid lumen. In comparison, measurements were performed in aggregated and trimeric light‐harvesting pigment‐protein complexes of photosystem II. We observed on all three levels of organization that a higher capacity of excitation energy dissipation was accompanied by a stronger red light‐induced detrapping of Z‐band thermoluminescence.
STIMULATORY AND INHIBITORY EFFECTS OF IRON ON PHOTODYNAMIC INACTIVATION OF LEUKEMIA CELLS
Authors: LIN FUBAO , GIROTTI ALBERT W.
Published: January 2008

Abstract—

The influence of exogenous iron on merocyanine 540 (MC540))sensitized photoinactivation of leukemia cells has been investigated. Irradiation of murine L1210 or human HL‐60 cells (‐lOh/mL in 1 % serumiRPM1 medium) with broadband visible light in the presence of MC540 (2 pIM) resulted in a progressive loss of clonally assessed cell viability. When added to cells 30 min before irradiation, the low polarity chelate, ferric 8‐hydroxyquinoline [Fe(HQ), 0.5 *MI stimulated dye‐sensitized pho‐ tokilling, whereas high polarity chelates such as ferric 8‐hydroxyquinoline‐5‐sulfonate [Fe(HQS)2, 0.5 p M] or ferric ethylenediaminetetraacetate (Fe.EDTA, 0.5 FM) had no effect. A striking reversal of Fe(HQ),‐enhanced photokilling was observed upon increasing the preirradiation incubation time with Fe(HQ)2 such that a marked resistance (relative to non‐iron‐treated controls) was evident after 24 h. Cells exposed for 24 h to Fe(HQS), or Fe.EDTA showed similar or even greater resistance to photo‐killing. Like phototoxicity, H,O,‐induced cytotoxicity was enhanced after a 30 min exposure of cells to Fe(HQ)2 but strongly repressed after 24 h. Immunoblot (western) analysis, using a polyclonal antibody to ferritin, revealed that cells exposed to Fe(HQ): for 24 h contained at least 12 times as much ferritin heavy chain as non‐Fe(HQ)?‐treated controls. Preincubating cells with emetine, an inhibitor of protein synthesis, prevented both ferritin induction and the development of hyperresistance. These findings, along with the observation that exogenous apoferritin protected LI 2 10 cells against photokilling, suggest a possible role for ferritin in iron‐stimulated photoresistance. It is conceivable that in photodynamic treatment of tumors, certain cells might resist inactivation via this mechanism, a possibility that has not been recognized heretofore.
PHOTOREPAIR OF NONADJACENT PYRIMIDINE DIMERS BY DNA PHOTOLYASE
Authors: Kim Sang‐Tae , Sancar Aziz
Published: January 2008

Abstract— Photolyases reverse the harmful effects of UV light on cells by converting pyrimidine dimers (Pyr[]Pyr) into two pyrimidine monomers by utilizing near‐UV and visible light. Previous work has shown that photolyase repairs T[c,s]T and T[t,s]T in DNA as well as U[]U in RNA, all of which are formed by joining the two adjacent pyrimidines in a light‐dependent reaction. In this report, we show that Pyr[]Pyr formed in nonadjacent pyrimidines are also substrates for DNA photolyase.
SOMATOSTATIN IN A WATER‐RESTRICTED ENVIRONMENT: FLUORESCENCE AND CIRCULAR DICHROISM STUDY IN AOT REVERSE MICELLES
Authors: Bhattacharyya Kasturi , Basak Soumen
Published: January 2008

