SYNERGISTIC LETHALITY OF PHAGE T7 BY NEAR-UV RADIATION AND HYDROGEN PEROXIDE: AN ACTION SPECTRUM

Abstract— Ozonation of valerophenone oxime o-methyl ether (4) produced a stereoisomeric mixture of crystalline dimeric valerophenone peroxides, 5a and 5b . N-n-butyl-N-methoxybenzamide (6) and N-methoxy-N-phenylvaleramide (7) along with valerophenone (1). Thermolysis of the higher melting peroxide 5a at 170–180°C, where a chemiluminescence was visible from added perylene. gave 1 and butyl benzoate (8) in addition to small amounts of the Norrish Type 11 products of 1. i.e. acetophenone (2) and cis- and trans- 2-methyl-1-phenylcyclobutanols. Biacetyl-sensitized photolysis of 5a in benzene yielded 2 in much higher yield in addition to 1, 8, and biphenyl. These results suggest that the triplet excited state of 1 is formed by the decomposition of 1 in low yield in thermolysis and in much higher yield in sensitized photolysis. although some of the Type II products may not arise from the triplet valerophenone.     

PROTEIN-RNA CROSSLINKS IN RIBOSOMES: ULTRAVIOLET ACTION SPECTRA. COMPARISON WITH INACTIVATION ACTION SPECTRUM

Abstract— RNA-protein crosslinking by UV of different wavelengths was studied in 70S E. coli ribosomes by three techniques: sucrose gradient centrifugation in the presence of sodium dodecyl sulfate (SDS), RNA solubilization in LiCI-urea concentrated solutions and RNA adsorption on nitrocellulose filters in the presence of SDS.
The centrifugational technique shows that the crosslinking reaction occurs in two steps, the first one corresponding to the fixation of a few protein molecules on 16 or 23 s RNAs and the second one corresponding to extensive RNA-protein crosslinking so that most protein molecules are no longer released by SDS from 30S and 50S subunits.
The initial rates for the first step of crosslinking were evaluated by the solubilization and adsorption techniques at 7 (or 6) wavelengths of irradiation between 223 and 290 mm. The action spectrum for RNA solubilization in LiCl-urea is perturbed at 223 nm by the breakage of protein chains. The action spectrum for retention on nitrocellulose filters seems to be exempt of this defect. It corresponds at high wavelengths to a nucleic chromophore and at low wavelengths to a proteic one. This means that RNA-protein crosslinking may occur through RNA and protein excitation. The similarity between the action spectrum for RNA retention on nitrocellulose filters and the action spectrum for inactivation of ribosomal synthesis activity suggests that RNA-protein crosslinking may be responsible for inactivation of ribosomes by UV.     

INACTIVATION OF THE LACTOSE PERMEASE OF ESCHERICHIA COLI BY MONOCHROMATIC ULTRAVIOLET LIGHT

Abstract— The lactose permease of E. coli is inactivated exponentially by seven wavelengths of monochromatic UV light. An action spectrum reveals that the shorter wavelengths (243, 290 and 313 nm) are much more efficient than longer wavelengths. Inactivation at 290 nm is most efficient and is not due to generalized membrane damage. The rate of counterflux of intracellular β-galactoside in response to externally added β-galactoside was slowed by 290 nm irradiation, indicating destruction of the facilitated diffusion mechanism. The induction of β-galactosidase and β-galactoside permease was co-ordinate both with and without pre-irradiation by 290 nm light. The β-galactosidase is approximately 26-fold more resistant to 290 nm than the permease. These results are discussed in terms of a greater sensitivity of membrane proteins to 290 nm light, which may be due to the role of aromatic amino acids in conferring stability to the permease in the membrane.     

