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.     

VARIATION IN THE PHOTOCHEMICAL REACTIVITY OF THYMINE IN THE DNA OF B. SUBTILIS SPORES, VEGETATIVE CELLS AND SPORES GERMINATED IN CHLORAMPHENICOL*,†

Abstract— The chief photoproduct of thymine produced in u.v. irradiated (2537Å) vegetative cells of B. subtilis is the cyclobutane-type dimer while in spores very little of this dimer is produced (maximum yield 2·6 per cent of thymine) but a new photoproduct is produced in high yield (maximum of 28·4 per cent of thymine). This difference in photochemical response appears to be due, at least in part, to a difference in uydration of the DNA. The photochemistry of thymine in isolated DNA irradiated in solution is similar to that of DNA in irradiated vegetative cells, but differs markedly from that of isolated DNA irradiated dry. The yield of cyclobutane-type thymine dimer is much reduced in isolated DNA irradiated dry but a new photoproduct of thymine. is produced which is chromatographically similar to the spore photoproduct. The yield of this photoproduct, however, is never as great as that obtained in irradiated spores. The photochemistry of the DNA thymine of spores germinated in the presence of chloramphenicol is very similar to that of normal vegetative cells. Except for hydration, the physical state of the DNA is probably not otherwise altered by germination in the presence of chloramphenicol since DNA replication is prevented by the presence of chloramphenicol. These results are also consistent with the hypothesis that the unique photochemistry of spores is due, at least in part, to the hydration state of the DNA. The acid stability of the spore photoproduct is indicated by the fact that it is isolated from irradiated spores after hydrolysis in trifluoroacetic acid at 155°C for 60 min. It still contains the methyl group of thymine as judged by the fact that for a given dose of u.v. the same yield of photoproduct was obtained whether the spores were labeled with thymine-2–C-14 or -methyl-C-14. This photoproduct is stable to reirradiation (2537Å) in solution under condiditions where thymine dimers of the cyclobutane-type are completely converted back to monomeric thymine. On a column of molecular sieve material (Sephadex-G10), the spore photoproduct elutes in a region intermediate between the cyclobutanetype thymine dimers and monomeric thymine. Of the numerous compounds tested by paper chromatography, the spore photoproduct is most similar (but not identical) in several solvents to 5–hydroxyuracil and 5–hydroxymethyluracil. Our data do not allow us to decide if the product is a monomer or a dimer. Although the photochemistry of thymine in the DNA of spores differs markedly from that for vegetative cells, several lines of evidence make it seem doubtful that the enhanced resistance of spores to u.v. relative to that of vegetative cells can be explained solely on the basis of this difference in the photochemistry of DNA thymine.     

PHOTOREACTIVITY OF HYDROXYPSORALENS AND THEIR PHOTOBIOLOGICAL EFFECTS IN BACILLUS SUBTILIS*

Abstract— Although psoralen and many substituted psoralens are potent skin-photosensitizing agents, hydroxypsoralens are not. A satisfactory molecular interpretation of this structural specificity has been given in terms of dissociation of the hydroxyl group of 5- and 8-hydroxypsoralens in their excited states. The dissociation process in the S₁ state effectively competes with S₁→T₁ intersystem crossing, thus reducing the photoreactive T₁ population. The T₁ states of the anions are more delocalized than those of neutral psoralens so that they are less reactive toward photocycloaddition with pyrimidine bases of DNA. The lack of significant phosphorescence of hydroxypsoralens in ionizing solvent or in the presence of base at low temperatures (14–77 K.) indicates ineffective S₁→T₁ and/or effective T₁→S₀ intersystem crossing. These factors make hydroxypsoralens unreactive, electronically and kinetically, as skin photosensitizers, which are known to react with DNA. In correlation with the hydroxypsoralens' spectroscopic characterization, they are also found to be ineffective or less effective photosensitizers in Bacillus subtilis.     

EFFECTS OF FLUENCE FRACTIONATION ON UV-INDUCED MALIGNANT TRANSFORMATION AND CELL KILLING IN NON-CYCLING PLATEAU PHASE MOUSE CELLS

Plateau phase C3H 10T 1/2 mouse cells were used to measure the response to split fluence UV light irradiation in the absence of any cell cycle effects. It was found that fluence fractionation with up to 24 h between the fluences resulted in no survival enhancement. The frequency of malignant transformation was potentiated 2.5-fold when the time interval between the fluences was greater than 4h. This potentiation of transformation was attributed to plateau phase holding rather than to fluence fractionation per se.     

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.     

