OYGEN DEPENDENCE AND REPAIR OF LETHAL EFFECTS OF NEAR ULTRAVIOLET AND VISIBLE LIGHT*

Abstract— –Lethality in a repairable strain (WP2) and an excision repair deficient strain (WP2hcr) of Escherichia coli was studied at wavelengths of 254, 313, 365, and 390–750 nm. Survival curves were empirically fitted to the expression S= 1 - (1-e^-kl)“, where S is the fraction surviving, D is the incident dose in ergs mm^-2, k is the inactivation constant in units of (erg mm^-2)^-1 and n is the ‘shoulder constant’. The repairable sector (k(hcr^-)–k(hcr^-)lk(hcr^-), a conservative estimate of the repair capability of E. coli WP2, was 0.91 at 254 nm, 0.92 at 313 nm, 0.60 at 365 nm, and 0.13 at 390–750 nm. Although there was no oxygen enhancement of inactivation at 254 nm and 313 nm, a strong enhancement was identified at 365 nm and 390–750 nm. These results suggest that oxygen-dependent damage induced by near u.v. (365 nm) can be partially repaired by the excision-repair system in E. coli.     

INTERFERENCE BY NEAR ULTRAVIOLET AND GREEN LIGHT WITH GROWTH OF ANIMAL AND PLANT CELL CULTURES*

Abstract— Near ultraviolet (365 nm) and green-yellow (546-579nm) radiations repress the growth of liquid cell cultures of Ginkgo pollen and of monolayer cultures of HeLa. The deleterious effects of green-yellow wavelengths can be negated by red radiation; near-u.v.-induced growth repressions are insensitive to visible light photorestoration. These wavelengths do not interact synergistically and evoke different kinetics of response.     

SENSITIVITY OF STRAINS OF ESCHERICHIA COLI DIFFERING IN REPAIR CAPABILITY TO FAR UV,NEAR UV AND VISIBLE RADIATIONS*

Abstract— In stationary phase, strains of Escherichia coli deficient in excision (B/r Her) or recombination repair (K.12 AB2463) were more sensitive than a repair proficient strain (B/r) to monochromatic near-ultraviolet (365 nm) and visible (460 nm) radiations. The relative increase in sensitivity of mutants deficient in excision or recombination repair, in comparision to the wildtype, was less at 365 nm than at 254 nm. However, a strain deficient in both excision and recombination repair (K12 AB2480) showed a large, almost equal, increase in sensitivity over mutants deficient in either excision or recombination repair at 365 nm and 254 nm. All strains tested were highly resistant to 650 nm radiation. Action spectra for lethality of strains B/r and B/r Her in stationary phase reveal small peaks or shoulders in the 330–340, 400–410 and 490–510 nm wavelength ranges. The presence of 5μg/ml acriflavine (an inhibitor of repair) in the plating medium greatly increased the sensitivity of strain B/r to radiation at 254, 365 and 460 nm, while strains E. coli B/r Her and K12 AB2463 were sensitized by small amounts. At each of the wavelengths tested, acriflavine in the plating medium had at most a small effect on E. coli K.12 AB2480. Acriflavine failed to sensitize any strain tested at 650 nm. Evidence supports the interpretation that lesions induced in DNA by 365 nm and 460 nm radiations play the major role in the inactivation of E. coli by these wavelengths. Single-strand breaks (or alkali-labile bonds), but not pyrimidine dimers are candidates for the lethal DNA lesions in uvrA and repair proficient strains. At high fluences lethality may be enhanced by damage to the excision and recombination repair systems.     

OXYGEN-DEPENDENCE OF NEAR UV (365 NM) LETHALITY AND THE INTERACTION OF NEAR UV AND X-RAYS IN TWO MAMMALIAN CELL LINES

Abstract— The colony-forming ability of Chinese hamster cells (V-79) and HeLa cells has been measured after near-ultraviolet (UV) irradiation, predominantly at 365 nm. To avoid the production of toxic photoproducts, cells were irradiated in an inorganic buffer rather than in tissue culture medium. Under these circumstances near-UV lethality was strongly oxygen-dependent. Both cell lines were approximately 10⁴ times more sensitive to 254 nm irradiation than to 365 nm radiation when irradiated aerobically. Pretreatment with 6 times 10⁵ Jm^-2 365 nm radiation sensitised the HeLa, but not the V-79 cell line to subsequent X-irradiation. Pretreatment of cells with 17 Jm^-2 254 nm radiation, a dose calculated to produce twenty times more pyrimidine dimers than the 365 nm dose, produced only slight sensitisa-tion to X-rays. It is suggested that the sensitisation to X-rays seen in the HeLa cells after 365 nm treatment is not the result of lesions induced in DNA by the near-UV radiation, but may reflect the disruption of DNA-repair systems.     

