DIFFERENT CHROMATOGRAPHIC BEHAVIOR OF A THYMINE-CONTAINING NEW ULTRAVIOLET PHOTOPRODUCT AND SPORE PHOTOPRODUCT

Abstract— One- and two-dimensional cochromatography in five different solvents of a near UV photo-product induced by 365 nm irradiation of DNA in aqueous solution and the 'spore photoproduct', 5-thyminyl-5,6-dihydrothymine, produced by 254 nm irradiation of dry DNA, indicate that the two photoproducts seem to have related structures but are different.     

RELATIONSHIP BETWEEN SURVIVAL, PHOTOPRODUCT PRODUCTION AND REPAIR CAPACITY IN A VARIANT OF BACILLUS CEREUS

Abstract —As sporulation progresses, there is an increased resistance to UV irradiation of the cells of Bacillus cereus var. alesti. This progressive increase is independent of post-irradiation treatment and appears to be a property of the stage of sporulation. In addition, the proportion of photoproducts formed is different for each stage of sporulation. Cells irradiated at Stage I (axial filament) of sporulation display relatively large amounts of spore photoproduct 'c' and less of photoproduct 'b'. As sporulation proceeds, UV irradiation results in the production of more spore photoproduct 'b' and less 'c', suggesting a progressive change in configuration of the DNA within the sporulating cell. If irradiated early in the process (Stage II), large amounts of cyclobutane-type dimers are also produced which, with the 'spore-specific' photoproducts, may be retained in the resultant spore. Although no excision-repair was detectable during germination of these spores, both vegetative and 'spore-specific' damage is reduced during this period. The 'spore-specific' repair mechanism may be able to remove vegetative damage from germinating spores.     

EFFECT OF DIPICOLINIC ACID ON THE ULTRAVIOLET RADIATION RESISTANCE OF BACILLUS CEREUS SPORES

Abstract— The ultraviolet radiation (UV) resistance of B. cereus spores was shown to depend on their content of dipicolinic acid (DPA). Wild-type spores with decreasing amounts of DPA exhibited increased UV resistance. Similarly, spores devoid of DPA (DPA-minus), produced by a mutant strain of B. cereus unable to synthesize DPA, were more resistant to UV than mutant spores (DPA-plus) produced in the presence of exogenously supplied DPA. Resistance of both the wild type and mutant strains to ionizing radiation, however, was unaffected by DPA content. Comparison of the resistance of DPA-minus and DPA-plus mutant spores to UV of various wavelengths showed that the greater sensitivity of the latter DPA-plus spores appeared at wavelengths corresponding to the region of the first molecular absorption band of the calcium chelate of DPA. In the wild type and mutant, thymine photoproducts were produced at a greater rate and to a greater extent in spores with high levels of DPA than in spores with low DPA.
The data indicate that DPA transfers energy to DN A in vivo , which leads to the conclusion that DPA occurs in the spore protoplast.     

ESTIMATION OF PHOTOPRODUCT FORMATION IN GERMINATING SPORES OF BACILLUS SUBTILIS

Abstract— Changes in UV sensitivity during spore germination of Bacillus subtilis mutants possessing various defects in DNA repair capacities were analysed in order to estimate the yield of the DNA photoproducts at the transient, UV resistant stage which occurs in the process of germination. It was concluded that the yield of the spore-specific photoproduct (5-thyminyl-5,6-dihydrothymine, TDHT) at the transient stage was only about 3% of that in dormant spores and the yield of the cyclobutane-type pyrimidine dimers at this stage was about 10% (or less) of that in germinated spores.     