Abstract—

Steady‐state and time‐resolved fluorescence emission from the single tryptophan residue of somatostatin, and the kinetics of quenching of this emission, were studied in aqueous solution and in reverse micelles of sodium bis (2‐ethylhexyl) sulfosuccinate (AOT)/water/isooctane, a system that mimics the water‐membrane interface well‐ Incorporation into micelles caused blue shifts and reduced band‐widths of the emission peaks and altered the quantum yields with respect to emission from bulk water. Steady‐state anisotropy values also increased considerably on micellization. These observations point to reduced polarity of the environment around the Tip residue of the peptide, as well as restricted freedom of its rotational motions, due to transfer from the aqueous to the micellar phase. Fluorescence emission kinetics of the Tip moiety followed biexponential decay laws in both aqueous and micellar media. Static and dynamic quenching constants were measured for acrylamide and CC14 quenchers localized in the micellar and organic pseudophases, respectively, using both steady‐state and time‐resolved experiments. The efficiency of dynamic quenching by acrylamide became vanishingly small in going from water to reverse micelles, in sharp contrast to the comparable quenching efficiencies exhibited by CC14 in micelles and acrylamide in water. The circular dichroic (CD) spectrum of the native peptide in water indicated the possibility of some amount of P‐type secondary structure being present. Conformational analysis of CD spectra in micelles showed that the relative amount of this structural feature was enhanced for the micellized peptide but was insensitive to the water content of micelles. The above results, put together, indicate that the Trp‐8 residue in somatostatin is likely to be located in the close neighborhood of the water‐AOT molecular interface, where the water molecules are strongly immobilized. This work also demonstrates the role of reverse micelles as a convenient membrane‐mimetic medium for the study of membrane interactions of bioactive peptides.
ENERGY TRANSFER‐ENHANCED CHEMILUMINESCENCE OF ADAMANTANONE (n,π*) AND ESTER (π,π*) SINGLET AND TRIPLET EXCITED STATES IN THE THERMOLYSIS OF SILYLOXYARYL‐SUBSTITUTED SPIROADAMANTYL DIOXETANES†
Authors: Trofimov Alexei V. , Vasil'ev Rostislav F. , Mielke Karsten , Adam Waldemar
Published: January 2008

Abstract—

The thermal generation of singlet and triplet excited states from silyloxyaryl‐substituted spiroadamantyl dioxetanes lab and the adamantylidineadamantane dioxetane (1c) was investigated by direct and enhanced chemiluminescence (CL). 9,10‐Diphenylanthracene (DPA) and 9‐fluorenone were used as energy acceptors in the singlet‐singlet (S‐S), naphthalene and europium chelate Eu(TTA)3Phen (TTA = thenoyltrifluoroacetone, Phen = 1,10‐phenanthroline) in the triplet‐triplet (T‐T) and 9,10‐di‐bromoanthracene (DBA) in triplet‐singlet (T‐S) energy transfer experiments. The direct chemiluminescence observed in the thermolysis of dioxetanes lab consisted of fluorescence derived from the singlet‐excited adamantanones 2a,b. In the presence of naphthalene, selective T‐S energy transfer with DBA (napthalene as quencher) displayed the adamantanone triplets 2a,b and with Eu(TTA)3Phen (naphthalene as mediator) also the silyloxyaryl ester 3 triplets. From the Stern‐Volmer constants (kTNTT0) the triplet lifetimes t0t of these triplet state products were assessed. By using the Hastings‐Weber standard, the total triplet excitation yield (φt) was estimated to be ca 20%. The energies of the first excited singlet and triplet states of the adamantanones 2a,b and the silyloxyaryl ester 3, the products of the thermally induced decomposition of dioxetanes la‐c, were determined by semiempirical calculations (AMI‐based configuration interaction), which included explicitly solvent effects on the excitation energies in terms of a self‐consistent reaction field approach. The calculations revealed that the first excited singlet and triplet states of the adamantanones 2a,b are expectedly n,π*‐type excitations while the silyloxyaryl ester 3 possesses π,π* character. The semiempirical computations suggest that excitation of the adamantanones 2a,b as well as the silyloxyaryl ester 3 is feasible in the thermolysis of the spiroadamantyl dioxetanes lab, which has been confirmed by the experimental energy transfer studies.
TARGETING ACTIVATED LYMPHOCYTES WITH PHOTODYNAMIC THERAPY: SUSCEPTIBILITY OF MITOGEN‐STIMULATED SPLENIC LYMPHOCYTES TO BENZOPORPHYRIN DERIVATIVE (BPD) PHOTOSENSITIZATION
Authors: Obochi Modestus O. K. , Canaan Alice J. , Jain Ashok K. , Richter Anna M. , Levy Julia G.
Published: January 2008

Abstract— Benzoporphyrin derivative monoacid ring A (BPD), a hydrophobic chlorin‐like porphyrin derivative, which fluoresces strongly at 690 nm, may have potential for both oncologic and nononcologic applications in photodynamic therapy (PDT). To study the influence of cellular characteristics on the uptake of BPD, the murine tumor cell line (P815), and in vitro and in vivo concanavalin A (Con A)‐stimulated and unstimulated murine splenic lymphocytes were incubated with 2 µg/mL BPD at 37°C for 0–60 min. At various times, cells were lysed and the amount of BPD taken up by the cells was quantified by fluorescence measurements. The subsets of cells taking up BPD were analyzed using a panel of monoclonal antibodies and the Coulter XL* fluorescence‐activated cell sorter. Furthermore, Con A‐stimulated and unstimulated spleen cells were incubated with 0–50 ng/mL of BPD for 1 h prior to exposure to red light (7.2 J/cm2). Cell survival 24 h post‐PDT was measured by the MTT assay. We found that the rapidly dividing tumor cell line and mitogen‐stimulated murine T cells (mainly CD4V IL‐2R+) took up significantly more BPD (5–10‐fold) than do unstimulated splenic lymphocytes. Increased BPD uptake correlated with greater photoinactivation when these cells were exposed to light at a wavelength of 690 nm. These findings suggest that activated cells of the immune system may be a target for photoinactivation by BPD.
SULFOPHTHALOCYANINES FOR PHOTODYNAMIC INACTIVATION OF VIRUSES IN BLOOD PRODUCTS: EFFECT OF STRUCTURAL MODIFICATIONS
Authors: Allen Cynthia M. , Weber Joseph M. , van Lier Johan E.
Published: January 2008

