EXCISION REPAIR OF PYRIMIDINE DIMERS IN MARSUPIAL CELLS

Monodelphis domestica was further characterized as a model for photobiological studies by measuring the excision repair capabilities of this mammal's cells both in vivo and in vitro. Excision repair capability of the established marsupial cell line, Pt K2 ( Potorous tridactylus ), was also determined. In animals held in the dark, we observed that ˜50% of the dimers were removed by 12 and 15 h after irradiation with 400 J m⁻² and 600 J m⁻², respectively, from an FS-40 sunlamp (280–400 nm). Cells from primary cultures of M. domestica excised ˜50% of the dimers by 24 h after irradiating with 50 J m⁻² and 36 h after exposure to 100 J m⁻² with no loss of dimers observed 24 h following a fluence of 300 J m⁻². Pt K2 cells were observed to have removed -50% of the dimers at -12 h after 50 J m⁻² with only -10% of the dimers removed at 24 h following 300 J m⁻². The observed loss of pyrimidine dimers from epidermal DNA of UV-irradiated animals and from fibroblasts in culture, held in the dark, suggests that these marsupial cells are capable of DNA excision repair.     

In situ PYRIMIDINE DIMER DETERMINATION BY LASER CYTOMETRY

By using antiserum against pyrimidine dimers and argon-laser imaging microspectrofluorometry, we established a new method to determine UV-induced pyrimidine dimers and their repair in individual human cells. The method was sensitive enough to determine dimers induced by UV dose as low as 2 J/m2. Normal human cells repaired 50 and 60% of total damage within 8 and 24 h after UV irradiation (20 J/m2), but Xeroderma pigmentosum cells (complementation group A) were unable to repair any within the same period. Therefore, the method proved to be a quick, easy, sensitive and accurate means to determine pyrimidine dimers in situ.     

SKH-1 hairless mice repair UV-induced pyrimidine dimers in epidermal DNA

The SKH-1 hairless mouse strain has been used extensively as a model for human photocarcinogenesis, photoimmunology and photoaging, but little is known about DNA repair in living mouse skin. Mice were irradiated with UV-B light at doses which produce mild to severe sunburn, and the frequency of pyrimidine dimers in epidermal DNA was measured immediately and 6 h after irradiation using T4 endonuclease V treatment and alkaline agarose gel electrophoresis. The results demonstrate significant removal of pyrimidine dimers in mouse skin in vivo, with a dimer half-life of 7.4 h. These findings are similar to the repair of dimers in human skin in vivo. The SKH-1 hairless mouse is thus a useful model for pyrimidine dimer repair in human skin.     

Dimeric Cyclodextrin Carriers with High Binding Affinity to Porphyrinoid Photosensitizers

The aim of our investigation was to develop carrier systems for an application of inert drugs in photodynamic cancer therapy. -Cyclodextrin dimers linked at their primary and secondary faces by spacers of varying lengths were synthesized as carrier systems. The binding constants of the inclusion complexes of these cyclodextrin dimers and porphyrinoid photosensitizers were determined by competitive spectrofluorometry. Particularly the secondary face linked dimers exhibited extremely high binding constants with values of 106-107 L/mol. Theoretical studies were carried out on these inclusion complexes to confirm the influence of spacer length and connecting side on complex stability.     

DNA DAMAGE AND REPAIR: INDUCTION AND REMOVAL OF THYMINE DIMERS IN ULTRAVIOLET LIGHT IRRADIATED INTACT WATER PLANTS

Abstract— The production of UV-induced thymine dimers and their fate upon post-irradiation incubation in the dark was studied in DNA of the intact water plants Wolffia microscopica and Spirodela polyrhiza. The results demonstrate production of thymine dimers, and the ability of the plant cells to remove the dimers from their DNA. The rate of removal is rapid during the first few h of post-irradiation incubation in the dark. It continues at a slower rate for the next 24–48 h, at which time it is essentially complete. The disappearance of thymine dimers in light or in the dark is analogous to the well-known processes in other biological systems, namely, photoreactivation and dark excision.     

Intermolecular 2hJNN coupling in multiply hydrogen-bonded ureidopyrimidinone dimers in solution

15N-Labeled ureido-4[1H]-pyrimidinones 4a and 5a were synthesized in order to investigate hydrogen bonding in the strongly hydrogen-bonded dimers in solution with intermolecular (2h)J(NN) coupling. Both direct-detection (15)N NMR and one-dimensional (15)N INADEQUATE (for smaller scalar coupling constants) were employed to determine the coupling constants. For dimers of 4 in CDCl(3), a temperature-dependent (2h)J(NN) was observed ranging from 2 Hz at +10 degrees C to 5.1 Hz at -20 degrees C. In dimers of more slowly exchanging bifunctional derivative 5, the coupling constants could be determined at room temperature from an inverse-gated (1)H-decoupled (15)N NMR experiment. Coupling constants in different isomers of the dimer of 5a (4.96, 5.13, 4.37, and 5.27 Hz) were used to test the relationship between (2h)J(NN) values and N-N distances as proposed by Del Bene et al. The N-N distances calculated with the aid of this relationship show excellent agreement with the distances observed in the X-ray crystal structures of 5b, particularly when the nonlinearity of the hydrogen bonds is taken into account.     