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.     

LIGHT ABSORPTION AND ENERGY TRANSFER IN POLYENE-PORPHYRIN ESTERS*

Two examples of a new class of compounds, polyenes covalently linked to meso-tetraarylporphyrins, have been prepared. Optical studies revealed that energy transfer from a 26 π electron polyene moiety to the porphyrin was zero, whereas energy transfer efficiency from a polyene containing 18 π electrons was ?80%. The results demonstrate that the polyene moiety can function as an efficient light gathering antenna for possible application to porphyrin-based solar energy conversion schemes. In addition, such polyene-porphyrin esters may serve as probes of the structural requisites for light harvesting by carotenoids in the photosynthetic membranes of green plants.     

ACUTE EFFECTS OF NEAR ULTRAVIOLET AND VISIBLE LIGHT ON THE CUTANEOUS ANTIOXIDANT DEFENSE SYSTEM

Reactive oxygen species are considered to play an important role in cutaneous pathology. Enzymic and non-enzymic antioxidants can prevent oxidative damage but may be overcome by strong pro-oxidative stimuli. The acute effect of a single exposure to near ultraviolet (UVA)/visible radiation (greater than 320 nm) on various skin antioxidants was examined in hairless mice immediately after irradiation. Impairment of cutaneous catalase and glutathione reductase activity was observed. Superoxide dismutase and glutathione peroxidase were not significantly influenced. Inhibition of catalase may render skin more susceptible to the damaging effects of hydrogen peroxide and its reaction products such as the hydroxyl radical. Partially diminished glutathione reductase activity is not accompanied by a change in reduced/oxidized glutathione level immediately after irradiation. There was a tendential (not statistically significant) decrease in cutaneous tocopherol, ubiquinol + ubiquinone 9 and ascorbic acid levels, either indicating direct photodestruction or consumption by reaction products of photooxidative stress. This partial impairment of the cutaneous antioxidant defense system by near ultraviolet/visible light, showing that the most susceptible component in skin is catalase, suggests possible pharmacological interventions.     

DIFFERENT EFFECTS OF RAD GENES OF SACCHAROMYCES CEREVISIAE ON INCISIONS OF INTERSTRAND CROSSLINKS AND MONOADDUCTS IN DNA INDUCED BY PSORALEN PLUS NEAR UV LIGHT TREATMENT

Abstract— The RAD1, RAD2, RAD3 and RAD4 genes of Saccharomyces cerevisiae are required for incising DNA containing UV induced pyrimidine dimers or psoralen plus 360 nm light induced interstrand crosslinks. We have now determined if these genes are also required for incising DNA at psoralen plus 360 nm light induced monoadducts. For distinguishing between incision breaks occurring at crosslinks and at monoadducts. we have used the cdc9-2 mutant, defective in DNA ligase activity at the restrictive temperature, and the radl-2 cdc9-2, rad2-5 cdc9-2 , rad3-2 cdc9-2 and rad4-4 cdc9-2 double mutant combinations. We conclude that the radl, rad2 , and rad4 mutants are defective in incising DNA both at crosslinks and monoadducts, whereas the rad3 mutant is proficient in incising DNA at monoadducts but not at crosslinks.     

LIGHT DISTRIBUTION AND ELECTRON DONATION IN THE Z SCHEME*

Abstract— Several recent reports indicate that the photosynthetic machinery possesses a device whereby the relative distribution of absorbed light energy into the two reaction centers can be varied in response to the environment in which the photosynthesizing cells or chloro-plasts are placed. Conditions reported which affect this distribution include the electron acceptor or electron donor system employed, the concentration of magnesium ions, and the preillumination history of the preparation. These observations are described and discussed in terms of their relevance to the interpretation of quantum yield and enhancement data. Several laboratories have presented evidence recently that chloroplasts, after undergoing a pretreatment which destroys their capacity to evolve oxygen, can still transfer electrons via both photosystems, provided an appropriate electron donor is added. It is shown that electron donors like manganous ions and ascorbate can compete effectively with water as electron donors to Photosystem II, in untreated chloroplasts.     