AN ACTION SPECTRUM IN THE BLUE and ULTRAVIOLET FOR PHOTOTROPISM IN ALFALFA*

Abstract Phototropism is a common property of plants, but it is not known if different species use the same photoreceptor for their response. We have determined fluence-response relations for phototropism in response to brief, broad-band blue irradiation for four plant species grown under red light (Amaranthus paniculatus, Linum usitatissimum, Vigna radiata and Medicago sativa) and compared these to ones previously obtained for Pisum sativum and Zea mays, grown under similar conditions. Curves for all species showed a bell-shaped dependence on fluence, a characteristic of first positive curvature as originally defined for the Avena coleoptile, and had a similar optimal fluence, near 3 H.mol m^?2. We have obtained an action spectrum in the blue and UV spectral regions for first positive phototropism of the hypocotyl of alfalfa grown under red light. Fluence-response curves at wavelengths between 300 and 500 nm were nearly identical in shape and magnitude; whereas below 300 nm, their slopes and maximum curvatures were reduced. The action spectrum showed that activity rose sharply at wavelengths below 500 nm, peaked at 450 nm with shoulders on either side of that peak, and had lesser peaks at 380 and, in the far ultraviolet, at 280 nm. This action spectrum was very similar to ones in the literature (obtained between 350 and 500 nm) for first and second positive phototropism of oat coleoptiles. We conclude that the same photoreceptor mediates phototropism in oat and alfalfa.     

ACTION SPECTRUM FOR INACTIVATION OF THE INFECTIVITY OF POTATO VIRUS X BY U.V. RADIATION

Abstract— Quantum yields for inactivation of infectivity of potato virus X by monochromatic ultraviolet radiation of wavelengths ranging from 230 to 290 nm, were measured with reference to energy absorbed by (a) the whole virus and (b) the virus RNA. The yields depended on the wavelength, but those with reference to energy absorbed by the RNA varied much less (with extreme values of 10^-3 and 1.9 ± 10^-3 than those with reference to whole virus. Consequently the action spectrum for inactivation of a dilute solution of the virus resembled the shape of the absorption spectrum of the RNA, but not closely enough to allow coincidence by adjusting the scales. The amount of photoreactivation increased as the wavelength increased and also as the year progressed from May to July; the extreme values of the photoreactivable sector were 0.43 and 0.86.     

ULTRAVIOLET ACTION SPECTRA FOR AEROBIC AND ANAEROBIC INACTIVATION OF Escherichia coli STRAINS SPECIFICALLY SENSITIVE AND RESISTANT TO NEAR ULTRAVIOLET RADIATIONS

Abstract— Action spectra for the lethal effects of ultraviolet light (254–434 nm) irradiation delivered under aerobic or anaerobic conditions to Escherichia coli RT2 (specifically sensitive to near-UV radiation; > 320 nm) and E. coli RT4 (near-UV resistant) were prepared. Negligible oxygen dependence was observed for both strains below about 315 nm. The oxygen enhancement ratio (OER) for RT4 increased above this wavelength to the longest wavelength used, whereas for RT2 there was a greater increase in the OER to a large peak at 365 nm, then a progressive decrease at longer wavelengths. The results are consistent with the possibility that the sensitivity of strain RT2 to near-UV radiation may be due to hyperproduction of photosensitizer, operating via photodynamic type reactions involving excited species of oxygen.     

THE SYNERGISTIC ACTION OF ULTRAVIOLET AND X RADIATION ON MUTANTS OF ESCHERICHIA COLI K-12

Abstract— Prior UV irradiation increased the X-ray sensitivity of wild-type E. coli K-12. This synergistic effect of combined UV and X irradiation was also observed, but to a reduced extent, in uvrA, uvrB, uvrC , and polA mutants, but was absent in exrA, recA, recB , or recC mutants of E. coli K-12. Alkaline sucrose gradient studies demonstrated that the wand err gene-controlled, growth-medium-dependent (Type III) repair of X-ray-induced DNA single-strand breaks was inhibited by prior UV irradiation. This inhibition probably explains the synergistic effect of these two radiations on survival.     

AN ACTION SPECTRUM FOR ULTRAVIOLET INDUCED ELASTOSIS IN HAIRLESS MICE: QUANTIFICATION OF ELASTOSIS BY IMAGE ANALYSIS

To determine an action spectrum for ultraviolet (UV)-induced elastosis, four groups of 24 albino hairless mice each were exposed to four different spectra emitted by a xenon arc solar simulator fitted with cut-off filters (Schott WG 320, 335, 345, and 360). These filters progressively removed more of the shorter wavelengths until, in the final spectrum, only long wavelength UVA (greater than 335 nm) remained. Exposures continued up to 62 weeks. A fifth group of mice served as controls. Skin biopsies were taken at pre-determined dose points and were processed for light microscopy. Elastosis was quantified by computerized image analysis, yielding dose-response curves for each spectrum. The total energy required for a 50% increase in elastic tissue compared to controls was determined graphically for each spectrum. These were: WG 320, 65 J/cm2; WG 335, 865 J/cm2; WG 345, 1230 J/cm2; and WG 360, 2000 J/cm2. Our results were tested against published action spectra for erythema, photocarcinogenesis and elastosis. The erythema spectrum was the most predictive for elastosis except that the longer UVA wavelengths were less effective for elastosis than for erythema. Solar simulating radiation (WG 320 filter) with its UVB component was the most effective in inducing elastosis. Full spectrum UVA (WG 345) required 20 times more energy while long wavelength UVA (WG 360) required 30 times more energy to induce equivalent elastosis.     