MICELLAR EFFECTS ON URACIL PHOTODIMERIZATION

Abstract —Initial quantum yields for the formation of all four uracil dimers, anti h, t ( I ); anti h, h ( II ); syn h, h ( III ) and syn h, t ( IV ), have been quantitatively determined in aqueous degassed and air-saturated solutions as well as in degassed and air-saturated solutions of micellar sodium dodecyl sulfate (NaLS) and hexadecyltrimethylammonium bromide (CTAB). In aqueous solutions the order of dimer yields is IV > III > II > I . Percentages of dimer III decreases from 18-9 per cent in water to 2–7 per cent in micellar CTAB and to 6.0 per cent in micellar NaLS, but those for dimers I and II increase in the micellar solutions.     

REPAIR OF CYCLOBUTANE DIMERS AND (6–4) PHOTOPRODUCTS IN ICR 2A FROG CELLS

Abstract— The removal of cyclobutane dimers and Pyr(6–4)Pyo photoproducts from the DNA of UV-irradiated ICR 2A frog cells was determined by radioimmunoassay. In the absence of photoreactivat-ing light, 15% of the cyclobutane dimers and 60% of the (6–4) photoproducts were removed 24 h post-irradiation with 10 J m⁻², Exposure to 30 kJ m⁻² photoreactivating light resulted in removal of 80% of the cyclobutane dimers and an enhanced rate of repair of (6–4) photoproducts, resulting in a loss of 50% of these lesions in 3 h. The preferential removal of (6–4) photoproducts by excision repair resembles previously published data for mammalian cells.     

Characterization of caffeic acid enzymatic oxidation by-products by liquid chromatography coupled to electrospray ionization tandem mass spectrometry

The products of tyrosinase-catalyzed caffeic acid oxidation at pH 6.5 were investigated using high-performance liquid chromatography (HPLC) coupled to electrospray ionization mass spectrometry (ESI-MS). Aliquots of reaction mixtures were withdrawn at different times, varying from 0 to 24 h, and directly analysed by HPLC-ESI-MS and, in the case of 1 and 5 h, by HPLC-ESI-MS/MS to obtain structural information on caffeic acid derivatives. In particular, two different classes of caffeic acid dimers were identified: caffeicins-like structures and dimers originated by CC coupling between the benzene rings. Evidences for the presence of trimeric derivatives of caffeic acid were also obtained from MS data.     

NATURE OF THE INTERACTION BETWEEN THE PHOTOACTIVE COMPOUND PHENYLHEPTATRIYNE AND ANIMAL VIRUSES

Abstract— The levels of DNA excision repair, as measured by unscheduled DNA synthesis (UDS) and the UV-endonuclease sensitive site assay, were compared in cells derived from human fetal brain and dermal tissues. The level of UDS induced following ultraviolet (UV) irradiation was found to be lower (approx. 60%) in the fetal brain cells than in fetal dermal cells. It was determined, using the UV-endonuclease sensitive site assay to confirm the UDS observation, that 50% of the dimers induced by UV in fetal dermal cells were repaired in 8h, while only 15% were removed in the fetal brain cells during the same period of time. Even after 24 h, only 44% of the dimers induced by UV in the fetal brain cells were repaired, while 65% were removed in the dermal cells. These data suggest that cultured human fetal brain cells exhibit lower levels of excision repair compared to cultured human fetal dermal cells.     

REPAIR OF PYRIMIDINE DIMERS IN ULTRAVIOLET-IRRADIATED CHLAM YDOMONAS

Abstract— A UV-specific endonuclease was used to monitor the presence of UV-induced pyrimidine dimers in the DNA of Chlamydomonas reinhardi . All of the dimers induced by 50 J/m² of 254 nm light are removed by a 2 h exposure to photoreactivating light. Nearly all of the dimers are removed by the wild-type strain of Chlamydomonas upon incubation for 24h in the dark. Two UV-sensitive mutants, UVS 1 and UVS 6, are deficient in removal of dimers in the dark. These results are interpreted to mean that Chlamydomonas has an excision-repair pathway for coping with UV-induced damage.     