EFFECTS OF ACRIDINE PLUS NEAR ULTRAVIOLET LIGHT ON ESCHERICHIA COLI MEMBRANES AND DNA IN VIVO

Results from a variety of experiments indicate that photodynamic damage to E. coli treated with the hydrophobic photosensitizer acridine plus near-UV light involves both cell membranes and DNA. Split-dose survival experiments with various E. coli mutants reveal that cells defective in rec A, uvr A, or pol A functions are all capable of recovery from photodynamic damage. Alkaline sucrose gradient analysis of DNA from control and treated cells revealed that acridine plus near-UV light treatment converts normal DNA into a more slowly sedimenting form. However, the normal DNA sedimentation properties are not restored under conditions where split-dose recovery is effective. Several lines of evidence suggest that membrane damage may be important in the inactivation of cells by acridine plus near-UV light. These include (a) a strong dependence of sensitivity on the fatty acid composition of the membranes; (b) a strong dependence of sensitivity on the osmolarity of the external medium; and (c) the extreme sensitivity of an E. coli mutant having a defect in its outer membrane barrier properties. Direct evidence that acridine plus near-UV light damages cell membranes was provided by the observations that (a) the plasma membrane becomes permeable to o-nitrophenyl-ß-D-galactopyranoside and (b) the outer membrane becomes permeable to lysozyme after treatment. A notable result was that cells previously sensitized to lysozyme by exposure to acridine plus near-UV light lose that sensitivity upon subsequent incubation. This strongly suggests that E. coli cells are capable of repairing damage localized in the outer membrane.     

TEMPERATURE AND MYCOCHROME SYSTEM IN NEAR-UV LIGHT INDUCIBLE AND BLUE LIGHT REVERSIBLE PHOTOINDUCTION OF CONIDIATION IN Alternaria tomato*

Abstract— When dark-grown colonies were exposed to near-UV light, conidiophore formation was induced, and conidia developed during a subsequent dark period. Simultaneous exposure to near-UV and blue light inhibited induction of conidiation. The inductive effect of near-UV light irradiation was greatly enhanced by treating colonies with low temperatures for 4 h during the 2nd-6th hour of incubation in the dark following inductive irradiation: the enhancement was greatest at 21^°C. The extent of inhibition by blue light increased with the temperature between 10 and 28^°C. This diminution by low temperature was greatest when colonies previously kept at a low temperature were exposed to inductive irradiation: the longer the duration between inductive irradiation and temperature treatment, the lower the diminution. Higher fluences of blue light were required to suppress conidial induction at lower temperature. Thus, it is evident that the inductive effect of near-UV light irradiation on conidiation and the suppressive effect of blue light irradiation are each responsive to different temperatures applied at different times.     

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.     

PHOTOCHEMICAL DAMAGE TO DNA TREATED WITH CHLORPROMAZINE AND NEAR UV RADIATION UNDER AEROBIC AND ANAEROBIC CONDITIONS

Double stranded salmon sperm DNA in a chlorpromazine (CPZ) solution is damaged when irradiated with near UV light. The damage of irradiated DNA can be estimated by measuring the increase in extinction at 260 nm following incubation at 60°C of the DNA with formaldehyde. Moreover, DNA irradiated in the presence of CPZ or kept in the dark separate quite differently in an aqueous polymer two-phase system. DNA irradiated in the presence of CPZ seemed to be susceptible to digestion by endonuclease S1, while the endonuclease of Neurospora crassa could not digest this DNA. Irradiation under aerobic conditions seemed to be less disastrous for DNA than under anaerobic conditions.     

