Reference #: SAR-1017-504495
Submit Date: 03/30/2002 09:40:53-0500

Interaction of Plasmenylcholine with Singlet Oxygen and Free Radicals

ABSTRACT:
Synthetic vinyl ether-based membrane fusogens that respond to low pH and oxidative environments are of considerable interest beacuse of their potential use in drug delievery. Indeed, photosensitized oxidation of plasmenylcholine (PlsC) vesicles has been shown to cause substantial increase in permeability of PlsC membranes and their fusion. On the other hand, a protective role of PUFA from reactive oxygen species (ROS) by PlsC has also been postulated. In this study, we investigated the interaction of singlet oxygen and other ROS with PlsC in solutions of PlsC in methanol and in multilamellar liposomes made of PlsC and the non-peroxidizable phospholipid DMPC. Singlet oxygen was generated by photosensitized reaction using rose bengal or merocyanine 540 as the sensitizers and free radicals were formed by decomposition of thermolabile azo-compounds. Progress of the reaction of PlsC with ROS was monitored by HPLC-EC(Hg) determination of singlet oxygen- and free radical-specific oxidation products of cholesterol, used as a mechanistic reporter molecule, by iodometric determination of lipid hydroperoxides and by electron spin resonance oximetry. Our data reveal that the vinyl-ether linked plasmenylcholine is unusually susceptible to oxidation, compared to other unsaturated lipids. Thus the rate of PlsC interaction of singlet oxygen in both homogenous and heterogenous systems is between one to two orders of magnitude faster than that of cholesterol. Although the mechanism of such an enhanced reactivity of PlsC with ROS remains to be determined, the apparent selectivity of singlet oxygen interaction with this phospholipid may have important practical and biological implications. Supported by NIH (FIRCA grant R03 TW01252-01A1) and by State Committe for Scientific Research (KBN).

Keywords: plasmenylcholine, photooxidation, singlet oxygen, free radicals

Reference #: MEL-1017-326978
Submit Date: 03/28/2002 07:42:46-0500

Writing on the Genome - Focused Induction of UV Photoproducts in Cell nuclear DNA by Three-Photon Infra-red Laser Radiation.

ABSTRACT:
Applications based on two and three-photon processes have recently generated renewed interest because of the ability of the technique to localize the photonic interaction within a femtolitre volume.So far the two and three photon phenomena in biological studies have been applied mainly to imaging microscopy where the techniques provide a unique three-dimensional detail with sub-micron resolution.The possibility of inducing DNA damage in cells by multiphoton processes has two major implications.Firstly, at cetain intensities of radiation, DNA damage could occur as a collateral effect of multiphoton imaging techniques and this may invalidate experiments on live cells. Secondly, highly localized induction of DNA damage in whole viable cells is a major goal of cell biologists who wish to study DNA repair protein dynamics, individual cell signaling and cell to cell communication. We have successfully used triple infra-red (IR) photon absorption to induce cyclobutane pyrimidine dimers (CPDs)in viable cell nuclear DNA. Three-photon near IR induction of the characteristic UV photoproducts, cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts, provides much higher spatial resolution and consequently fewer numbers of lesions. The damage which is identified by immunostaining with monoclonal antibody, can be generate in a precise and distinct pattern within nanometer three-dimensional resolution (i.e. in spot sizes of less than 300nm dimension). The estimated intensity of the IR light which generates the 3-photon absorption in the DNA and leads to the induction of CPDs was approximately 200GW/cm2. Repair of the damage induced at this intensity was observed which confirmed that collateral heating by one-photon IR absorption was insignificant. We are therefore able to assess the probabilities of DNA damage induction by radiation intensities used for multiphoton imaging processes as well as induce very highly focused damage in cell nuclei.