ENHANCEMENT OF PHOTOREPAIR OF ULTRAVIOLET-DAMAGE BY PREILLUMINATION WITH FLUORESCENT LIGHT IN CULTURED FISH CELLS

Fluorescent light (FL) illumination of RBCF-1 cells, derived from a goldfish, prior to 254 nm UV-irradiation enhanced their ability to photorepair. The cells were illuminated with FL for 1 h (29 W/M2) and incubated for 8 h in the dark before being irradiated with 10 J/m2 UV. The surviving fraction of FL-treated cells after UV-irradiation rose about 7-fold (from 3 to 20%) by 20 min photorepair treatment with the same FL source, whereas 4-fold (from 1.6 to 6%) in the FL non-treated cells. Flow cytometric analysis showed that FL treatment did not affect the distribution of cell cycle phase at the time of UV-irradiation (8 h after FL treatment). Pyrimidine dimers induced by UV were measured by the use of UV endonuclease of Micrococcus luteus and alkaline agarose gel electrophoresis. Initial yields of dimers after exposure to 10 J/m2 UV were almost the same (about 0.11 dimer/kb) between FL treated and non-treated cells. But after 20 min photorepair treatment, about 70% of dimers were removed in the FL treated samples, while less than 20% were removed in the non FL-treated ones.     

DETECTION OF CYCLOBUTANE THYMINE DIMERS IN DNA OF HUMAN CELLS WITH MONOCLONAL ANTIBODIES RAISED AGAINST A THYMINE DIMER- CONTAINING TETRANUCLEOTIDE

Abstract— A hybrid cell line (hybridoma) has been isolated after fusion between mouse-plasmacytoma cells and spleen cells from mice immunized with a thymine dimer-containing tetranucleotide coupled to a carrier protein. Monoclonal antibodies produced by this hybridoma were characterized by testing the effect of various inhibitors in a competitive enzyme-linked immunosorbent assay (ELISA). The antibodies have a high specificity for thymine dimers in single-stranded DNA or poly(dT), but do not bind UV-irradiated d(TpC)₅. Less binding is observed with short thymine dimer-containing sequences. In vitro treatment of UV-irradiated DNA with photoreactivating enzyme in the presence of light, or with Micrococcus luteus UV-endonuclease results in disappearance of antigenicity. Antibody-binding to DNA isolated from UV-irradiated human fibroblasts (at 254 nm) is linear with dose. Removal of thymine dimers in these cells during a post-irradiation incubation, as detected with the antibodies, is fast initially but the rate rapidly decreases (about 50% residual dimers at 20 h after 10 J/m²). The induction of thymine dimers in human skin irradiated with low doses of UV-B, too, was demonstrated immunochemically, by ELISA as well as by quantitative immunofluorescence microscopy.     

PHOTOREACTIVATION and EXCISION REPAIR OF UV INDUCED PYRIMIDINE DIMERS IN THE UNICELLULAR CYANOBACTERIUM GLOEOCAPSA ALPICOLA (SYNECHOCYSTIS PCC 6308)

Abstract— The survival curve obtained after UV irradiation of the unicellular cyanobacterium Synecho-cystis is typical of a DNA repair competent organism. Inhibition of DNA replication, by incubating cells in the dark, increased resistance to the lethal effects of UV at higher fluences. Exposure of irradiated cells to near ultraviolet light(350–500 nm) restored viability to pre-irradiation levels. In order to measure DNA repair activity, techniques have been developed for the chromatographic analysis of pyrimidine dimers in Synechocystis. The specificity of this method was established using a haploid strain of Sacchar-omyces cerevisiae. In accordance with the physiological responses of irradiated cells to photoreactivating light, pyrimidine dimers were not detected after photoreactivation treatment. Incubation of irradiated cells under non-photoreactivating growth conditions for 15 h resulted in complete removal of pyrimidine dimers. It is concluded that Synechocystis contains photoreactivation and excision repair systems for the removal of pyrimidine dimers.     

EXCISION REPAIR OF UVR-INDUCED PYRIMIDINE DIMERS IN CORNEAL DNA

Abstract— We measured excision repair of ultraviolet radiation (UVR)-induced pyrimidine dimers in DNA of the corneal epithelium of the marsupial, Monodelphis domestica , using damage-specific nucleases from Micrococcus luteus in conjunction with agarose gel electrophoresis. We observed that 100 J ^-2 of UVR from aFS–40 sunlamp(280–400 nm) induced an average of 2.2 ± 0.2 times 10^-2 endonuclease-sensitive sites per kilobase (ESS/kb) (pyrimidine dimers) and that ∼ 50% of the dimers were repaired within 12 h after exposure. We also determined that an exposure of 400 J m^-2 was needed to induce comparable numbers of pyrimidine dimers (2.5 times 10^-2) in the DNA of skin of M. domestica in vivo . In addition, we found that 50% of the dimers were also removed from the epidermal cells of M. domestica within 12 h after exposure. A dose of 100 J m^-2 was necessary to induce similar levels of pyrimidine dimers (2.0 ± 0.2 times 10^-2) in the DNA of the cultured marsupial cell line Pt K2 ( Potorous tridactylus ).     