PHYSIOLOGICAL EFFECTS OF ULTRAVIOLET LIGHTON DICTYOSTELIUM DISCOIDEUM SPORE GERMINATION

Abstract— The spore germination in Dictyostelium discoideum consists of four stages: activation, postactivation lag, swelling and emergence. Ultraviolet irradiation (total fluence of 250 J/m²) of spores at any time prior to late spore swelling allows full swelling, but inhibits the emergence of myxamoebae. In the case of freshly activated spores, a UV exposure time of 30 s (total fluence of 50 J/m²) is sufficient to reduce emergence to about 6% when measured after 24 h of incubation. This same fluence results in about 10% viability as measured by plaque forming ability. Experiments utilizing 'fractionated exposures' result in the same percentage inhibition of emergence as that found for 'single exposures' provided the total fluence is equivalent. The higher fluences (250 J/m²) which completely prevent emergence, do not affect the endogenous oxygen uptake of spores during swelling. Ultraviolet light irradiated spores respond to the same activation and deactivation treatments as control unirradiated spores. Ultraviolet irradiation after late spore swelling allows emergence to occur in only a small fraction of the population. This fraction of cells which can emerge after UV treatment is said to have passed a 'competence point', which is believed to be the time when all the events necessary for emergence have been completed. Though the sites of UV inactivation in spores can only be postulated at present, it is apparent that the initial stages of germination (activation, postactivation lag and spore swelling) occur independently of the UV sensitive sites. The final stage of germination (emergence), however, is dependent on UV sensitive functions.     

WAVELENGTH DEPENDENCE OF THE FORMATION OF SINGLE-STRAND BREAKS AND BASE CHANGES IN DNA BY THE ULTRAVIOLET RADIATION ABOVE 150 nm

The wavelength dependence of the formation of two types of DNA damage, single-strand breaks and base changes, was investigated in the UV region from 150 nm to 254 nm using superhelical closed circular (form I) colicin El DNA with synchrotron radiation. Single-strand breaks were measured by agarose gel electrophoresis as a direct conversion of form I DNA to form II DNA (open circular). Base damages were defined as sensitive sites to a crude extract of endonuclease from Micrococcus luteus. They also were estimated using the same conversion, from form I to form II after the DNA was treated with endonuclease. The fluence-effect relationship could be fitted by a simple exponential function for both types of damage. Action spectra were constructed based on the reciprocal of the 37% fluence. The action spectrum for strand breaks increased rather monotonically over three decades from 254 nm to 150 nm in a logarithmic scale, while that for base damages showed a breaking point at 190 nm, being relatively flat above 190 nm. The characteristics of the action spectra are compared with the absorption spectra of the DNA and its main chain moiety calculated on the basis of data on calf thymus DNA and synthetic polynucleotides. Our main conclusions are (1) that the majority of single-strand breaks were induced by the absorption of photon in the sugar-phosphate group in the vacuum-UV region and (2) that the base changes were induced equally well by absorption in the vacuum-UV and in the far-UV region.     

FORMATION OF SEMIQUINONE RADICALS BY ULTRAVIOLET IRRADIATION OF PYRIMIDINES IN METHANOL

Abstract —Ultraviolet irradiation of pyrimidine and 4- and 5-methylpyrimidine in methanol at 113 K gave the corresponding semiquinone radicals in a monophotonic process. It is likely that this process involves an n * triplet state of the photoexcited pyrimidines.     

CHANGES IN TOTAL DIFFUSE SPECTRAL REFLECTANCE OF AMINO ACIDS AND PROTEINS AFTER ULTRAVIOLET IRRADIATION

Abstract— Total diffuse reflectance spectra of air-dried surfaces of free and neutralized amino acid preparations before and after irradiation in vitro are reported. It was found that some free or neutralized amino acid surfaces underwent modification which resulted in changes in the diffuse reflectance spectra after U.V. exposure. It is suggested that these reflectance changes result from transformations in the side chains of the amino acids and that the transformations may differ from those occurring when amino acids in solution are irradiated. Histidine, cystine, hydroxyproline and some protein surfaces showed changes in reflectance of 330–400 nm light similar to those reported in skin after U.V. irradiation in viuo.     

