SINGLE-STRAND DNA BREAKS INDUCED BY 365 NM RADIATION IN ESCHERICHIA COLI STRAINS DIFFERING IN SENSITIVITY TO NEAR and FAR UV

The gene mutation nur has been shown specifically to sensitize Escherichia coli stationary phase cells to inactivation by broad spectrum near-UV (NUV) radiation. In the work reported here, E. coli strains RT1. RT2, RT3, and RT4, carrying the 4 possible combinations of recA1, recA⁺, nur , and nur⁺ , were exposed to monochromatic NUV (365 nm). The strains carrying the nur allele (RT1 and RT2) were more sensitive to inactivation by this wavelength and exhibited considerably more single strand break's (SSB's) than the strains carrying the nur⁺ allele (RT3 and RT4). As predicted, following X-irradiation the strains carrying the recA1 allele (RT1 and RT3) were more sensitive than the recA⁺ strains (RT2 and RT4). We conclude that the enhanced SSB's observed in strains RT1 and RT2 following monochromatic NUV irradiation correlated with the nur mutation and are unrelated to the recA1 mutation.     

HYDROGEN PEROXIDE INDUCED RESISTANCE TO BROAD-SPECTRUM NEAR-ULTRAVIOLET LIGHT(300–400 nm) INACTIVATION IN Escherichia coli

Abstract— Strains of Escherichia coli carrying the four possible combinations of the alleles nur, nur⁺, uvrAb, and uvrA ⁺ were either untreated or pretreated with a sublethal dose of H₂0₂ prior to inactivation with NUV radiation. Pretreated cells exhibited a greater resistance to NUV than did untreated cells. Pretreatment with H₂O₂ did not induce resistance to FUV radiation. The induction of resistance to NUV inactivation by H₂O₂ pretreatment apparently leads to protection against the damage caused by NUV radiation. Although pretreatment of cells with H₂0₂ leads to resistance of such cells to inactivation by H₂O₂ and NUV, survival of H₂O₂ treated bacteriophage PI cml clr100 is not enhanced when assayed on H₂O₂ pretreated E. coli host cells.     

THE INTERACTION OF A GENE (nur) CONTROLLING NEAR-UV SENSITIVITY AND THE polA1 GENE IN STRAINS OF E. COLI K12

Abstract— When stationary cell populations of the Escherichia coli W3110 strain and the polA1 mutant (p3478) derived directly from it were compared for their sensitivity to near-UV (NUV, 300–400 nm) inactivation, the polA1 strain proved to be more sensitive. By appropriate matings and transductions, four essentially isogenic strains have been developed which carry all four possible combinations of genes conferring far-UV (FUV, 200-300 nm) sensitivity ( polA1 vs. polA ⁺) and NUV sensitivity ( nur vs. nur ⁺). Stationary cells of strains carrying either the polA1 or polA ⁺ allele in combination with the nur allele are indistinguishable in their sensitivity to NUV inactivation and are equivalent in their NUV sensitivity to the original polA1 mutant strain (p3478). With the two strains carrying the nur ⁺ allele, stationary cell populations of the polA1 strain are clearly more sensitive to NUV inactivation than is the polA ⁺ strain. The NUV sensitizing effect of the polA1 mutation in a nur ⁺ genetic background is about the same as that of the nur mutation at the 0.37 survival level. This may mean that the polA1 and nur mutations sensitize E. coli stationary cell populations to NUV inactivation by a common mechanism.     

THE PHOTOTOXICITY OF PHENYLHEPTATRIYNE: OXYGEN-DEPENDENT HEMOLYSIS OF HUMAN ERYTHROCYTES AND INACTIVATION OF Escherichia coli