THE ACTION SPECTRUM OF AN IN VITRO DNA PHOTOREACTIVATION SYSTEM

Abstract— The wavelength-dependence of in vitro photoreactivation of transforming DNA by yeast extract has been determined. There is an intensity-dependent lag at the beginning of the biological reaction. There is a similar lag in the splitting of thymine dimers by the yeast extract in the light, a process known to account for most or all of the increase in transforming activity of photoreactivated DNA. The most efficient wavelengths for photoreactivation are around 3550 and 3850 Å. Although the action spectrum is not very similar to flavin absorption, riboflavin at very low concentration inhibits photoreactivation, as it also inhibits a number of flavoenzymes, suggesting that the photoreactivating enzyme might be a flavoprotein.     

AN ACTION SPECTRUM OF PHYTOCHROME BINDING TO A SUBCELLULAR FRACTION OF MAIZE COLEOPTILES

Abstract— An action spectrum was constructed for the photo-induced pelletability of phytochrome in excised coleoptiles of etiolated maize seedlings. It closely resembled the absorption spectrum of purified phytochrome in the P, form as reported in the literature. The spectral dependence of phytochrome pelletability effected by sustained irradiation (4 h) was also determined and it appeared remarkably similar to the high irradiance response (HIR) action spectrum reported for the inhibition of lettuce hypocotyl lengthening. The induction action spectrum was held to support the conclusion that phytochrome itself is the photoreceptor for its own binding to the subcellular fraction of maize coleoptiles and that the binding phenomenon is an early, if not the first, physiological consequence of irradiation. Also a modified version of Hartmann's interpretation of the high irradiance response was given.     

PHOTOSENSITIZED INACTIVATION OF E. COLI CELLS IN TOLUIDINE BLUE-LIGHT SYSTEM

E. coli cells were inactivated with visible light in the presence of toluidine blue as a photo-sensitizer. This photodynamic effect was partially protected with α-tocopherol. Not only pH but the concentration of the buffer during irradiation also affected the survival. The addition of osmotic stabilizers such as KCI, glycerol and polyethyleneglycol to the buffer increased the survival. The difference in singlet oxygen production in these reaction mixtures could not be related to these features. Furthermore, the survival was also dependent upon both irradiation temperature and cultivation temperature of the cells. These results with E. coli cells support the notion that one of the primary targets of toluidine blue sensitized photodynamic inactivation is cytoplasmic membrane, although other factors than cytoplasmic membrane also influence the survival of the cells.     

SINGLET OXYGEN: A MAJOR REACTIVE SPECIES IN THE FUROCOUMARIN PHOTOSENSITIZED INACTIVATION OF E. COLI RIBOSOMES

Abstract— The skin photosensitizing furocoumarins, 8-methoxypsoralen (MOP) and 4,5',8-trimethylpsoralen (TMP), inactivate E. coli ribosomes in vitro , on UV irradiation at 313 nm. Purging the solutions with N₂ protects the ribosomes considerably against photoinactivation (75% with MOP and 80% with TMP). In air, the ribosome photoinactivation is mainly due to singlet oxygen (¹O₂), since the presence of NaN₃ and other ¹O₂ quenchers protects the system and the inactivation is enhanced in D₂O. Although ¹O₂ dominates as the inactivating species, the possibility of additional (∼15%) minor mechanisms involving free radicals exists. However, O^-₂ does not appear to be the damaging species, since superoxide dismutase does not provide any protection.
Photosensitization of the partially purified enzyme, phe-tRNA-synthetase with MOP or TMP shows inactivation and protection curves similar to those seen with the ribosomes. On the other hand, unfrac-tionated tRNA^phc is not photosensitized under similar conditions, although it shows self-photosensitization. It is likely that in the furocoumarin-sensitized ribosomes, the primary events of photoinactivation are associated with the proteins.     