THREE LIGHT REACTIONS AND THE TWO PHOTOSYSTEMS OF PLANT PHOTOSYNTHESIS*

Abstract— Recent work in our laboratory yielded new evidence that noncyclic electron transport in chloroplasts from water to ferredoxin (Fd) and N ADP is carried out solely by System II which, unexpectedly, was found to include not one but two photoreactions (IIa and IIb). The evidence suggests that these operate in series, being joined together by a ‘dark’ chain of electron carriers that includes (but is not limited to) cytochrome b₅₅₉ and plastocyanin (PC): H₂O → IIb_hv→ C550 → Cyt b₅₅₉ rarr;PC→IIa_hv→ Fd → NADP. Photoreaction IIb involves an electron transfer from water to C550, a new chloroplast component distinct from cytochromes, whose photoreduction is observed as a decrease in absorb-ance with a maximum at 550 nm. The photoreduction of CSSO proceeds effectively only in short-wavelength System II light, is insensitive to low temperature (at least down to — 189°C). does not require plastocyanin, and is the first known System II photoreaction which is resistant to inhibition by DCMU or o-phenanthroline. Photoreaction IIa involves an electron transfer from cytochrome b₅₅₉ to ferredoxin-NADP and also proceeds effectively only in System II light. The photooxidation of cytochrome b₅₅₉ requires plastocyanin. Cytochrome b₅₅₉ is reduced by C550 in a reaction that is readily inhibited by DCMU or o-phenanthroline. Thus, the site of DCMU (and o-phenanthroline) inhibition of System II appears to lie between C550 and cytochrome b₅₅₉. System I, comprising a single long-wavelength light reaction and a cyclic electron transport chain that includes cytochromes b₆ and f, is viewed as operating in parallel to System II. The photoreduction of NADP by artificial electron donors via System I involves a portion of the cyclic electron transport chain and appears to be independent of plastocyanin. Chloroplast fragments have been prepared which either (a) exhibit System II activity (water → NADP) and lack functional cytochrome f and P700 or (b) exhibit System I activity and lack plastocyanin. The present concept is consistent with the following: (i) No enhancement effect was found for NADP reduction by water where only System II is thought to be involved, but a large enhancement effect was observed in chloroplasts engaged in complete photosynthesis where both cyclic (System I) and noncyclic photophosphorylation (System II) are needed for CO₂ assimilation. (ii) The transfer of one electron from water to ferredoxin via System II requires optimally two quanta, but the transfer of one electron from reduced dye to ferredoxin via System I requires optimally only one quantum of light.     

VISIBLE LIGHT IRRADIATION OF HUMAN AND BOVINE SERUM ALBUMIN-BILIRUBIN COMPLEX*

Abstract— The UV absorption and the fluorescence emission spectra of both bovine (BSA) and human (HSA) serum albumin underwent noticeable changes upon irradiation of their 1:1 complexes with bilirubin; both these phenomena are suggestive of the photosensitized modification of aromatic amino acid residues. Amino acid analysis showed that after relatively short irradiation times of both albumins, only histidyl and tryptophyl residues appeared to be affected to a significant extent. After 60min of irradiation, some decrease in the tyrosine content was also observed, especially for HSA.
Conformational studies, obtained by exposing unirradiated and irradiated BSA and HSA to denaturing agents, showed that the three-dimensional organization of the 15 min irradiated samples was slightly different from that of the native proteins. On the other hand, after 15 min of irradiation, the association constant of the bilirubin-albumin complexes decreased from 2.07 to 0.54×10⁸ M ^-1 for HSA and from 2.16 to 0.87×10⁷ M ^-1 for BSA.
These data indicate that the histidyl residues are relatively unimportant for maintaining the native tertiary structure of BSA and HSA, but they are critical for determining the binding capacity of the albumins. Our data also imply that the tertiary structure of the BSA molecule is more labile than that of HSA.     

ACTION SPECTRA FOR SUPEROXIDE GENERATION AND UV AND VISIBLE LIGHT PHOTOAVOIDANCE IN PLASMODIA OF Physarum polycephalum

Abstract— The initial photochemical process leading to photoavoidance by plasmodia of an albino strain of Physarum Plasmodium was studied. Superoxide (O), detected as superoxide dismutase (SOD)-inhibitable electron spin resonance (ESR) signal of a spin trap (tBN), was formed upon irradiation. The amount of O formed increased linearl) with log fluence rate above the threshold. The photoavoidance to radiation at wavelengths between200–800 nm also showed the similar linear relationship in log fluence rate-response curves. Thresholds for photoavoidance and O generation agreed with each other and the action spectra showed peaks at about 260, 370, and 460 nm. Thus, active oxygen generated by photosensitization seems to trigger the UV and blue light photoavoidance.     

FLASH PHOTOLYSIS OF SEVERAL AROMATICS BY ULTRAVIOLET LIGHT AND VISIBLE LIGHT IN THE PRESENCE OF EOSIN Y*

Abstract— A flash photolysis investigation was made of the photo-oxidation of aqueous aniline, resorcinol, βnaphthol, p-sulfanilic acid, and p-bromophenol induced by ultraviolet and visible light irradiation in the presence of eosin Y. The transient spectra show that u.v. irradiation generates the hydrated electron (except in p-bromophenol) and the radical products of one-electron oxidation. The initial products of the eosin-sensitized oxidations are the dye semi-quinone and aromatic radicals which coincide with the u.v. photolysis products in at least several cases. The investigation of the reaction kinetics by rapid spectrophotometry with analog computer analysis shows that the aromatics quench the triplet state of eosin and also react with it in a slower electron-transfer process, in competition with ‘dye-dye’ quenching and electron-transfer reactions. The u.v. and dye-sensitized oxidations are discussed in terms of their energetics.