Synthesis and characterisation of thermotropic liquid-crystalline properties of azomethine dimers

A series of azomethine dimers were prepared by condensation reactions of benzaldehyde, biphenylcarboxaldehyde and 9-anthraldehyde with various aromatic diamines of varying flexibility in ethanol in the presence of tosic acid. Their chemical structures were determined by Fourier transform infrared and ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopies, as well as elemental analysis. Their thermal properties were also examined by using a number of experimental techniques, including differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), polarising optical microscopy (POM) and variable temperature X-ray diffraction (VTXRD). Azomethine dimer, prepared from benzaldehyde and 1,9-bis(4-aminophenoxy)nonane, exhibited a monotropic, nematic liquid-crystalline (LC) phase. The majority of the azomethine dimers containing biphenyl moieties exhibited enantiotropic, nematic LC phase on melting at relatively low temperatures, since they developed typical Schlieren, threaded or marbled textures in their LC phase. They also had accessible isotropisation temperatures well below their decomposition temperatures. Azomethine dimers containing anthracene moieties did not exhibit LC properties, but exhibited polymorphism as determined by POM and VTXRD in two cases. All of these azomethine dimers in the series had excellent thermal stability that was in the broad range of temperatures of 307–400°C depending on their degrees of aromaticity index.     

PHOTOREVERSIBILITY OF UV-INDUCED THYMINE DIMERS AND ABNORMAL MORPHOGENESIS IN SEA-URCHIN EMBRYOS

Abstract— Irradiation of synchronously dividing 16-cell embryos of a sea-urchin ( Hemicentrotus pul-cherrimus ) with 200 J m⁻² of UV light (254 nm) resulted in the complete inhibition of normal pluteus-larva formation when the embryos were cultured in the dark after UV-irradiation. Illumination of the UV-irradiated embryos with visible light (11 W m⁻²) for 1 h immediately after the UV-irradiation reversed the abnormal morphogenesis. Measurement of thymine dimers indicates that the degree of UV-induced abnormal morphogenesis is greatly correlated with the amount of thymine dimers in the DNA of the embryos. The degree of the photoreversal decreased with an increase in the interval between UV-irradiation and exposure to visible light. Visible light was ineffective as to the reversibility of both thymine dimers and the abnormal morphogenesis at 60 min after the UV-irradiation, when the UV-irradiated 16-cell embryos entered the next cell cycle.     

Correlation of photoreactivation of ultraviolet-induced killing with dimer splitting before and after DNA replication in an excisionless strain of Escherichia coli

Abstract— Splitting of thymine-containing dimers was compared quantitatively with photoreactivation (PR) of killing induced by ultraviolet radiation (254 nm) in a uvrA (excisionless) strain of E. coli. Immediately after irradiation, the splitting rate (number of dimers split/genome/unit PR dose) agreed well with the PR rate of the cells (rate of recovery from photoreactivable lethal damage converted into an ‘estimated’ number of dimers split/genome/unit PR dose). After 4 h of incubation of cells in nutrient medium, the maximal fraction of splittable dimers decreased, as did the maximal fraction of photoreactivable lethal damage. However, the initial splitting rate after incubation was equal to that before incubation. During the 4-h incubation, the heavily irradiated uvrA cells did not divide but became filamentous and their DNA increased about 70 per cent. It is concluded that roughly half of the dimers in DNA that has replicated after ultraviolet irradiation are split as efficiently as those in DNA that has not replicated.     

Structure and thermodynamics of alpha-, beta-, and gamma-cyclodextrin dimers. Molecular dynamics studies of the solvent effect and free binding energies

The alpha-, beta-, and gamma-cyclodextrin (CyDs) dimers were studied by molecular dynamics (MD) simulations in water as an explicit solvent. The relative stability of dimers and the involved molecular interactions were determined. Three possible starting orientations were considered for the dimers: head-to-head, head-to-tail, and tail-to-tail. MD simulations were performed over a period of 5 ns to ensure the stability of the system for both the CyD dimers and monomers. The MM-PBSA methodology was used to obtain the free binding energy of the dimers and to determine the most stable arrangement for each solvated CyD. In a vacuum, MD simulations provided the head-to-head orientation as the most stable orientation for the three CyDs, while in aqueous solution the, the head-to-tail orientation was found to be the most stable for the alpha-CyD dimer and the tail-to-tail orientation the most stable for the beta- and gamma-CyD dimers.