Keywords: three-photon infra-red laser radiation, DNA damage, UV photoproducts, DNA repair

Reference #: SIM-1017-674626
Submit Date: 04/01/2002 08:50:50-0500

Primary Photophysics Following UV-A Photoexcitation of Pheomelanin

ABSTRACT:
Epidemiological data indicate that fair skin individuals are more susceptible to skin cancers; this observation is commonly explained by invoking the enhanced photoreactivity of the red melanin, pheomelanin, compared to the black melanin, eumelanin. Scanning electron microscopy images of synthetic pheomelanin have shown a structural morphology different than that observed for eumelanins. Pump-probe spectroscopic measurements reveal photoexcitation of pheomelanin by UVA light generates a transient absorption centered at 720 and 350 nm within the instrument response. These features exhibit multi-exponential decay dynamics. The time constants from a multi-exponential fit to the absorption data are similar to those obtained from an analysis of the corresponding emission decay of the pigment. While both transient absorption bands show the same ultrafast decay component (~1 ps), the 720 nm completely vanishes on the 10s of picosecond time scale, but the UV band shows a kinetic evolution to a subsequent intermediate. The kinetics of formation of this species is determined from the time-resolved data recorded at 390 and shows this species forms with a ~1 ps time constant. The action spectra between 300 nm and 380 nm for the generation of the 720 nm transient were measured for the samples with the different distribution of molecular sizes. Similar action spectral features are found for molecular weight (MW) < 10000 and MW > 10000 fractions of pheomelanin, suggesting that the reactive molecules has a low molecular weight but is present in the bulk pigment. The shapes of the action spectra differ from the absorption spectra of bulk melanin, but resemble that of benzothiazines, oxidation products of 5-S-cysteinyl-dopa, the biosynthetic precursor of pheomelanin. The peak of the action spectrum matches the absorption maximum of a recently identified 1,4-benzothiazine dimmer isolated from red hair. The collective data are discussed in terms of the molecular heterogeneity of pheomelanins, and the role of aggregation in determining physicochemical properties of the pigment.

Keywords: pheomelanin, ultrafast spectroscopy, aggregation

Reference #: HAM-1017-433652
Submit Date: 03/29/2002 14:05:48-0500

Targeted photodynamic therapy for Staphylococcus aureus soft tissue infections in vivo monitored by bioluminescence imaging

ABSTRACT:
Although many workers have used PDT to kill bacteria in vitro, the use of this approach has seldom been reported in vivo in animal models of infection. We have previously described the first use of PDT to treat excisional wound infections in living mice. We used a polycationic photosensitizer conjugate designed to penetrate the Gram-negative bacterium cell wall, topically administered into the infected wound followed by illumination with red light. We showed that both a non-pathogenic strain of Escherichia coli and a highly pathogenic strain of Pseudomonas aeruginosa could be effectively and rapidly killed in wounds in living mice. In the latter case treated mice were saved from death caused by the bacteria invading the bloodstream. However both these infected wound models used a short time after infection (30 min) before PDT. We now report on the use of PDT to treat an established soft-tissue infection in mice. Methicillin resistant S. aureus is a significant cause of morbidity in post-surgical infections. We used S. aureus transfected with the Photorhabdus luminescens lux operon (luxABCDE) that was genetically modified to be functional in Gram-positive bacteria and that emitted bioluminescence allowing the progress of the infection to be monitored in both space and time with a low-light imaging camera. One million cells were injected into the thigh muscles of mice that had previously been rendered neutropenic by cyclophosphamide administration. Twenty-four h later the bacteria had multiplied one hundred-fold, and poly-L-lysine chlorin(e6) conjugate was injected into the area of infected muscle as imaged with the luminescence camera. Thirty min later red light from a diode laser was delivered as a surface spot onto the skin overlying the infection. There was a light-dose dependent loss of luminescence from the infection not seen in mice that received conjugate alone or light alone. Treated mice recovered well and their infected thighs healed faster than control infected mice.

