THE WAVELENGTH DEPENDENCE OF THE EFFECT OF 8-METHOXYPSORALEN PLUS ULTRAVIOLET RADIATION ON THE INDUCTION OF LATENT SIMIAN VIRUS 40 FROM A MAMMALIAN CELL

Abstract— The effect of 8-methoxypsoralen (8-MOP) plus ultraviolet radiation (UV) of different wavelengths in the region 238–365 nm on the induction of SV40 from SV40-transformed Syrian hamster kidney cells was investigated. Results indicate that 8-MOP + UV treatment activates as much as 1000-fold more virus than UV alone at wavelengths in the region 302–365 nm. At wavelengths below 302 nm, 8-MOP addition to cells prior to irradiation shows little, if any, effect. A wavelength dependence for this viral induction is presented.     

ULTRAVIOLET ENHANCED REACTIVATION OF HERPES SIMPLEX VIRUS INACTIVATED BY DIFFERENT WAVELENGTHS OF UV RADIATION

The degree of ultraviolet enhanced reactivation (UVR) exhibited by mammalian cells when infected with Herpes simplex virus inactivated by different wavelengths of far ultraviolet (UV) radiation was measured. A wavelength dependence for this effect is presented over the wavelength region 238–297 nm. Within the limits of the deviations obtained, the degree of UVR exhibited is similar at each wavelength. This suggests that virus irradiated with different wavelengths of UV radiation received the same type of damage or that cells repaired the different types of viral damage with the same efficiency.     

LIQUID PHASE ELECTRON SPIN-ECHO STUDIES: DIRECT DETECTION OF THE TRANSVERSE RELAXATION TIME OF PHOTOPRODUCED TETRAMETHYLBENZIDINE CATION IN AQUEOUS MICELLAR SOLUTIONS OF SODIUM DODECYLSULFATE

Abstract— The inactivation of cellular viral capacity for Herpes simplex type I growth at six separate wavelengths (254–302 nm) was measured in five human cell lines. These consisted of one "normal" skin fibroblast line (KD), and four photosensitive lines. Two lines of Xeroderma pigmentosum, one of Bloom's syndrome, and one of Cockayne's syndrome cells were used. Similar relative sensitivities were observed for the Bloom's syndrome, Xeroderma pigmentosum, and normal cell lines. The Cockayne's syndrome line became relatively more sensitive at 289 nm and longer wavelengths. Absolute sensitivities varied. Some divergence in response was noted at the longest wavelength tested, 302 nm.     

PHOTOREACTIVATING ENZYME FROM Streptomyces griseus—VI. ACTION SPECTRUM AND KINETICS OF PHOTOREACTIVATION

Abstract— A high resolution action spectrum for photoreactivation was determined using purified photoreactivating enzyme from Streptomyces griseus. Conversion of pyrimidine dimers in UV-irradiated DNA, the substrate for photoreactivating enzyme, was measured with a Haemophilus influenzae transformation assay. A high similarity was found between action spectrum (max. at 445 nm) and the long wavelength absorption band (max. at 443 nm)of photoreactivating enzyme. In addition to the400–470 nm region considerable photoreactivation was found with wavelengths between 280 and 320 nm. No evidence was obtained for the presence of nonenzymatic photoreactivation. Comparison of in vitro and in vivo action spectra revealed that the sharp peak at 313 nm found in vivo is probably the result of counteracting photoreactivation and inactivation effects. Comparison of the action spectrum with the absorption spectrum of 8-hydroxy-10-methyl-5-deazaisoalloxazine in an aprotic dipolar solvent (which serves as a model for the 8-hydroxy-5-deazaflavin chromophore in photoreactivating enzyme) indicates the possible presence of other chromophore(s) involved in the photorepair process. From kinetic measurements and flash experiments values were obtained for the rate constants of the photoreactivation reaction. The quantum yield of photoreactivation was estimated to be approximately 1.     