ACTION SPECTRUM FOR THE OXYGEN-INDEPENDENT INACTIVATION OF HAEMOPHILUS INFLUENZAE TRANSFORMING DNA WITH NEAR ULTRA-VIOLET LIGHT*

Abstract— The oxygen-independent inactivation of Haemophilis influenzae transforming DNA by near UV light (300–380 nm) has an action spectrum in which the efficiency of inactivation drops rapidly between 313 and 334 nm and more slowly between 334 and 405 nm, with a shoulder between 334 and 365 nm.     

REDUCTION BY NEAR-ULTRAVIOLET (334 nm) LIGHT OF E. COLI CAPACITY FOR PHAGE GROWTH DEPENDS UPON THE rel GENE AND 4-THIOURIDINE

Abstract— Near-ultraviolet radiation (near UV; 300–380 nm) has long been known to produce a transient reduction of the capacity of bacteria to support phage growth. The present work shows that, at high fluenœs (40–100 kJ/m²), 85% of 334-nm-induced reduction of capacity in Escherichia coli B/r requires the rel gene; that is, it results from rel -gene activity caused by the near-UV treatment. This rel -gene activity leads to (1) a bacterial growth delay and concomitantly lowered bacterial metabolism, and (2) a parallel delay in phage development, with a considerable depression of burst size. We propose that the observed effects on phage development are a consequence primarily of the lowered bacterial metabolism, but they may also result partly from a direct inhibition of phage DNA synthesis by the rel gene product, these effects together leading to the observed reduction of capacity in a rel ⁺ strain. The remaining 15% of capacity reduction, observed in a rel strain, has an unknown mechanism, but does appear to involve a delay in phage development.
At least 95% of the total capacity reduction observed in the rel ⁺ strain in the range 40–100 kJ/m² requires the presence of 4-thiouridine, an unusual base in E. coli transfer RNA, which is presumably both the chromophore and the target for near-UV-induced capacity reduction.     

ACTION SPECTRUM STUDIES FOR INDUCTION OF IMMUNOLOGIC UNRESPONSIVENESS TO DINITROFLUOROBENZENE FOLLOWING IN VIVO LOW DOSE ULTRAVIOLET RADIATION

Abstract— Topical application of the contact sensitizer dinitrofluorobenzene to the skin of C3H mice previously exposed in vivo to low doses of UVB radiation from FS20 sun lamps resulted in the acquisition of antigen-specific unresponsiveness to that hapten. When narrow band radiation at various wavelengths between 260 and 320 nm was employed, increasing doses of radiation were found to produce increasing amounts of immunosuppression. Construction of an action spectrum curve revealed that radiation between 260 and 300 nm was most efficient in producing unresponsiveness. Wavelengths above 300 nm were much less efficient than those below 300 nm. These results indicate that there are major differences in the effectiveness of various components of the short and middle wavelength UV spectrum in eliciting immunologic unresponsiveness to a topically administered hapten. Potential chromophores for this UVB-induced immunosuppressive effect include DNA and urocanic acid.     

THE INFLUENCE OF NUTRITION ON THE DNA CONTENT OF ESCHERICHZA COLI AND ITS RESPONSE TO ULTRAVIOLET LIGHT

Abstract— The influence of nutrition on the sensitivity of Escherichia coli 15 T- to ultraviolet light (u.v.) and the synthesis of DNA has been studied. Growth in media containing glucose or NH,⁺ has been found to endow cells with a greater resistance to lethal u.v. damage than those grown in media containing succinate or amino acids, respectively. In addition, the sensitivity of the lactose ( lac ) locus of the DNA to mutagenic damage has been found to be altered by changes in the carbon supply but not by changes in the nitrogen source, while the sensitivity of loci controlling amino acid synthesis was altered by changes in the nitrogen source but not in the carbon source. Cells fed with glucose or NH₄⁺ have been found to possess more DNA than cells fed with succinate or amino acids, respectively. The data indicate that the type of carbon and nitrogen supplied to the cells will determine whether or not set regions of the DNA will undergo more than one round of replication. The presence in the cell of identical genetic loci either in duplicate or in multiples, directed by the particular types of carbon and nitrogen supplied, is suggested to be, in part, the reason why an alteration in nutrition is able to influence the sensitivity of bacterial cells to radiation.     

