PHOTOREVERSION VON VIER GRUPPEN UV-INDUZIERTER MUTATIONEN ZUR GIFTRESISTENZ IM NICHTPHOTOREAKTIVIERBAREN E. COLI

Abstract— In the non-photoreaclivable bacterial strain E. coli B/phr^-/MC2 the photoreversion of four groups of u.v.-induced mutations were investigated. They lead to resistance to Chloramphenicol (2 mg/l; "C"), Penicillin (13 or 16 mg/l; "P13" and "P16") or Streptomycin (3 mg/l; "S"). The u.v.-dose curve is concave for the C-mutations (two to three hits), about linear for P13 and S, and they reach peaks and decrease at high u.v.-doses. Though no photoreactivation of killing (PR) is present there is photoreversion of all four types of mutations (PRM). At u.v.-doses below the peaks in average about 43 per cent mutations are photoreversible. At high u.v.-doses the curves with light-post treatment (L) cross the darkcurves (D). In the photoreactivable strain B/r (by the spontaneous mutation MC2 to Mitomycin-resistance strain B/phr^- was made about as u.v.-resistant as B/r is) the photoreversion of the mutation groups C, P13 and P16 (S was not investigated here) was much higher, in average about 77 per cent at low doses. It is assumed that the difference in PRM of about 34 per cent between both strains is due to a PRM-mechanism present in B/r but not in B/phr^-/MC2; this mechanism may be the photoreactivating enzyme that opens thymine-dimers. The PRM in B/phr-/MC2 must then be due to a second mechanism which is probably not the dimer opening enzyme. It may be the same mechanism as in the case of mutations of phage kappa which are induced by u.v. and reversed partially by light, both extra cellularly. The premutations giving this second type of PRM may perhaps be cytosine-hydrate in the DNA. Tn average about 23 per cent mutations of B/r are photostable. Since this ratio decreases with low u.v.-doses in the C-mutations and increases in P13 and in P16 probably two types of photostable premutations seem to exist.     

SUPPRESSION OF PHAGE T4 BY CO-INFECTION WITH U.V.-INACTIVATED HOMOLOGOUS PHAGE

Abstract— Unirradiated phage T4v₁ may fail to produce viable progeny in cells which are co-infected with u.v.-irradiated homologous particles. The extent of this effect, called suppression , is positively correlated with the multiplicity of infection of the irradiated phage and with the U.V. dose. The suppression reaches a maximum level at about 30–600 lethal hits. Quantitative evaluation of the results shows that in some complexes one irradiated phage particle is sufficient to suppress an unirradiated phage. Two hypotheses are discussed to explain the results. (a) Lethal u.v.-damages are incorporated into the unirradiated phage genome by genetic recombination; ( b ) Genetic subunits (e.g. cistrons or operons) of the u.v.-irradiated phages produce informationally incorrect messenger RNA molecules, which compete with the correct ones from the unirradiated phage in the protein-synthetizing system. Hypothesis (6) appears to be more adequate to explain the experimental results.     

CHEMICAL FILTERS FOR NARROW BAND U.V. IRRADIATION BETWEEN 235 AND 300 nm

Abstract— Six chemical filters are described. Bands of 10–20 nm half width are isolated at the following wavelengths: 235–240–252–260–277–294 nm. The filters transmit at the center of their band pass between 1.5 and 30 per cent of the incident radiation. Average photon fluence rates of 15–150 ergs mm^-2 sec^-1 (0.3 × 10^-3 to 3.5 × 10^-3μE cm^-2 sec^-1) are obtained with a 500 W high pressure mercury arc focussed on a 10 cm^-2 area. Under these conditions, the flux transmitted in the near i.r., visible and near u.v. does not exceed 1 per cent of the total flux transmitted. The flux transmitted outside a 40 nm band centered at the maximum does not exceed 1 per cent of the total transmitted flux for four of the filters, this flux goes up to 4 per cent of the total for the 294 nm filter and to 10 per cent for the 235 nm filter.     

RELATIONSHIP BETWEEN SURVIVAL AND THYMINE-DIMER EXCISION IN ULTRAVIOLET-IRRADIATED ESCHERICHIA COLI

Abstract— The influence of amino acid prestarvation on both the resistance to u.v. light and excision of thymine dimers of bacterial strains E. coli B/r hcr ⁺ thy- trp ^-, E. coli B/r hcr ^-thy^- trp ^-, and E, coli 15 T^- 555–7 thy ^- meth ^- trp ^- arg ^- has been studied.
The prestarvation increased the resistance of all the strains but reasonably inhibited excision of thymine dimers. Thus the enhancement of u.v. resistance after amino acid prestarvation was not due to more complete excision of thymine dimers.     