ESTABLISHMENT and CHARACTERIZATION OF A MONOCLONAL ANTIBODY RECOGNIZING THE DEWAR ISOMERS OF(6–4)PHOTOPRODUCTS

Abstract— We established a monoclonal antibody(DEM–1) that recognizes UV-induced DNA damage other than cyclobutane pyrimidine dimers or(6–4)photoproducts. The binding ofDEM–1 antibody to 254 nm UV-irradiated DNA increased with subsequent exposure to UV wavelengths longer than 310 nm, whereas that of the 64M-2 antibody specific for the(6–4)photoproduct decreased with this treatment. Furthermore, the increase inDEM–1 binding was inhibited by the presence of the 64M-2 antibody during the exposure. We concluded that theDEM–1 antibody specifically recognized the Dewar photoproduct, which is the isomeric form of the(6–4)photoproduct. TheDEM–1 antibody, however, also bound to DNA irradiated with high fluences of 254 nm UV, suggesting that 254 nm UV could induce Dewar photoproducts without subsequent exposure to longer wavelengths of UV. Furthermore, an action spectral study demonstrated that 254 nm was the most efficient wavelength for Dewar photoproduct induction in the region from 254 to 365 nm, as well as cyclobutane dimers and(6–4)photoproducts, although the action spectrum values in the U V-B region were significantly higher compared with those for cyclobutane dimer and(6–4)photoproduct induction.     

Ultraviolet action spectra for DNA dimer induction, lethality, and mutagenesis in Escherichia coli with emphasis on the UVB region

Abstract —Ultraviolet (UV) action spectra were obtained for lethality and mutagenesis (reversion to tryptophan independence) in Escherichia coli WP2s for wavelengths 254–405 nm with detailed analysis in the UVB region (290–320 nm). Parallel chemical assay yields of pyrimidine dimers in DNA of E. coli RT4 were determined at the same wavelengths. Spectral regions isolated from a Xe arc and resonance lines from a high-pressure Hg-Xe arc lamp were both used for irradiation. In all cases, precise energy distributions throughout the isolated Xe bands regions were defined.
Lethality, mutagenesis, and dimer induction all decreased in efficiency in a similar fashion as the wavelengths of the radiation increased. Between 300 and 320 nm, all characteristics measured showed differences of about two and a half orders of magnitude. Between these wavelengths, the values of the three end points used either coincide with or parallel the absorption spectrum of DNA. The mutagenesis action spectrum coincides closely with the absorption spectrum of DNA. The lethality spectrum is closely parallel to the mutagenicity spectrum; the points, however, consistently occur at about 2 nm longer wavelengths. A calculation derived from the slope of the UVB spectra reveals that a 1-nm shift of the solar UV spectrum to shorter wavelengths would result in a 35% increase in its mutagenic potential. At 325 nm, both biological action spectra show sharp decreases in slope. In addition, above 325 nm the spectra for lethality. mutagenicity, and dimer formation diverge sharply; lethalities at these UVA wavelengths were approximately tenfold greater relative to mutagenicity than at shorter wavelengths. The relative yield of dimer formation by 365 nm radiation is intermediate between the yields for lethality and mutagenesis.     

THE WAVELENGTH DEPENDENCE OF ULTRAVIOLET INACTIVATION OF HOST CAPACITY IN A MAMMALIAN CELL-VIRUS SYSTEM

Abstract. The ability of UV-irradiated African green monkey kidney cells (CV-1) to support the growth of unirradiated herpes simplex virus type 1 as measured by plaque forming ability has been investigated. The lowering of plaque formation by the virus when the host cell was irradiated was examined at thirteen different wavelengths. An action spectrum for this cellular parameter (capacity) was obtained in the wavelength region of 235–302 nm. This action spectrum points to nucleic acid as the critical target molecule for this effect.     