TIME COURSE OF PHOTOREACTIVATION OF UV-INDUCED CHROMOSOMAL ABERRATIONS AND LETHAL DAMAGE IN INTERPHASE XENOPUS CELLS

Abstract— Sets of G1, S, and G2 phase Xenopus cells were exposed to 15.0 Jm⁻² UV and their ability to photoreactivate the induced cell killing (loss of colony forming ability) and chromosomal aberrations was determined as a function of time following the UV exposure. Most of the lesions induced in G1 cells that lead to cell death were converted to a non-photoreactivable state before the cells entered the S phase, while lesions leading to chromosomal aberrations were converted to a non-photoreactivable state as the cells entered the S phase. In S phase cells the UV-induced lesions leading to aberrations appeared to be converted to a non-photoreactivable state at a much faster rate than those leading to cell death. A significant fraction of the lesions induced in G2 cells, that lead to cell death, were converted to a non-photoreactivable state before the progeny of the exposed cells reached the next succeeding S phase. Few, if any, lesions were induced in G2 cells that were expressed as aberrations at the first mitosis following exposure. Some of the lesions induced in the G2 cells led to aberrations that were observable in the progeny that progressed to the second mitosis following exposure. These lesions were converted to a non-photoreactivable state as the progeny of the exposed G2 cells progressed through the first S phase following exposure. These results suggest that the intracellular mechanism which expresses photoreactivable UV-induced lesions as cell death is not identical to the mechanism which expresses such lesions as chromosomal aberrations, and the two mechanisms operate with different efficiencies in different phases of the cell cycle.     

反应条件对稻杆热解产物分布的影响

以稻杆为研究对象，在温度６５０℃－１０００℃，考察了热解因素包括最终温度，析出挥发性产物停留时间，传热与传质强化措施，原料颗粒尺寸和原料种类对稻杆在间壁式加热固定床上热解产物分布的影响。结果表明，温度，停留时间，传热与传质强化措施对热解产物分布影响显著，转化率随各种反应条件的变化可高达８０％。     

THE EFFECT OF TEMPERATURE AND WAVELENGTH ON PRODUCTION AND PHOTOLYSIS OF A UV-INDUCED PHOTOSENSITIVE DNA LESION WHICH IS NOT REPAIRED IN XERODERMA PIGMENTOSUM VARIANT CELLS

Abstract— Ultraviolet light causes a type of damage to the DNA of human cells that results in a DNA strand break upon subsequent irradiation with wavelengths around 300 nm. This DNA damage disappears from normal human fibroblasts within 5 h, but not from pyrimidine dimer excision repair deficient xeroderma pigmentosum group A cells or from excision proficient xeroderma pigmentosum variant cells. The apparent lack of repair of the ultraviolet light DNA damage described here may contribute to the cancer prone nature of xeroderma pigmentosum variant individuals. These experiments show that the same amount of damage was produced at 0^°C and 37^°C indicating a photodynamic effect and not an enzymatic reaction. The disappearance of the photosensitive lesions from the DNA is probably enzymatic since none of the damage was removed at 0^°C. Both the formation of the lesion and its photolysis by near ultraviolet light were wavelength dependent. An action spectrum for the formation of photosensitive lesions was similar to that for the formation of pyrimidine dimers and(6–4) photoproducts and included wavelengths found in sunlight. The DNA containing the lesions was sensitive to wavelengths from 304 to 340 nm with a maximum at 313 to 317 nm. This wavelength dependence of photolysis is similar to the absorption and photolysis spectra of the pyrimidine(6–4) photoproducts     

INACTIVATION ACTION SPECTRA OF BACILLUS SUBTILIS SPORES IN EXTENDED ULTRAVIOLET WAVELENGTHS (50–300nm) OBTAINED WITH SYNCHROTRON RADIATION

Five types of Bacillus subtilis spores (UVR, UVS, UVP, RCE, and RCF) differing in repair and/or recombinational capabilities were exposed to monochromatic radiations at 13 wavelengths from 50 to 300 nm in vacuum. An improved biological irradiation system connected to a synchrotron radiation source was used to produce monochromatic UV radiation in this extended wavelength range with sufficient fluence to inactivate bacterial spores. From the survival curves obtained, the action spectra for the inactivation of the spores were depicted. Recombination-deficient RCE (recE) and RCF (recF) spores were more sensitive than the wild-type UVR spores in the entire range of wavelengths. This was considered to mean that DNA was the major target for the inactivation of the spores. Vacuum-UV radiations of 125-175 nm were effective in killing the spores, and distinct peaks of the sensitivity were seen with all types of the spores. Insensitivities at 190 and 100 nm were common to all five types of spores, indicating that these wavelengths were particularly impenetrant and absorbed by the outer layer materials. The vacuum-UV peaks centering at 150 nm were prominent in the spores defective in recombinational repair, while the far-UV peaks at around 235 and 270 nm were prominent in the UVS (uvrA ssp) and UVP (uvrA ssp polA) spores deficient in removal mechanisms of spore photoproducts. Thus, the profiles of the action spectra were explained by three factors; the penetration depth of each radiation in a spore, the efficiency of producing DNA damage that could cause inactivation, and the repair capacity of each type of spore.     