Abstract— Phenylheptatriyne (PHT) plus near-ultraviolet light(320–400 nm; NUV) hemolyzed human erythrocytes in an oxygen dependent manner. When the phototoxicity of PHT plus NUV was tested with a series of Escherichia coli strains carrying all four possible combinations of genes controlling excision proficiency ( uvrA6 vs uvrA ⁺) and catalase activity (HPII, katF vs katF *), the membrane was found to be an important lethal target. Consistent with this observation. PHT plus NUV did not induce histidine independent ( his-4 ⁺) mutations in the four tester strains (RT7h-RT10h). Using tester strain RT10h, it was shown that there was no inactivation by PHT plus NUV in nitrogen. Results of experiments with an E. coli fatty acid auxotroph (K1060) treated with PHT plus NUV are also consistent with membrane proteins being the chief targets for attack. Radicals were formed during the photolysis of PHT plus NUV in aqueous solutions, both in the presence of air and under nitrogen. Since PHT plus NUV did not hemolyze erythrocytes or inactivate E. coli cells under nitrogen, these radicals are not cytotoxic.     

INTERACTIONS AMONG PHOTOSYNTHETIC ANTENNA EXCITED STATES

Abstract. The data of Kung and DeVault (1978) showing high-order fluorescence from chromatophores of photosynthetic bacteria are analyzed in relation to other data on first-order fluorescence of photosynthetic systems, particularly that of Monger and Parson (1977). The wavelengths of emission observed (down to 445 nm) require energy equivalent to two lowest singlet-excited states. The dependence on excitation intensity is best explained by any of the following third-order processes: (a) 3 S ₁→3 S ₀; (b) 2 S ₁+ T , → 2 S ₀+ T ₁; (c) S ₁+ 2 T ₁→ 3 S ₀. However, (c) is ruled out because it predicts heavy T ₁-destruction which is not observed. Contribution from the second order process: 2 S ₁→ S ₀ is probable, but even the data of Monger and Parson show that it is insufficient by itself. Two-photon absorption: S ₀+ hv ₁→ S ₁; S ₁+ hv ₁→ S _n; S _n S ₀+ hv ₂ could also account for the high-order fluorescence and its dependence on excitation intensity. [ S ₀, S ₁ S _n are ground, first excited and a higher excited singlet states, respectively, of antenna bacteriochlorophyll, T _t is the lowest triplet state, c/v , is the exciting wavelength (694 or 868 nm) and c/v ₂ the wavelength of the high-order fluorescence (445, 535. or 600 nm), where c = velocity of light.] Maximum values are estimated for some of the rate constants.     

SENSITIVITY OF HemA MUTANT Escherichia coli CELLS TO INACTIVATION BY NEAR-UV LIGHT DEPENDS ON THE LEVEL OF SUPPLEMENTATION WITH δ-AMINOLEVULINIC ACID

Abstract— Four strains carrying all four possible combinations of the alleles nur, nur⁺, uvr A6 and uvr A ⁺ were transduced to hemA8 . The hemA8 mutation blocks the synthesis of δ-aminolevulinic acid (δ-ALA), one of the first steps in the synthesis of porphyrin and, ultimately, cytochromes essential for aerobic respiration. The cells were grown either with or without δ-ALA and treated with broad-spectrum near-ultraviolet light (NUV; 300–400 nm). hemA8 defective cells grown without δ-ALA were resistant to inactivation by NUV while hemA8 cells were sensitive to such inactivation when supplemented with δ-ALA. The sensitivity to NUV inactivation conferred by the nur gene was retained in the hemA8 derivatives. The sensitivity of such cells to NUV inactivation can be controlled by varying the level of δ-ALA supplementation. The level of δ-ALA supplementation did not influence the sensitivity of the cells to inactivation by far-UV light (FUV; 200–300 nm). The near-UV sensitivity of hemA⁺ cells was not significantly altered when grown with δ-ALA suppiementation suggesting that endogenously formed δ-ALA supports the normal, regulated level of porphyrin synthesis. These results can be interpreted to mean that porphyrin components of the respiratory chain in E. coli represent chromophores involved specifically in broad-spectrum NUV inactivating events.     