ACTION SPECTRA FOR LETHALITY IN RECOMBINATION-LESS STRAINS OF SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI*

Abstract— Action spectra for lethality of both stationary and exponentially growing cells of recombinationless (recA) mutants of Salmonella typhimurium and Escherichia coli were obtained. Maximum sensitivity was observed at 260nm which corresponds to the maximum absorbance of DNA. However, a shoulder occurred in the 280–300 nm range that departed significantly from the absorption spectrum of DNA. At wavelengths longer than 320nm, the shapes of inactivation curves departed significantly from those at wavelengths shorter than 320nm and survival curves at wavelengths longer than 320nm had a large shoulder. A small peak or shoulder occurred in the 330–340nm region of the action spectra. The special sensitivity of recA mutants to broad spectrum near-UV radiation may be due to synergistic effects of different wavelengths. Parallels between the inactivation of recA mutants and the induction of a photoproduct of l -tryptophan toxic for recA mutants (now known to be H₂O₂) suggest that H₂O₂ photoproduct from endogenous tryptophan may be involved in the high sensitivity of these strains to broad spectrum near-UV radiation.     

THE WAVELENGTH DEPENDENCE OF 8-METHOXYPSORALEN PHOTOSENSITIZATION OF HOST CAPACITY INACTIVATION IN A MAMMALIAN CELL-VIRUS SYSTEM

Abstract— The addition of 8-methoxypsoralen to cultures of African green monkey cells (CV-I) sensitized the inactivation by near UV radiation (302–370 nm) of the ability of the cells to host herpes simplex virus. No sensitizing effect by drug addition was noted for far UV radiation (232–297 nm). An action spectrum for the photosensitized inactivation of this cellular parameter was obtained. This action spectrum is consistent with the absorption spectrum of 8-methoxypsoralen.     

COMPARATIVE ANALYSES OF ACTION SPECTRA OF GLYCOLATE EXCRETION ("PHOTORESPIRATION") AND PHOTOSYNTHESIS IN THE BLUE-GREEN ALGA AN ACYSTIS NIDULANS

Abstract. The action spectra were determined by measuring photosynthetic H¹⁴CO^-₃-fixation and ¹⁴C-glycolate excretion to the medium during 15 min exposure to light at 15 different wavelengths in the visible region using interference filters and a 2500 W high pressure Xe lamp at a constant photon flux of about 1.51 × 10¹⁹ quanta m^-2.s^-1 at all wavelengths.
When plotted on relative scales the action spectrum of glycolate excretion lies below that of photosynthesis at all wavelengths shorter than 517 nm. As glycolate excretion had an exponential relationship to photosynthetic rates, different methods were used to analyze for a specific blue light effect which demonstrated that the relative amount of glycolate excretion was depressed by blue light compared with that by green and red. The greatest difference was observed around 460–480 nm. However, on statistical grounds it is not permitted to draw a difference spectrum which might indicate the absorption characteristics of pigment(s) involved.
A hypothesis is discussed assuming that some glycolate is consumed in an oxidation process for supply of electrons to Photosystem I when Photosystem II is poorly excited in the blue region of the spectrum, which was the case for Anacystis used in the present investigation.     

SPECTRAL ALBEDO MEASUREMENTS IN THE UV and VISIBLE REGION OVER DIFFERENT TYPES OF SURFACES

Abstract— The spectral albedo of the earth's surface, i.e. the ratio between spectral irradiance reflected by the ground to all directions and global irradiance, was measured by a spectroradiometer in the UV and visible region from 290 nm to 800 nm with a spectral resolution of 1.5 nm at steps of 2 nm in the UV (290–400 nm) and 10 nm in the visible (400–800 nm) region. The measurements were performed over bare fertile soil, sand at the beach, concrete (autobahn) and snow as well as over different types of vegetation (grass, oats, rye, sugar-beet, stubble). As the albedo increases with increasing wavelengths for most types of surfaces considered, it is smaller in the UV than in the visible region. In the UVB region (λ < 315 nm) the measured albedo is as small as 0.016-0.017 over vegetation, 0.04-0.05 over bare fertile soil, 0.07-0.10 over concrete ("autobahn") and 0.62-0.76% over polluted snow with a small wavelength dependence. A somewhat higher albedo occurs in the UVA region (315 < λ < 400 nm) with values ranging from 0.02 over vegetation to 0.05 to 0.08 over bare soil. The albedo over dry bright sand, which is typically found at the beach, is significantly higher (0.14 at 300 nm to 0.24 at 400 nm) than over other snow-free surfaces, thus leading to an enhanced dose of biologically effective radiation at the beach.     