INTERACTION OF 8-METHOXYPSORALEN AND NEAR-UV LIGHT CAUSES MUTATION AND CYTOTOXICITY IN MAMMALIAN CELLS

Abstract The interaction of near-UV light and a photosensitizer, 8-methoxypsoralen (8-MOP), was studied in the Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyl transferase system; cell survival (cloning efficiency) and mutation induction (resistance to 6-thioguanine) were quantified. Exposure of cells to either 8-MOP up to 20 μg/m l (93 μ M ) or near–UV light up to 40000 J/m² had no effect on either survival or mutation frequency. Preincubation of cells with 8–MOP from 5 to 120 min prior to irradiation with various fluences did not affect cell survival or mutation frequency. Survival decreased and mutation frequency increased linearly when either the 8-MOP concentration or fluence was increased while the other factor was held as a constant. Mutation frequency appears to show reciprocity relative to the product of 8-MOP concentration times fluence of near–UV light [(μg/m l )·(J/m²)] throughout a range apparently limited by high cell lethality. The observed pooled data on mutation, f (x), as a function of (μg/m l )·(J/m²), x , fit a linear dose–response line, f (x) = (34.2 + 0.05 x ) × 10^-6. Cell survival, however, does not appear to exhibit such reciprocity.     

STUDY OF THE EFFECTS OF ULTRAVIOLET LIGHT ON BIOMEMBRANES—IV. THE EFFECT OF OXYGEN ON UV-INDUCED HEMOLYSIS AND LIPID PHOTOPEROXIDATION IN RAT ERYTHROCYTES AND LIPOSOMES

Abstract— Hemolysis induced by irradiation with ultraviolet (UV) light at 254 nm showed a pronounced oxygen effect: under irradiation in vacuum, the rate of hemolysis was decreased by an order of magnitude. Irradiation at 254 nm in air but not under vacuum caused the peroxidation of erythrocyte membrane lipids. These results suggest that membrane lipid photoperoxidation is one of the causative factors of UV hemolysis. Irradiation at different wavelengths showed that UV-induced lipid photoperoxidation in erythrocyte membranes developed while the antioxidant α-tocopherol was directly photooxidized. It is shown that the process of lipid photolysis in erythrocyte membranes involves sensitization, possibly by protoporphyrin, whose presence in liposomes accelerates the photoperoxidation at 254 and 365 nm of unsaturated fatty acid residues in lecithin. Possible mechanisms of photochemical damage to erythrocyte membranes are discussed.     

APPLICATION OF TIME-RESOLVED DIFFUSE REFLECTANCE TECHNIQUES IN STUDIES OF REACTION INTERMEDIATES IN SUSPENSIONS OF BACILLUS SUBTILIS

Short lived reaction intermediates such as triplet states and free radicals can be detected in vivo using laser photolysis techniques with time-resolved diffuse reflectance detection. This novel approach is illustrated for bacterial suspensions of Bacillus subtilis.     

UV-INDUCED ALKALINE LABILE DNA DAMAGE IN HUMAN ADENOVIRUS AND ITS REPAIR AFTER INFECTION OF HUMAN CELLS

Abstract— UV-induced alkaline labile viral DNA damage was detected following irradiation of adenovirus type 2 and found to be repaired following the infection of human KB cells. Human adenovirus type 2 was irradiated with various doses of UV and subsequently used to infect human KB cells in tissue culture at approximately 2 × 10³ particles per cell. Before, and at various times after infection, the viral DNA was examined on alkaline sucrose gradients. Irradiated free virus DNA showed a dose dependent decrease in molecular weight compared to unirradiated virus DNA, indicating the presence of UV-induced alkaline labile lesions. Furthermore, an increase in the molecular weight of the irradiated virus DNA was found after infection indicating that alkaline labile lesions were removed from the viral DNA by a host mediated repair mechanism. After infection, the molecular weight of the irradiated virus DNA reached a value similar to that of unirradiated virus DNA for all the UV doses studied.     

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.     