Role of Enterobactin and Intracellular Iron in Cell Lethality During Near-UV Irradiation in Escherichia coli

Abstract— In Escherichia coli, fur mutants that constitutively express their native iron chelating agent, enterobactin, are significantly more sensitive to near-UV radiation (NUV) than wild type. An entA mutant, which is incapable of synthesizing enterobactin, is equal to wild type in resistance to NUV irradiation. However, the addition of Fe⁺³ enterobactin but not Al⁺¹ enterobactin to entA cell suspensions just prior to irradiation results in an increased sensitivity to NUV irradiation. A fes mutant, which is unable to reduce and release iron from enterobactin, is significantly more sensitive to NUV irradiation than wild type. The addition of nontoxic levels of H₂O₂ (5 μ M ) just prior to irradiation significantly increases sensitivity of both fur and fes mutants. These results suggest that one mechanism by which NUV irradiation leads to cell lethality is by creating a transient iron overload, producing very favorable conditions for the production of highly deleterious free radicals through a variety of mechanisms that lead to oxidative stress and DNA damage including lethal and mutagenic lesions. These results are consistent with the hypothesis that enterobactin is an endogenous chromophore for NUV and contributes to cell lethality via the destruction of its ligand, releasing Fe⁺² into the cytoplasm to catalyze the production of highly reactive hydroxyl radicals and other toxic oxygen species via the Haber-Weiss reaction.     

INVESTIGATION OF DAMAGE TO FOREST BY EPR SPECTROSCOPY in vivo

Spruce needles collected from several trees of the Black Forest were investigaled by EPR spectroscopy. These needles show in the g = 2.00 region a signal II_S(Tyr D ₊) and a light-induced signal I(P700₊) and a Mn₂₊ hyperfine structure which superimposes the other absorptions. Difference spectra, light minus dark, partly eliminate the manganese hyperfine structure, and P700₊ can be observed. By comparison of these EPR signals with those of spinach chloroplast or thylakoid membranes described in the literature, significant deviations were observed, whereas several trees grown in the vicinity of Tubingen exhibit the well known D₊ and P700₊ EPR spectra. After treatment of branches of these 'normal' trees with herbicides like Amitrol and Roundup or chemicals like toluene or trichlormethane the EPR signals obtained are comparable with those observed with needles of the Black Forest.     

CHEMILUMINESCENT REACTIONS OF SO

Abstract— The rapid bimolecular reaction SO + O₃= SO₂+O₂+ 106 kcal/mole
yields electronically excited SO₂ in the ³B ₁ and ¹B₂ states with some vibrational excitation, as well as SO₂ in its electronic ground state. It is shown that k₁ = 1.5 x 10¹² exp (-2100/ RT ) cm² mole^-1 s-¹ and that the formation of electronically excited SO₂ involves higher activation energies.     

INACTIVATION OF PHAGE BY NEAR-ULTRAVIOLET RADIATION AND HYDROGEN PEROXIDE

A series of phage with different genomes (both single-stranded and double-stranded RNA and DNA) was inactivated with hydrogen peroxide (H₂O₂) in various combinations with far-ultraviolet (FUV) and near-ultraviolet (NUV) radiations. In every case but one (a lipid-coated phage), a sublethal H₂O₂ concentration greatly enhanced killing by NUV but not FUV. Moreover, this NUV/H₂O₂ synergism was oxygen independent and there was little if any host cell reactivation upon NUV plus H₂O₂ inactivation. These results suggest that these phage are inactivated by a common mechanism irrespective of nucleic acid composition, but that some phage genomes may be more vulnerable to NUV/H₂O₂ inactivation than others.     

PROTECTION OF Ustilago violacea FROM TOLUIDINE BLUE PHOTOSENSITIZATION and HYDROGEN PEROXIDE INDUCED KILLING and MITOTIC RECOMBINATION BY CAROTENES

Abstract— The accumulation of (J-carotene in the ph/ph ⁺ y diploid strain of the smut fungus Ustilago violacea was associated with reduced killing and lower levels of induced mitotic recombination compared to the β-carotene lacking ph/ph⁺ w strain in response to both incandescent photosensitization and treatment with H₂0₂. The ph/ph⁺ y strain was only slightly more resistant to killing by exogenous toluidine blue (TB) photosensitization. The ph/ph⁺ y strain exhibited significantly greater levels of survival when exposed to incandescent radiation and 1.5 μ.M TB for 15 min, as well as 3.0. 0.3, 0.03, 0.003% H₂0₂ in the dark. The ph/ph⁺ y strain also exhibited lower levels of mitotic recombination after endogenous TB photosensitization and the latter two H₂0₂ treatments. Similar survival results were obtained for the carotene accumulating haploid strain l.C2y and the carotene lacking haploid strain l.C2iv in response to H₂0₂ exposure.     