Abstract— Transmission of infectious diseases through blood transfusions is well known. Ultraviolet irradiation, solvents and detergents provide a means of sterilizing noncellular blood components. However these harsh methods are not applicable to cellular blood products. Recently, attempts have been made to sterilize biological fluids using photodynamic treatment and phthalocyanine (Pc) dyes have been advanced as photosensitizers for this purpose. We have evaluated a series of water‐soluble Pc, chelated with different central metal ions, substituted to different degrees with sulfonato and r‐butyl groups, for their effectiveness to reduce virus infectivity in red blood cell suspensions. Vaccinia virus cytopathogenicity was determined by endpoint serial dilutions in the CV‐1 cell line. Anti‐viral activity increased with the central metal ion in the following order: Ga(III) < Al(III) < Zn(II), and varied inversely with the degree of sulfonation. Furthermore, addition of a t‐butyl group onto the trisulfonated dyes (PcS3[t‐Bul) resulted in a 5–40‐fold increase in anti‐viral potency, suggesting that amphiphilicity enhances the photodynamic activity of the dye. Strong anti‐viral photosensitizing properties cannot be the sole selection criterion. Of equal importance is the preservation of blood component integrity. Accordingly, the photohemolytic activity of the dyes was evaluated using the rate of hemolysis as a parameter and a toxicity index was defined. Among the most active dyes, the AlPcS3(t‐Bu) complex exhibited the most favorable anti‐viral properties combined with a low toxicity index. Our results suggest that trisulfophthalocyanines, bearing an additional t‐butyl group to enhance amphiphilicity, are particularly promising dyes for photodynamic blood sterilization.
MAGNETIC FIELD EFFECTS ON THE PHOTOHEMOLYSIS OF HUMAN ERYTHROCYTES BY KETOPROFEN AND PROTOPORPHYRIN IX
Authors: Chignell C. F. , Sik R. H.
Published: January 2008

Abstract— Application of a static external magnetic field (3350 G) during UV‐irradiation (>300 nm) reduced the time for 50% photohemolysis of human erythrocytes by the phototoxic drug ketoprofen (3‐benzoyl‐α‐methylbenzoacetic acid) from 96 min to 78 min. This observation can be attributed to a magnetic field induced decrease in the rate of intersystem crossing (kISC) of the geminate triplet radical pair generated by the reduction of ketoprofen in its triplet excited state by erythrocyte membrane constituents, probably lipids. The decrease in kJSC results in an increase in the concentration and/or lifetime of free radicals that escape from the triplet radical pair. Thus the critical radical concentration needed to cause membrane damage and cell lysis is reached sooner in the presence of the magnetic field. In contrast, the photohemolysis induced by the photodynamic agent protoporphyrin IX was not affected by the magnetic field. Protoporphyrin IX photohemolysis, which is initiated by singlet oxygen, does not involve the initial generation of a triplet radical pair and so is not influenced by the magnetic field. The enhancement of ketoprofen‐induced photohemolysis by an externally applied magnetic field is the first example of a magnetic field effect on a toxicological process involving free radicals.
AGE‐RELATED CHANGES IN THE HUMAN LENS AS MONITORED BY DETECTION OF PORPHYRIN EXCITED STATES
Authors: Roberts Joan E. , Atherton Stephen J. , Gaillard Elizabeth R. , Dillon James
Published: January 2008