FORMATION OF SINGLE AND DOUBLE STRAND BREAKS IN DNA ULTRAVIOLET IRRADIATED AT HIGH INTENSITY

The induction of single-strand breaks (SSB) by two quantum processes in DNA is well established. We now report that biphotonic processes result in double-strand breaks (DSB) as well. pUC19 and bacteriophage M13 RF DNA were irradiated using an excimer laser (248 nm) at intensities of 10(7), 10(9), 10(10) and 10(11) W/m2 and doses up to 30 kJ/m2. The proportion of DNA as supercoil, open circular, linear and short fragments was determined by gel electrophoresis. Linear molecules were noted at fluences where supercoiled DNA was still present. The random occurrence of independent SSB in proximity to each other on opposite strands (producing linear DNA) implies introduction of numerous SSB per molecule in the sample. If so, supercoiled DNA that has sustained no SSB should not be observed. A model accounting for the amounts of supercoiled, open circular, linear and shorter fragments of DNA due to SSB, DSB and Scissions (opposition of two independently occurring SSB producing an apparent DSB) was developed, our experimental data and those of others were fit to the model, and quantum yields determined for SSB and DSB formation at each intensity. Results showed that high intensity laser radiation caused an increase in the quantum yields for both SSB and DSB formation. The mechanism of DSB formation is unknown, and may be due to simultaneous cleavage of both strands in one biphotonic event or the biased introduction of an SSB opposite a preexisting SSB, requiring two biphotonic events.     

TIME-RESOLVED ULTRAVIOLET ABSORPTION CHANGES IN THE PHOTOCYCLE OF BACTERIORHODOPSIN*

Abstract– The kinetics of the absorption changes associated with the perturbation of aromatic acids during the photocycle of bacteriorhodopsin (bR) were studied at room temperature with microsecond time-resolution. Flash experiments with nanosecond excitation at 532 nm were performed on the purple membrane suspension at a number of measuring wavelengths in the spectral range250–630 nm (to monitor both non-chromophore changes and the photocycle kinetics). The kinetic data collected at different wavelengths were simultaneously fitted with a sum of exponentials to obtain time-resolved UV-VIS difference spectra of photocycle intermediates. This approach allowed us to separate kinetically distinct contributions coupled with tryptophan(s) and tyrosine(s) perturbations. Contributions associated with a reversible perturbation of tryptophans appeared with complex (multistep) kinetics during the bRM transitions and relaxed in a single step during the M0 transition. A contribution associated with perturbation of the local environment of tyrosine appeared before the L and relaxed during the Ob̊ transition.     

TRANSMISSION OF HUMAN EPIDERMIS AND STRATUM CORNEUM AS A FUNCTION OF THICKNESS IN THE ULTRAVIOLET AND VISIBLE WAVELENGTHS

Abstract The dependence of radiation transmission on sample thickness was studied in isolated samples of human stratum corneum and full-thickness epidermis. The investigation also included samples of skin repeatedly exposed to UV-B. Transmission was measured in the ultraviolet and in the visible from 248–546 nm. Two methods, one microscopic and the other mechanical, were used to measure thickness. There was a good correlation between the results.
The dependence of transmission on thickness in these samples could be described satisfactorily by an exponential function, implying that the Lambert-Beer law is approximately valid. Thus, a single parameter, such as the half-value layer ( d _½), is sufficient to characterize absorption in the skin samples.
Water content of the isolated stratum corneum was influenced by maintenance conditions: samples floating on water containing a small amount of NaCl were more hydrated than samples floating on a more concentrated salt solution, or stored in air. Changes in water content of the samples resulted in changes of thickness and, to a lesser extent, of transmission. Approximate in vivo values of d _½ were computed after estimating the in vivo water content of stratum corneum.
Differences found in the shape of the transmission spectra of stratum corneum and full-thickness epidermis may reflect differences in chemical composition. The influence of wetting of the skin on its sensitivity to sunlight is explained in a new way.     

NEAR ULTRAVIOLET INDUCTION OF GROWTH DELAY STUDIED IN A MENAQUINONE-DEFICIENT MUTANT OF BACILLUS SUBTILIS

Abstract— The induction by near UV light of growth delay in Bacillus subtilis was studied utilizing a menaquinone-deficient ( men ^–) strain. Menaquinone appears to be a target molecule in this bacterial species, in view of the following: (i) the men ^– strain requires menaquinone precursors to terminate growth delay; (ii) the menaquinone synthesis inhibitor diphenylamine prolongs growth delay; (iii) the men ^– strain must be phenotypically Men* at the time of near UV irradiation to induce growth delay. These findings suggest that growth delay in B. subtilis may be associated with a prerequisite removal of photochemically altered menaquinone from the cytoplasmic membrane, rather than simply the time required for resynthesis of menaquinone. Alternatively, the altered menaquinone may inhibit some critical reaction(s) of intermediary metabolism or macromolecular synthesis.     