OXYGEN-DEPENDENT PHOTOSENSITIVITY and MITOTIC RECOMBINATION IN THE SMUT FUNGUS Ustilago violacea

Abstract— The effect of high intensity incandescent radiation on survival and mitotic recombination in the phlph + diploid strain of Ustilago violacea was studied with and without atmospheric O₂. In the presence of atmospheric O₂, strain phlph+ was characterized by photokilling to approximately 50% survival, and induction of mitotic recombination to about 60% by 90 min of light exposure. No photokilling and little induction of mitotic recombination were observed when light exposure was carried out in an 0₂ depleted environment. Photokilling and photo-induced mitotic recombination in U. violacea may be due to DNA damage or repair in response to a photosensitized reaction, involving an endogeneous photosensitizer.     

SOLID PHASE PHOTOREDUCTION OF METHYLVIOLOGEN ON CELLULOSE SENSITIZED BY TRIS(2,2'-BIPYRIDYL)RUTHENIUM COMPLEX

–Methylviologen (MV²⁺) adsorbed on cellulose could be reduced photochemically in the solid phase sensitized by tris(2,2'-bipyridyl)ruthenium(II) complex, [Ru(II)(bpy)₃], using disodium ethylene-diaminetetraacetic acid (Na₂EDTA) as a reducing agent. Formation of the cation radical (MV ⁺.) was confirmed by visible and EPR spectra. The MV⁺. formed on cellulose was remarkably stable against air oxidation and rapidly accumulated even by the irradiation under air. Water adsorbed on the cellulose greatly retarded the photoreaction. Action spectrum showed that the excitation of Ru(II)(byp)₃ is responsible for the photochemical reaction. The quenching of the emission from Ru(II)(bpy)*₃ by MV²⁺. indicated that a primary photochemical reaction occurs between Ru(II)(bpy)*₃ and MV²⁺. The main reaction path is the reduction of MV²⁺ by Ru(II)(bpy)₃, giving MV⁺. and Ru(III)(bpy)₃, followed by the reduction of Ru(III)(bpy)₃ to Ru(II)(bpy)₃ with Na₂EDTA, which in turn is oxidized to give carbon dioxide.     

THE EFFECTS OF PHOTOACTIVATED PROTOPORPHYRIN ON RETICULOCYTE MEMBRANES, INTRACELLULAR ACTIVITIES and HEMOGLOBIN PRECIPITATION

Abstract— Photoactivated protoporphyrin effects were studied on reticulocyte membranes and distinct intracellular activities. Membrane bound (Na ₊ -K ₊)-ATPase activity and incorporation of ₅₅Fe into heme were almost 80% inhibited at a low concentration of protoporphyrin (3 fiM). On the other hand, a much higher protoporphyrin concentration (15 nM) was needed to cause 80% inhibition of protein synthesis. By 15 JXM protoporphyrin and treatment with light, an initial leak of hemoglobin out of the cells was observed. Electron microscopic examination showed that the lytic effects seem to be a result of membrane damage which appeared as holes in the membrane. Heinz-body-like particles of condensed hemoglobin were observed in the protoporphyrin-treated cells. The condensed hemoglobin spheres were shown to be bound to disrupted membranes prepared from photoactivated protoporphyrin-treated reticulocytes     