EFFECTS OF UV, SUNLIGHT AND X-RAY RADIATION ON QUIESCENT HUMAN CELLS IN CULTURE

Nondividing human diploid fibroblasts (HDF) in culture have been used to study the effect on cell lethality of ultraviolet light, natural sunlight and X-rays. A lethal effect is defined as cellular degeneration, loss from the culture and inability to exclude vital strains. Far-and mid-UV have a readily observable lethal effect (cell loss), with DNA and DNA damage as the critical target and critical damage respectively. In part, natural sunlight kills cells by a similar mechanism but has an additional lethal effect at longer exposure times. This additional effect is expressed by the retention of the dead cells in culture, in contrast to the UV-induced promotion of cell degeneration and loss. Relatively large doses of X-rays that destroy proliferative capacity, have no detectable lethal effect on the maintenance of nondividing cells. The biological response of nondividing HDF to radiations from different parts of the electromagnetic spectrum is dissimilar.     

ULTRAVIOLET EFFECTS ON KILLING, FRUITING BODY FORMATION AND THE SPORES OF DICTYOSTELIUM DISCOIDEUM

Abstract— -Ultraviolet effects on amoeboid cells of three strains of Dictyosrrlium discoidruin , NC-4. γs-13 and γs-18. for killing. fruiting body formation, spore formation and viability of the spores were studied.
The strain of γs-13 was more sensitive to UV light for killing than NC-4 at 10% survival. In addition. γs-13 was the most sensitive strain among the three for fruiting body formation and spore formation. The developmental process of this organism, however, showed a large resistance to UV light when compared with the killing. The spores of γs-13 formed after UV irradiation were mostly non-viable, though those of γs-18 and NC-4 were fully viable     

EFFECTS OF ULTRAVIOLET LIGHT ON THYMIDINE INCORPORATION AND DNA CHAIN ELONGATION IN PHOTOREACTIVABLE INSECT CELLS

An insect cell line, IAL-PID2, was exposed to UV and analyzed for its ability to incorporate [3H]-thymidine and to elongate replicon-sized DNA fragments. After exposure to 5 or 10 J/m2 UV, the cells exhibited a rapid and prolonged depression in the rate of thymidine incorporation. Photoreactivation reduced this depression but did not entirely reverse it. For exposures of 5 J/m2 or above, full recovery did not occur until 18 h after exposure. The blockage of fork progression after UV exposure was fluence-dependent, with replication segments after exposure to 20 J/m2 being shorter than those observed after exposure to 10 J/m2. Immediately after exposure to either 10 or 20 J/m2, photoreactivation reversed blockage of fork progression, indicating that the (5-6) cyclobutyl pyrimidine dimer is responsible for blockage. This also indicates that blockage of fork progression may not be the only factor responsible for the prolonged depression seen in thymidine incorporation. Three hours after exposure to either 10 or 20 J/m2, replication segments were still significantly shorter than control segments. Photoreactivation completely reversed blockage after exposure to 10 J/m2, but did not completely reverse blockage after exposure to 20 J/m2, indicating that at such fluences, other lesions may play a role in UV-induced blockage of fork progression.     

STIMULATORY AND INHIBITORY EFFECTS OF IRON ON PHOTODYNAMIC INACTIVATION OF LEUKEMIA CELLS

The influence of exogenous iron on merocyanine 540 (MC540))sensitized photoinactivation of leukemia cells has been investigated. Irradiation of murine L1210 or human HL-60 cells (-lOh/mL in 1 % serumiRPM1 medium) with broadband visible light in the presence of MC540 (2 pIM) resulted in a progressive loss of clonally assessed cell viability. When added to cells 30 min before irradiation, the low polarity chelate, ferric 8-hydroxyquinoline [Fe(HQ), 0.5 *MI stimulated dye-sensitized pho- tokilling, whereas high polarity chelates such as ferric 8-hydroxyquinoline-5-sulfonate [Fe(HQS)2, 0.5 p M] or ferric ethylenediaminetetraacetate (Fe.EDTA, 0.5 FM) had no effect. A striking reversal of Fe(HQ),-enhanced photokilling was observed upon increasing the preirradiation incubation time with Fe(HQ)2 such that a marked resistance (relative to non-iron-treated controls) was evident after 24 h. Cells exposed for 24 h to Fe(HQS), or Fe.EDTA showed similar or even greater resistance to photo-killing. Like phototoxicity, H,O,-induced cytotoxicity was enhanced after a 30 min exposure of cells to Fe(HQ)2 but strongly repressed after 24 h. Immunoblot (western) analysis, using a polyclonal antibody to ferritin, revealed that cells exposed to Fe(HQ): for 24 h contained at least 12 times as much ferritin heavy chain as non-Fe(HQ)?-treated controls. Preincubating cells with emetine, an inhibitor of protein synthesis, prevented both ferritin induction and the development of hyperresistance. These findings, along with the observation that exogenous apoferritin protected LI 2 10 cells against photokilling, suggest a possible role for ferritin in iron-stimulated photoresistance. It is conceivable that in photodynamic treatment of tumors, certain cells might resist inactivation via this mechanism, a possibility that has not been recognized heretofore.