ACTION SPECTRA FOR KILLING NON-DIVIDING NORMAL HUMAN AND XERODERMA PIGMENTOSUM CELLS

Abstract— Non-dividing human cells degenerate and eventually detach from a culture vessel surface when exposed to UV light. Action spectra for this kind of cell inactivation were determined using eight monochromatic wavelengths from 240 to 313 nm and both a normal DNA excision-repair-proficient strain and a repair-deficient Xeroderma pigmentosum (XP12BE) strain. The action spectra for both strains have similar shapes with a broad peak between 254 and 280 nm followed by a steep decline at wavelengths greater than 280 nm. The relative action spectra are similar to those for inactivation of reproductive capacity and pyrimidine dimer formation in rodent cells suggesting that the critical target and critical damage for inactivation of non-dividing human cells is DNA and damage to DNA, respectively. Normal repair-proficient cells are 5–7 times more resistant at all wavelengths, based on a comparison of D_o values, than repair-deficient XP12BE cells, supporting the conclusion that the inactivating damage at all wavelengths is to DNA.     

Action spectrum for the suppression of arylalkylamine N-acetyltransferase activity in the two-spotted spider mite Tetranychus urticae

An action spectrum was obtained for the suppression of arylalkylamine N -acetyltransferase (NAT) activity in the two-spotted spider mite Tetranychus urticae by irradiating the mite with monochromatic lights of various wavelengths using the Okazaki Large Spectrograph at the National Institute for Basic Biology, Okazaki, Japan. Fluence–response curves were obtained for wavelengths between 300 and 650 nm by irradiating the mite for 4 h day⁻¹. The samples were frozen after the third exposure. A negative correlation between the logarithmic fluence rate and NAT activity was detected in the range of 0.01–1 μmol m⁻² s⁻¹ for wavelengths between 300 and 500 nm and in the range of 0.1–10 μmol m⁻² s⁻¹ for wavelengths between 550 and 650 nm. The constructed action spectrum indicated that the photoreceptors mediating the circadian and/or photoperiodic systems might be UV-A- and blue-type photoreceptors with absorption peaks at 350 and 450 nm.     

FORMATION OF THYMINE DIMERS IN MAMMALIAN SKIN BY ULTRAVIOLET RADIATION IN VIVO

Abstract— Ultraviolet radiation of 220–300 nm is known to produce cyclobutyl pyrimidine dimers in extracellular DNA, in bacteria, and in mammalian cells in culture. The formation in vivo of such dimers in mammalian skin has remained inferential. We report that one of the important and recognizable biologic events that occurs in mammalian skin during irradiation is the formation of thymine dimers. [³H]-labelled thymidine was applied to the epilated skin of guinea pigs to label their DNA. Animals were irradiated individually, using wavelengths of either 254, 285–350, or 320–400 nm. Immediately after irradiation, epidermis was separated from the rest of the skin and homogenized; DNA and RNA were isolated. Irradiation with wavelengths of 285–350 nm, which included the sunburn-producing spectrum (i.e., 290–320 nm), produced thymine dimers (1·7–2·6 per cent of the total [³H]-thymine incorporated into DNA). Irradiation with 254nm also produced fewer dimers (0·46–1·2 percent); and 320–400 nm produced none. The dimer could be cleaved by 250 nm radiation to form thymine. The epidermal cell damage by ultraviolet radiation, particularly by the sunburn-producing spectrum (290–320 nm), may be related to the formation of such dimers.     