CARBOXYLATION OF FORMIC ACID IN AQUEOUS SOLUTIONS UNDER THE INFLUENCE OF U.V.-LIGHT

Abstract— Carboxylation of various organic substances by dissolved carbon dioxide or bicarbonate is achieved by the action of U.V. -Light of 184.9 and 253.1 mμ on water. The transformation of formic acid to oxalic acid was investigated in more detail as a function of u.v.-dose, pH-values and the formate concentration. For an irradiation period of 12 hr of 0.1 M formate, a yield of about 2 m mol.1^--¹ oxalic acid was obtained. A reaction mechanism for the photochemical carboxylation process is presented. The results are of some interest in respect of the synthesis of organic compounds under the primitive earth conditions.     

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.     

PHYTOCHROME CONVERSION BY ULTRAVIOLET LIGHT

Abstract— Light absorbed primarily by the protein of phytochrome is active in transforming both the red and far-red absorbing forms. P _r and P _fr. The ratio of quantum yields for the conversions of P _r and P _fr by u.v. radiation (φ_r/φ_fr)_u.v.= 1.5 and does not differ significantly from the ratio obtained with red and far-red light absorbed directly by the chromophores (φ_r/φ_fr)v_vis. Thus, the efficiency of energy transfer from protein to chromophore is essentially the same for both forms of the chromoprotein. The ratio of the relative quantum yields for u.v. and visible light (φ^r)^u.v./(φ^r)^vis was 0.32 indicating that 30–35 per cent of the light energy absorbed by the protein was transferred to the chromophore.     

ON SOME POSSIBILITIES OF PROTECTING CELLS FROM DAMAGING EFFECTS OF ULTRAVIOLET IRRADIATION

Abstract. The possibility of increasing the cell resistance to short-wave u. v. rays has been investigated with some chemicals. Ciliated epithelium cells of gills of pearly mussel Unio crassus Phillipsson have been shown to become more resistant after treatment with 0^.25–0.50 per cent solutions of calcium chloride during 24 hr or by 0.001– 0.001 per cent solutions of 2-benzyl-benzimidazole during 3–7 days or by 0.0001 per cent solution of 5–6-di-methyl-benzimidazole during 7 days. When irradiated with u. v. light (λ=2537 Å) these cells survive for a longer time than irradiated control cells.     

A STUDY OF THE RELATIONSHIP BETWEEN SURVIVAL AND REPAIR SYNTHESIS'' OF DNA AFTER ULTRAVIOLET LIGHT EXPOSURE

Abstract— The influence of amino acid prestarvation on both the resistance to u.v. and the postirradiation repair synthesis of E. coli 15 T^- 555-7 thy meth arg trp and E. coli B/r (HCR⁺) was followed. Prestarvation increased the number of survivors about 30–100 fold in both strains at doses 600-1200ergs/mm². In contrast to survival no increase in repair synthesis was observed. Thus, the increase in survival has to be brought about by a mechanism which seems to be independent of additional repair synthesis.     

PHOTOLESIONS IN DNA UPON 193 nm EXCITATION

Aqueous solutions of plasmid (pBR322 and pTZ18R) and calf thymus DNA were excited by 20 ns laser pulses at 193 nm. The quantum yields of single- and double-strand break formation, interstrand cross-links, locally denatured sites, (6–4)photoproducts and biological inactivation (Φ_ssb, Φ_dsb, Φ_icl, Φ_ids, Φ_6–4 and Φ_ina, respectively) were measured. The quantum yields are virtually independent of intensity, demonstrating a one-quantum process. The obtained values in aerated neutral solution in the absence of additives are Φ_ssb= 1.5 × 10^--³, Φ_dsb, = 0.06 × 10^--³ (dose: 10–200 J m^-2), Φ_icl=Φ_Ids= 0.1 × 10³ and Φ_6–4= 0.5 × 10^--³ Both Φ_ssb and Φ_dsb decrease strongly with increasing concentrations of TE buffer (0.01–10 m M ). Biological inactivation of the pTZ18R plasmid was determined from the transformation efficiency of Escherichia coli bacteria strains AB1157, AB1886 uvr and A82480 uvr rec; the Φ_ina values are 1.4 × 10³, 2.1 × 10³ and 3 × 10^--³, respectively. The monoexponential survival curves in all cases show that a single damage site leads to inactivation (one single hit). The biological consequences of different photoproducts are discussed.     