FORMATION OF PROTEIN-BOUND 3,4-DIHYDROXYPHENYLALANINE IN COLLAGEN TYPES I AND IV EXPOSED TO ULTRAVIOLET LIGHT

Abstract— Collagen was exposed to an ultraviolet (UV) lamp that emitted predominantly in the UVB range. The cross-linking of collagen type I and type IV by UV irradiation was observed. Amino acid analyses revealed that Tyr residues in both collagen types I and IV were decreased by irradiation. In collagen type IV, losses of His and Met residues were also observed. These losses of collagen type IV may be due to the degradation of Trp, which exists in collagen type IV and decreased drastically during UV irradiation. To clarify the mechanism of Tyr modification in both types of collagen, the degradation products of Tyr were analyzed. Dityrosine, which is a dimer of the Tyr residue, could not be detected in the acid hydrolysates of UV-irradiated collagen. However, 3,4-dihydroxyphenylalanine, DOPA, was detected in the hydrolysates using HPLC with an electrochemical detector. The amounts of DOPA in the acid hydrolysates of collagen exposed to UV light for 24 h were approximately 350 pmol/mg protein (collagen type IV) and 80 pmol/mg protein (collagen type I). The DOPA formed may partially contribute to photoaging of the skin.     

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.     

REPAIR OF PSORALEN PLUS NEAR ULTRAVIOLET LIGHT DAMAGE IN BACTERIOPHAGES T3 AND T4

Abstract— Repair of T3 and T4 DNA damaged by treatment with 8-methoxypsoralen plus near UV (PNUV) has been investigated. It is shown that the excision repair mechanisms of the host cell can repair a substantial fraction of the psoralen-DNA mono-adducts in T3 DNA, but cannot by themselves repair crosslinks. In contrast neither the excision repair system of the host nor the phage coded v gene endonuclease is involved in the repair of psoralen adducts in T4 DNA. Multiplicity reactivation is effective in the recovery of T4 DNA containing psoralen-DNA mono-adducts, but is ineffective in the recovery of crosslinked phages. Comparisons of the lethality of PNUV treatment and the number of crosslinks induced in T4 DNA show clearly that mono-adducts are lethal to this phage. Both T3 and T4, however, appear to effectively repair many mono-adducts by postreplicational repair.     

ULTRAVIOLET DOSIMETRY IN OUTDOOR MEASUREMENTS BASED ON BACTERIOPHAGE T7 AS A BIOSENSOR

A simple method has been worked out for measuring the biologically effective dose (BED) of solar radiation The method uses phage T7 as a biosensor and it includes field measurements of global and direct UV radiation from the sun in the air; it has been applied to underwater measurements as well. Results of field measurements are presented with discussion of the angle-dependent sensitivity of the biosensor. A model of spectral irradiance based on the measured values is presented. Relevance of the H^T7 unit—derived earlier by us from T7 phage inactivation upon UV radiation—as a measure of the BED is also discussed.     

BIOLOGICALLY EFFECTIVE DOSE OF SOLAR ULTRAVIOLET RADIATION ESTIMATED BY SPORE DOSIMETRY IN TOKYO SINCE 1980

Abstract— The biologically effective dose of solar UV radiation has been measured in Tokyo since 1980 using Bacillus subtills spores. To determine the cumulative dose in a half day, several samples of UV-sensitive spores were exposed in successive intervals from the solar-noon time. Because fluence-survival curves were exponential, the number of lethal hits received by the spores was calculated for each interval and termed inactivation dose (ID). The total number of hits obtained in a half day (half-day ID) was correlated with the amount of global insolation by a power-function regression. The regression analyses were performed for the data collected on 35 days from 1980 to 1986 and for the data collected on 53 days from 1989 to 1991. The latter data set yielded significantly larger estimates of half-day ID relative to the insolation than the former. These analyses suggested that the biologically effective dose relative to the insolation increased about 30% at some time in the later part of 1980s at this location. Changes of solar activity, air pollution and stratospheric ozone layer were considered as potentially responsible for this increase, but identification of the causative factors requires further efforts.