Abstract— Previous studies have shown that the triplet state lifetimes of various porphyrins are increased by several orders of magnitude when they are bound to lens proteins. Flash photolysis studies of me‐sotetra (p‐sulfonatophenyl)porphyrin (TPPS) on intact bovine lenses indicated a biexponential decay of the triplet state with lifetimes of 160 μs and 1.6 ms. Here we extend those measurements to TPPS associated with intact human lenses. Steady‐state fluorescence measurements indicate that TPPS binds to both young and old human lenses. In an intact young human lens, the TPPS triplet state is observed to decay biexponentially with lifetimes of 50 and 680 μs. As the age of the lens increases, the lifetime of the shorter‐lived component lengthens while that of the longer‐lived component decreases slightly. In older human lenses, the two lifetimes coalesce and the triplet decay exhibits purely monoexponential behavior. These photophysical characteristics apparently are due to age‐related modification(s) of the protein in the human lens resulting in an increasingly more homogeneous environment around the porphyrin.
PHOTOSTATIONARY STATE COMPOSITIONS OF RETINAL and RELATED COMPOUNDS INCLUDED IN ß‐LACTOGLOBULIN. EFFECTS OF PROTEIN HOST ON ISOMER DISTRIBUTION OF POLYENE SUBSTRATES
Authors: Li Xiao‐Yuan , Liu Robert S. H.
Published: January 2008

Abstract— The UV‐visible absorption spectra and photostationary state compositions of retinal (or the related C‐18 ketone, 3‐dehydroretinal and the C‐22 aldehyde) imbedded in the binding cavity of (3‐lactoglobulin (BLG) are consistent with the view that the carbonyl group of these polyenes are hydrogen‐bonded with the protein host, most likely with the lone protonated lysine residue in the binding pocket. Patterns of variation in photochemical behavior of the imbedded chromophore versus that in solution are discussed in terms of possible specific protein‐substrate interactions. The results are also compared with that of the methyl ether of retinol where similar hydrogen bonding is not possible.
TRAPPING OF NITRIC OXIDE FORMED DURING PHOTOLYSIS OF SODIUM NITROPRUSSIDE IN AQUEOUS AND LIPID PHASES: AN ELECTRON SPIN RESONANCE STUDY
Authors: Singh Ravinder Jit , Hogg Neil , Neese Frank , Joseph Joy , Kalyanaraman B.
Published: January 2008

Abstract— Photolytic decomposition of sodium nitroprusside (SNP), a widely used nitrovasodilator, produced nitric oxide (NO), which was continuously monitored by electron spin resonance (ESR) spectroscopy. The NO present in the aqueous or the lipid phase was trapped by either a hydrophilic or a hydrophobic nitronyl nitroxide, respectively, to form the corresponding imino nitroxide. The conversion of nitronyl nitroxide to imino nitroxide was monitored by ESR spectrometry. The quantum yield for the generation of NO from SNP, measured from the rate of decay of nitronyl nitroxide, was 0.201 ± 0.007 and 0.324 ± 0.01 (¯± SD, n = 3) at 420 nm and 320 nm, respectively. The action spectrum for NO generation was found to overlap the optical absorption spectrum of SNP closely. A mechanism for the reaction between SNP and nitronyl nitroxide in the presence of light is proposed and computer‐aided simulation of this mechanism using published rate constants agreed well with experimental data. The methodology described here may be used to assay NO production continuously during photoactivation of NO donors in aqueous and lipid environments. Biological implications of this methodology are discussed.
SELECTIVE DNA THYMINE DIMERIZATION DURING UVA IRRADIATION IN THE PRESENCE OF A SATURATED PYRIDOPSORALEN
Authors: Guillo LidiaAndreu , Blais Jocelyne , Vigny Paul , Spassky Annick
Published: January 2008

Abstract— It has been recently shown that UVA (320–400 nm) irradiation of DNA in the presence of pyridopsoralens induces the formation of thymine cyclobutane dimers in addition to monoadducts. In this work, we measured the potency of a saturated pyridopsoralen to photosensitize DNA, despite its inability to covalently attach to DNA. First, from spectroscopic fluorescence measurements, we have shown that both analogs, saturated and unsaturated pyridopsoralens, namely 4′,5′‐dihydro‐7‐methyl‐pyrido[3,4‐clpsoralen (DH‐MePyPs) and 7‐methylpyrido[3,4‐c]psoralen, exhibit a similar global affinity for DNA. Secondly, we demonstrated, by footprinting experiments, that exposure of a DNA sequence to 365 nm UV radiation in the presence of DH‐MePyPs results in selective cyclobutane thymine dimerization. Thymines located in the immediate proximity of the 5′‐TA‐3′ step are exclusively affected and the frequency of this photoprocess depends on flanking sequences. We thus probe a selective thymine dimer photosensitizer. Results are discussed in terms of drug affinity and physical properties of the helix at the binding site.