PHOTOBIOLOGICAL ACTIVITY OF MARMESIN (5-B-HYDROXYISOPROPYL-4–5 DIHYDROFUROCOUMARIN) IN CHINESE HAMSTER V79 CELLS

Abstract— Marmesin was isolated from the medicinal plant, Afraegle paniculata. Its cytotoxicity and mutagenicity in Chinese hamster V79 cells when sensitized to near ultraviolet (NUV) and long wavelength ultraviolet light or black light (BL) were assayed.
Marmesin was extremely cytotoxic in the dark. This cytotoxicity was photoenhanced in NUV and BL; the photoenhanced lethality being higher in NUV than in BL. The LD₅₀ of marmesin under NUV and BL photosensitization were 0.002 μ M and (0.012 μ M ), respectively. In the absence of NUV and BL, marmesin's LD₅₀ was 0.013 μ M . NUV and BL without marmesin were not significantly cytotoxic at the fluence rates of 0.29 W/m² and 4.2 W/m², respectively, for up to 20 min. In contrast to the observed high cytotoxicity of marmesin, its mutagenicity at the HGPRT locus (A_zG^r) was weak. The implication of this result in the high incidence of skin cancer in Nigeria in which A. paniculata is used as a medicinal plant is discussed.     

GENETIC CONTROL OF NEAR-UV (300–400nm) SENSITIVITY INDEPENDENT OF THE recA GENE IN STRAINS OF ESCHERICHIA COLI K12

Abstract— Stationary cells of isogenic pairs of Escherichia coli K12 strains presumably differing only in the recA function have been inactivated with near-UV (300–400 nm) radiation. Based on near-UV inactivation kinetics, the strains can be divided into two discrete categories in which near-UV sensitivity does not necessarily correlate with far-UV sensitivity conferred by two different recA alleles. Lack of overlap between near-UV and far-UV ( recA ) sensitivity can be explained hy assuming that a different chromosomal gene ( nur ) controls near-UV sensitivity. Support for this hypothesis comes from a mating experiment in which four selected recombinants, isogenic with respect to auxotrophic markers, were identified exhibiting all four possible combinations of far-UV ( recA 1 vs recA ⁺ ) and near-UV sensitivity ( nur vs nur⁺ ). Transduction with phase P1 has shown that introduction of the recA 1 allele into a recA⁺ recipient does not affect the near-UV sensitivity of the recipient. Additional matings together with transduction experiments suggest that the nur gene is located at a position on the E. coli linkage map clearly separable from recA (minute 58).     

HYDROGEN PEROXIDE MEDIATES UV-INDUCED IMPAIRMENT OF ANTIGEN PRESENTATION IN A MURINE EPIDERMAL-DERIVED DENDRITIC CELL LINE

Abstract— Ultraviolet-B (290–320 nm) radiation is known to impair the antigen-presenting cell (APC) function of Langerhans cells (LC), skin-specific members of the dendritic cell (DC) family. We sought to address mechanisms of this effect, focusing on the role played by hydrogen peroxide. For this purpose, we used a newly established murine DC line, XS52, which resembles epidermal LC in several respects. The APC capacity of XS52 cells, using two different CD4* T cell clones as responders, was inhibited significantly (>50%) by exposure to UV radiation (unfiltered FS20 sunlamps) at relatively small fluences (50–100 J/m²). Ultraviolet radiation also inhibited growth factor-dependent proliferation of XS52 cells. On the other hand, cell surface phenotype was relatively well preserved after irradiation; expression levels of B7-1 and B7-2 were reduced slightly, while other molecules ( e.g. Ia, CD54, CD1 la and CD18) were not affected. With respect to the role played by hydrogen peroxide, pretreatment with purified catalase (900 U/mL) prevented UV-induced inhibition of APC function. Short-term exposure to 3 miM H₂0₂ or f-butyl H₂0₂ mimicked UV radiation by inhibiting APC function. Finally, intrinsic catalase activity was substantially lower in XS52 cells compared with Pam 212 keratinocytes. These results indicate that the generation of hydrogen peroxide alone is sufficient to produce some, but not all, of the deleterious effects of UV radiation on DC derived from the skin.     