Keywords: photodynamic therapy, localized infection, bacterial targeting, bioluminescence

Reference #: RED-1017-959548
Submit Date: 04/04/2002 16:15:13-0500

Role of secondary ROS in transducing membrane photosensitization to nuclear damage

ABSTRACT:
For a type II photosensitizer, the subcellular localization determines the sites of primary photochemical reactions and initial damage to the cell. Laser scanning confocal fluorescence microscopy showed that the tetrapyrrolic photosensitizer, deuteroporphyrin (DP), localized in the plasma membrane and the Golgi system of WTK1 lymphoblasts. However, upon excitation with green light, a dose-dependent formation of DNA single strand breaks was observed by comet assay. In tandem, DP also increased the mutation frequency at the autosomal tk locus of WTK1 cells. To identify the mechanisms responsible for these nuclear alterations, the intracellular ROS content was monitored using a reduced fluorescein probe. A dose-dependent increase in the ROS level was observed, with confocal microscopy revealing that these ROS were initially generated in sites where DP accumulates before spreading throughout the cell. Increased ROS levels could still be detected tens of minutes following irradiation. Dose-dependent lipid peroxidation was also observed by TBARs assay, and was inhibited by pretreatment with the lipophilic antioxidant -tocopherol, or with the iron-chelator, desferioxamine. -tocopherol and/or its water soluble analog, Trolox, also reduced the extent of comet formation and photocytotoxicity of DP in this cell line. DNA damage was also reduced when the samples where kept at 4 C during and after irradiation. Photoexcitation of DP induces singlet oxygen, a short-lived species that should not reach the nucleus during its lifetime. Results suggest that these nuclear alterations arise from secondary oxidative species generated after the formation and initial reactions of singlet oxygen. The temperature dependence of the DNA damage suggests that enzymatic pathways may also be involved in the generation of the observed DNA modifications

Keywords: mutagenesis, photosensitization, DNA damage, reactive oxygen species

Reference #: GAN-1017-517454
Submit Date: 03/30/2002 12:57:20-0500

Triplet vs. Singlet and Energy-Transfer vs. Charge-Transfer Interactions of Metallo-Phthalocyanines with (Hydro)Peroxides

ABSTRACT:
Light-activated triplet photosensitizers can be quenched by organic (hydro)peroxides, ROOR', followed by an efficient, but usually non-quantitative homolytic cleavage of the peroxide O-O bond via energy transfer (EnT) to low-laying repulsive state(s) of the peroxide. Hydrogen atom abstraction (R' = H) and intricate charge transfer (CT) mechanisms (ground/excited (exciplex) mediated, partial CT, etc.) add to the perplexity of both, singlet and triplet interactions of ROOR' with variety of photosensitizers, thus often hampering the analysis of the elementary processes underlying heterolytic and/or homolytic breakdown of the peroxides. Here we report our results from photophysical measurements of metallo-phthalocynaine (MePc, Me = Zn, Al) triplet and singlet excited state quenching by a series of (hydro)peroxides and related compounds. The ensuing photochemical processes (primary decomposition products of peroxides) were assessed using photo-EPR and spin-trapping. It was found that triplet state interactions are weak (highest bimolecular rate constant, log k ~ 4). Fluorescence quenching, however, was several orders of magnitude more effective, with a dynamic rate constant, log k ~ 8-9, e.g. for PhCMe2OOH and (PhCMe2O)2. In all cases, in apolar solutions and relatively low (< 2 mM) ZnPc concentrations alkoxyl free radicals (RO) were positively recognized as primary products of (hydro)peroxide breakdown. However, at high MePc concentrations the main products of ROOH decomposition were peroxyl radicals (ROO) indicating a switchover to a new predominant interaction pathway, identified to proceed via intermediacy of MePc exciplexes and involving H-atom abstraction from ROOH by MePc-cation radicals. Further theoretical analysis is performed in terms of frontier molecular orbitals and thermodynamic calculations, with a special emphasis on the efficiency of MePc* - ROOR' CT within the frame of Marcus-Saveant theory for concerted (one-step) dissociative electron transfer. Consequences and implications of the above described MePc-mediated photochemistry in the presence of (hydro)peroxides during photodynamic cell inactivation will be discussed.