NEAR ULTRAVIOLET INACTIVATION STUDIES ONESCHERICHIA COLI TRYPTOPHANASE AND TRYPTOPHAN SYNTHETASE

Abstract— –The response of two pyridoxal-phosphate-requiring enzymes of E. coli, tryptophanase and tryptophan synthetase, to near UV light (320–400 nm) has been studied. Tryptophanase is inactivated both in vivo and in vitro, but tryptophan synthetase is resistant to near UV under both conditions. This shows that near UV inactivation is not general for pyridoxal-phosphate-requiring enzymes. Substrate protection against light inactivation is demonstrated for tryptophanase. It is furthermore shown that pyridoxal phosphate is required for inactivation of this enzyme. However, the action spectrum for this inactivation does not coincide with the absorption spectrum of tryptophanase or of pyridoxal phosphate.     

Action spectra for inactivation of normal and xeroderma pigmentosum human skin fibroblasts by ultraviolet radiations

—Action spectra for UV-induced lethality as measured by colony forming ability were determined both for a normal human skin fibroblast strain (lBR) and for an excision deficient xeroderma pigmentosum strain (XP4LO) assigned to complementation group A using 7 monochromatic wavelengths in the range 254-365 nm. The relative sensitivity of the XP strain compared to the normal skin fibroblasts shows a marked decrease at wavelengths longer than 313 nm. changing from a ratio of about 20 at the shorter wavelengths to just greater than 1.0 at the longer wavelengths. The action spectra thus indicate that the influence on cell inactivation of the DNA repair defect associated with XP cells is decreased and almost reaches zero at longer UV wavelengths. This would occur, for example, if the importance of pyrimidine dimers as the lethal lesion decreased with increasing wavelength. In common with other studies both in bacterial and mammalian cells, our results are consistent with pyrimidine dimers induced in DNA being the major lethal lesion in both cell strains over the wavelength range 254-313 nm. However, it is indicated that different mechanisms of inactivation operate at wavelengths longer than 313 nm.     

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.     

DNA PHOTOREACTIVATING ENZYME FROM SILKWORM

An ammonium-sulfate-precipitable (33–70%) fraction in extracts from eggs of silkworm Bombyx mori contains photoreactivating enzyme that reactivates the transforming activity of UV inactivated Hemophilus influenzae DNA. The action spectrum for in vitro photoreactivation with the enzyme has a broad peak around 365–385 nm, with a shoulder extending to 460 nm. This relatively higher photoreactivation efficiency at wavelengths longer than 450 nm seems to be a unique feature of DNA photoreactivating enzyme of silkworm. Using gel filtration, a mol wt of 42,000 was estimated for the enzyme. Optimum and isoionic pH of the enzyme were 7.2 and 5.4, respectively. These properties of silkworm enzyme are within the range of variations in reported biochemical characteristics of photoreactivating enzymes from different species.     

LEAKAGE OF 86Rb+ AFTER ULTRAVIOLET IRRADIATION OF Escherichia coli K-12

Abstract— Stationary phase cultures of a DNA repair proficient Escherichia coli K-12 strain showed a release of intracellular material as assessed by three different methods (260 nm absorption; [methyl-³H]thymidine leakage and ⁸⁶Rb⁺ leakage) after broad-band (Black-Light Blue) near-UV radiation but not after far-UV (254 nm) radiation. As a control response for membrane damage to cells, this leakage of intracellular material was also determined by each method after mild-heat (52°C) treatment of E. coli K-12. An action spectrum for the release of ⁸⁶Rb⁺ from E. coli K-12 after irradiation with monochromatic wavelengths, from 254 to 405 nm, is also presented. The action spectrum for lethality (F₃₇ values) obtained for this strain, shows that leakage of ⁸⁶Rb⁺ occurs at fluences equivalent to or slightly less than fluences causing inactivation at wavelengths above 305 nm. In contrast, at wavelengths below 305 nm, leakage of ⁸⁶Rb⁺ from irradiated cells can be induced but only at fluences significantly greater than was required to cause cell inactivation. These results indicate, therefore, that near-UV radiation can induce a damaging effect on the cell's permeability barrier which may be significant in causing the death of the cell, whereas the effect is not significant in causing the death of cells by far-UV radiation where DNA damage is known to be the main cause of lethality.