Association of Ceramide Accumulation with Photodynamic Treatment-Induced Cell Death

Abstract— Ceramide, a stress-induced second messenger, has been associated with apoptosis in several malignant and non-malignant cell lines. We have shown that photodynamic treatment (PDT), using the phthalocyanine photosensitiz-er Pc 4 (HOSiPcOSi[CH₃]₂[CH₂]₃N[CH₃]₂), causes increased ceramide generation and subsequent induction of apoptosis in L5178Y-R (LY-R) mouse lymphoma cells. To test further if ceramide generation accompanies photocytotoxicity, we treated various cell lines with a PDT dose producing a 99-99.9% loss of clonogenicity. Like LY-R cells, human leukemia (U937) cells underwent rapid DNA fragmentation initiating within 1 h after PDT. Similarly, Chinese hamster ovary (CHO) cells showed rapid DNA laddering, beginning 1 h following the treatment. In contrast, mouse radiation-induced fibrosarcoma (RIF-1) cells showed no apoptosis within 24 h post-PDT, as judged by the absence of 50 kbp and oligonucleosome-size DNA fragments, as well as no annexin V binding to cells with preserved membrane integrity. Using the same doses of PDT, we observed a time-dependent ceramide accumulation in all three cell lines. While a significant increase in ceramide levels was reached within 1 and 10 min in U937 and CHO cells, respectively, elevated ceramide production was measured only after 30 min in RIF-1 cells. In addition, exogenous N-acetyl-sphingosine was able to mimic PDT-induced apoptosis in U937 and CHO cells. We suggest that ceramide accumulation is associated with PDT-induced apoptosis and photocytotox-icity.     

COOPERATION OF CHARGES IN PHOTOSYNTHETIC O2 EVOLUTION–I. A LINEAR FOUR STEP MECHANISM

Abstract— Using isolated chloroplasts and techniques as described by Joliot and Joliot[6] we studied the evolution of O₂ in weak light and light flashes to analyze the interactions between light induced O₂ precursors and their decay in darkness. The following observations and conclusions are reported: 1. Light flashes always produce the same number of oxidizing equivalents either as precursor or as O₂. 2. The number of unstable precursor equivalents present during steady state photosynthesis is ∼ 1.2 per photochemical trapping center. 3. The cooperation of the four photochemically formed oxidizing equivalents occurs essentially in the individual reaction centers and the final O₂ evolution step is a one quantum process. 4. The data are compatible with a linear four step mechanism in which a trapping center, or an associated catalyst, ( S ) successively accumulates four + charges. The S ⁴⁺ state produces O₂ and returns to the ground state S ₀. 5. Besides S ₀ also the first oxidized state S ⁺ is stable in the dark, the two higher states, S²⁺ and S³⁺ are not. 6. The relaxation times of some of the photooxidation steps were estimated. The fastest reaction, presumably S *₁← S ₂, has a (first) half time ≤ 200 μsec. The S *₂ state and probably also the S *₀ state are processed somewhat more slowly (˜ 300–400 μsec).     

PHOTOCHEMISTRY OF trans-[Rh(BIS(DIPHENYLPHOSPHINO)ETHANE)2X2][PF6]: TRANSIENT ABSORBANCE KINETIC STUDIES OF METAL-HALOGEN BOND HOMOLYSIS

Abstract— Laser flash photolysis of trans -[Rh(dppe)₂X₂][PF₆] (X=Br and I; dppe=bis(diphenylphosphino)ethane) in CH₂Cl₂ or CH₃CN produces the d⁷ Rh(II) radicals, [Rh(dppe)₂X]⁺, and halogen atoms. The kinetics of the disappearance of [Rh(dppe)₂X]⁺ radicals in CH₂Cl₂ or CH₃CN were mixed order: H-atom abstraction from solvent to produce the rhodium hydrides, [RhH(dppe)₂X][PF₆], and Rh/X recombination. In the poor H-atom donor solvent, benzonitrile, Rh/Br recombination was observed to be uncomplicated by competing H-atom abstraction. The hydride complexes [RhH(dppe)²X][PF₆], formed by H-atom abstraction were completely characterized by ³¹P{¹H}-NMR, ¹H-NMR, and mass specrometry. Cyclohexene was used as an effective trap for photogenerated Br atoms and yielded bromocyclohexane and 3-bromocyclohexene in a relative yield, 1:9. The photochemical mechanism is discussed in light of the transient absorbance and trapping studies.