Keywords: phthalocyanine, (hydro)peroxide, alkoxyl (peroxyl) free radicals

Reference #: KAN-1017-335979
Submit Date: 03/28/2002 10:53:08-0500

Structural requirements for efficient cellular photoprotection by carotenoid derivatives.

ABSTRACT:
We have synthesized four carotenoid derivatives, GRP-carotenal from the reaction of Girard's Reagent P with -apo-8'-carotenal, GRT-carotenal from the reaction of Girard's Reagent T with -apo-8'-carotenal, GRT-canthaxanthin from the reaction of two moles of Girard's Reagent T with one mole of canthaxanthin, and dansyl-carotenal from the reaction of dansylhydrazine with -apo-8'-carotenal. The first two derivatives are cations, while the third is a dication and the fourth is a weak base. The subcellular distributions and the photoprotective properties of these derivatives were compared with two uncharged natural carotenoids, -carotene and -apo-8'-carotenal. Each carotenoid was incubated with K562 cells for 30 min. For measurements of the subcellular carotenoid distribution, the cells were disrupted and the post-nuclear supernatant fractionated using ultracentrifugation along a density gradient. The two natural carotenoids separated mainly with the cell fraction containing the cell membranes. The other carotenoid derivatives were more broadly distributed among the cell fractions. The three cationic derivatives had higher concentrations in the mitochondrial fractions than did the other derivatives. The distribution of dansyl-carotenal in the two most dense fractions was similar to that of lysosomes. For measurement of photoprotection, both carotenoid-labeled and control K562 cells were incubated with a photosensitizer (hypericin, protoporphyrin-IX or cis-di[4-sulfonatophenyl]diphenylporphine). The cells were then exposed to light with cell membrane integrity being used as a measure of cell damage. All of the cationic carotenoid derivatives were significantly better photoprotectors than dansyl-carotenal or the two natural carotenoids. These data support the hypothesis that potent singlet-oxygen quenchers that localize within critical cell organelles are more effective photoprotectors than quenchers that do not localize in these organelles.

Keywords: photoprotection, singlet oxygen, carotenoid derivatives

Reference #: GOY-1017-342563
Submit Date: 03/28/2002 12:55:03-0500

Subcellular photodynamics of porphyrins using Two-photon Excitation

ABSTRACT:
Photodynamic therapy (PDT) is a treatment modality finding use in the arsenal of cancer therapeutics, as well as skin and eye diseases. Two-photon excitation photodynamic therapy (TPE-PDT) is a treatment option whereby very small, highly defined treatment volumes can be generated. While the mechanics of PDT on the macroscale are well documented, there are several unanswered questions as to exactly how a photosensitizer works on a cellular level. This is of particular interest when considering TPE-PDT, as the two-photon excitation provides a means to treat tissue on a cell-by-cell basis. Of particular interest is the photosensitizer location within a cell and the conditions of the local environment. Data related to both of these variables can be measured, within the limits of optical microscopy, by direct observation of fluorescence from a single cell. Alternatively, one can observe the altered fluorescence behaviour of a photosensitizer in an unknown environment and compare the resultant fluorescent signature with those created under known conditions. The fluorescence behaviour of a photosensitizer provides an interesting window into the local environment, as the position of the fluorescence band, the lifetime of the sensitizer, and the photobleaching rate will all be affected by the local conditions. In this work, we present our forays into determining the subcellular location of photosensitizers using two-photon fluorescence spectroscopy.