PHOTODYNAMIC INACTIVATION OF E. COLI BY ROSE BENGAL IMMOBILIZED ON POLYSTYRENE BEADS

Abstract. The photodynamic inactivation of E. coli by visible light and O₂ was found to occur in the presence of the sensitizer rose bengal, immobilized by covalent bonding to polystyrene beads. The demonstrated absence of significant amounts of dissolved rose bengal indicated that an inactivation mechanism based on penetration of sensitizer molecules into the cell's interior could not be operating. Survival curves typically exhibited induction periods followed by rapid exponential death, with 99.99% kill requiring 1–2 h depending on conditions. A mechanism involving the participation of photo-generated singlet excited oxygen O₂(¹δ) in inactivation of E. coli is proposed. The photodynamic inactivation rate increased significantly in H₂O compared with H₂O, which is evidence supporting singlet oxygen as an active intermediate, since O₂(¹δ) has a much longer lifetime in H₂O than in H₂O. H₂O did not act as a short term poison in the absence of sensitizer.     

ULTRAVIOLET (254-405 nm) ACTION SPECTRUM AND KINETIC STUDIES OF ALANINE UPTAKE IN ESCHERICHIA COLI B/R*

Abstract Uptake of ala in exponentially grown and starved cells of Escherichia coli B/r is inhibited by monochromatic far–UV (254–310 nm) and near UV (310–405 nm) light. The action spectrum for inhibition of ala uptake is similar to that found earlier for gly uptake, showing a maximum at 280 nm and a significant but much lower action throughout the near–UV region. The action spectra suggest that the chromophores for inactivation of ala and gly uptake lie in the carrier proteins and that these proteins are similar. Kinetic studies, in unirradiated bacteria, show that (a) the K_m for ala uptake (11 μM) is about twice that for gly uptake (4.9 μM), (b) the K_m for ala uptake does not change in the presence of gly, although the K_max does decrease, and (c) other amino acids, such as ser and phe, have no effect on the K_m or V_max of the ala uptake system. These data suggest that ala and gly are transported by the same carrier protein, with the binding sites for ala and gly on different subunits.     

ULTRAVIOLET INACTIVATION OF THE MIDIVARIANT OF Qß RNA: THE SITES OF UV-INDUCED REPLICATION INHIBITION

Abstract— MDV-1 RNA is a small variant of bacteriophage Qß, and consists of complementary 'plus' (+) and 'minus' (-) strands. It is an active template in replication reactions catalyzed by Qß replicase. Using high-resolution polyacrylamide gel electrophoresis, we have located the sites of replication inhibition caused by irradiation of (+) and (-) MDV-1 template RNA with 254-nm light. Presumably, this inhibition was caused by uridine hydrates and pyrimidine cyclobutane dimers, which previously were shown to be formed at levels of 3.5 and 0.3 per (+) MDV-1 strand, respectively, at the dose used here (2500 Jm^-2). Each of the ten inhibition sites correspond to regions in the template which contain two or more pyrimidines, including at least one uridine. At each site, replication inhibition occurred at two or three adjacent bases. The sites of the photolesions with respect to the known primary sequence and proposed secondary structure of MDV-1, and the implications of this work for locating the sites of lesions produced by other agents which inhibit the replication of a small RNA molecule are discussed.     

INDUCTION OF DNA-PROTEIN CROSSLINKS IN HUMAN CELLS BY ULTRAVIOLET and VISIBLE RADIATIONS: ACTION SPECTRUM

Abstract— DNA-protein crosslinking was induced in cultured human P3 teratocarcinoma cells by irradiation with monochromatic radiation with wavelengths in the range254–434 nm (far-UV, near-UV, and blue light). Wavelength 545 nm green light did not induce these crosslinks, using the method of alkaline elution of the DNA from membrane filters. The action spectrum for the formation of DNA-protein crosslinks revealed two maxima, one in the far-UV spectrum that closely coincided with the relative spectrum of DNA at 254 and 290 nm, and one in the visible light spectrum at 405 nm, which has no counterpart in the DNA spectrum. The primary events for the formation of DNA-protein crosslinks by such long-wavelength radiation probably involve photosensitizers. This dual mechanism for DNA-protein crosslink formation is in strong contrast to the single mechanism for pyrimidine dimer formation in DNA, which apparently has no component in the visible light spectrum.