Keywords: photodynamic therapy, two photon, porphyrin, fluorescence

Reference #: SIM-1017-693731
Submit Date: 04/01/2002 14:37:31-0500

Atomic Force Microscopy Studies of the Structural Morphology of Sepia Eumelanin

ABSTRACT:
Atomic force microscopy is used to investigate the structural organization of eumelanin isolated from the inks sacs of the cuttlefish Sepia officinalis. The natural eumelanin granules are found to be comprised of uniform-sized spherical structures with a diameter of ~150 nm. Mechanical manipulation of these structures using the AFM tip show that these particles, while stable, are not a fundamental structural unit but are aggregates of oligomeric constituents. The effect of iron addition or iron depletion on the melanin morphology is examined. The structural organization is found to vary with iron content. We found Fe-content in melanin correlates with the p-stacking of the melanin aggregates. Removal of Fe decreases the extension of the p-stacking. However, the addition of too much iron can destroy the natural spherical morphology. Without external mechanical forces, the natural morphology still remains in the Fe-depleted melanin. Therefore, -stacking is not required in maintaining the natural morphology of the ~150 nm spheres. Thus some other forces must maintain the spherical structures, possibly by melanin-protein interactions. These AFM imaging studies on the morphology changes of melanin correlate well with photoreactivity studies. These consistent results suggest a structure-function relationship for melanin exists. The switch of the function of melanin from protective to destructive upon metal-ion binding possibly results from changes in the aggregation morphology through melanin-metal complexation.

Keywords: melanin, atomic force microscopy

Reference #: VOG-1016-649758
Submit Date: 03/20/2002 12:11:07-0500

Chloroplast movement in Alocasia does not shorten gas diffusion pathlength as measured by photoacoustics

ABSTRACT:
Light-mediated chloroplast movements are common throughout the plant kingdom. In leaves of Alocasia macrorrhiza (L.), when dim light is normal to the leaf surface, chloroplasts spread along periclinal walls, absorbing light maximally. Under high-light, chloroplasts move to anticlinal walls. One hypothesis concerning the adaptive advantage of movement to the high-light position is that it might in some way shorten the diffusion path for CO₂, thus reducing CO₂ limitation of photosynthesis. To test this hypothesis, we used oxygen diffusion as a proxy for CO₂ diffusion, and employed pulsed photoacoustics to measure lag times for oxygen diffusion out of leaf tissues. By coupling pulsed photoacoustic measurements with measurements of the diffusion distance from chloroplasts to the intercellular space made with a microscope, we calculated an overall diffusion coefficient for oxygen of 5.5 x 10^-6 cm² s^-1, a value that is notably lower than for water (2.1 x 10^-5 cm² s^-1). We also estimated the corresponding viscosity of the diffusion medium, primarily the chloroplast, of 3.9 mPa s, which is higher than for water (1.0 mPa s). When temperature was increased from 5 to 40°C, the diffusion coefficient for oxygen increased while the viscosity decreased with most change occurring between 5 and 20°C. These data are consistent with nonlinear effects of temperature upon the physical properties of the chloroplast. There was no evidence that chloroplast movement to the high light position enhanced gas diffusion. When leaves were pre-treated with white light to induce chloroplast movement, lag times for oxygen diffusion did not shorten compared to leaves pre-treated with yellow light, which did not induce movement.

Keywords: Alocasia macrorrhiza, chloroplast movement, pulsed photoacoustics, oxygen diffusion

Reference #: SON-1016-519661
Submit Date: 03/19/2002 00:05:21-0500

Inter-domain crosstalks within the phytochrome A protein: Functional implications

ABSTRACT:
Phytochromes (phy) are plant's red/far-red light-responding photoreceptor for photomorphogenesis. The phy molecules are bifunctional with a two-domain structure consisted of the N-terminal photosensory and the C-terminal regulatory domains. The light-induced Pr-Pfr phototransformation triggers subtle conformation changes in the apoprotein. The "light signals" thus stored are subsequently transmitted to the regulatory motifs of the C-terminal domain through as yet ill-defined inter-domain crosstalks. The corssltalks elicit activation of the C-terminal domain to prepare its interactions with phy-interacting proteins such as NDPK2, PIF3, PKS1, etc. Phytochrome phosphorylation/dephosphorylation may serve as a molecular switch to modulate the inter-domain crosstalks. For example, phosphorylation of the Pfr-phy attenuates its regulatory activity. Finally, biotechnological implications of the phy structure-function will be discussed.

Keywords: photomorphogenesis, phytochrome, plants, inter-domain crosstalk