Measurement of Tumor Vascular Damage in Mice with 99m Tc-MIBI Following Photodynamic Therapy

Abstract— The clinical perfusion agent ^99mTc-MIBI was used to monitor changes in tumor vascular perfusion (TVP) induced by Photofrin® (Pll)-mediated photodynamic therapy (PDT). BALB/c mice bearing an EMT-6 tumor on each hind thigh were given an intravenous injection of 1, 2 or 5 mg kg⁻¹ PII. Twenty-four hours later, one tumor was illuminated (600–650 run, 200 mW cm⁻² 400 J cm⁻²) while the other served as a control. At various time intervals after PDT (0, 2 and 24 h) mice received an intravenous injection of ^99mTc-hexakismethoxy(sobutyusonitri-le (MIBI) (0.18 MBq g⁻¹) and were sacrificed 2 min later. The light-treated and the untreated tumors were then dissected, the radioactivity was counted and the percentage of the injected dose per gram of tumor (%ID g⁻¹) was calculated as a measure of TVP. We observed that TVP is drug dose dependent, develops progressively with time post-PDT and is inversely related to PDT efficacy. Our data show that early tumor retention of ^99mMIBI is a simple method to assess TVP and vascular damage induced by PDT.     

In vitro survival of nonsmall cell lung cancer cells following combined treatment with ionizing radiation and photofrin-mediated photodynamic therapy

It has been suggested that combination high dose rate (HDR) intraluminal brachytherapy and photodynamic therapy (PDT) in nonsmall cell lung cancer (NSCLC) may improve efficacy of treatment, reduce toxicity and enhance quality of life for patients. To provide a cellular basis for this we examined the in vitro sensitivity of MRC5 normal lung fibroblasts and four NSCLC cell lines following HDR radiation, PDT and combined HDR radiation and PDT. HDR radiation was cobalt-60 gamma rays (1.5–1.9 Gy min⁻¹). For PDT treatment, cells were exposed to 2.5 μg mL⁻¹ Photofrin for 18–24 h followed by light exposure (20 mW cm⁻²). For combined treatment cells were exposed to Photofrin and then either exposed to light and 15–30 min later exposed to HDR radiation or exposed to HDR radiation and 15–30 min later exposed to light. D₃₇ values calculated from clonogenic survival curves indicated a six-fold difference in HDR radiation sensitivity and an eight-fold difference in PDT sensitivity. The effect of combined treatment was not significantly different from an additive effect of the individual treatment modalities for the NSCLC cells, but was significantly less than additive for the MRC5 cells. These results suggest an equivalent tumor cell kill may be possible at reduced systemic effects to patients.     

PHOTOCHEMINE ANOMALER NUCLEINSÄUREBAUSTEINE-I. PHOTODEGRADATION VON AZAPYRIMIDINEN UND AZAPURINEN

Abstract— The photodegradation of Azapyrimidines and Azapurines by intensive u.v.-irradiation has been investigated and compared with some pyrimidines and purines. The main output at 253.7 nm of our special low pressure mercury lamp which surrounded the cylindrical cell, was 6.7 times 10^-6 einstein cm^-2 min^-1. The concentrations (5 times 10^-4 M in phosphate buffer pH 7) of the irradiated substances decrease exponentially with half-lifetimes between 2 and more than 60 min. In contrast to the very high stability of adenine, 8-Azaadenine was degradated in a few minutes only. 6-Azauracil also reacts rapidly in its excited state with solvent and the first stable photoproduct one can enrich is its hydratation product. By heating of the hydratation product the 6-Azauracil was regenerated. The changes in stability:
Azauracil > Uracil
Azaadenine < Adeninc
Azaguanine < Guanine
are of great interest in the field of photomutagenis of DNA, modifies by azabases.     

PHOTODYNAMIC EFFECTS OF PROFLAVINE ON BACTERIOPHAGE φ times 174 AND ITS ISOLATED DNA

Abstract. Proflavine-mediated photoinactivation of φ times 174 phage and its isolated DNA was studied under identical irradiation conditions. The inactivations followed single-hit kinetics and a linear relationship was obtained in reciprocal plots of the inactivation rates vs the proflavine concentrations for both phage and isolated DNA. The phage photoinactivation rate was increased with an increase in the amount of proflavine bound to the phage DNA in a strong binding range (0.01-0.04 proflavine/ nucleotide) as the total proflavine concentration was increased or the ionic strength decreased. Further, a phage-specific factor was also found to affect the inactivation rate. The photodynamic treatment induced mutations in three phage strains from "amber" to "wild type" at a mutation rate per lethal hit of 0.3 times 10^-5 to 2.6 times 10^-5. In contrast to phage infectivity, the φ times 174 DNA infectivity was measurable only at a high multiplicity of infection, and its photoinactivation occurred only at high proflavine concentrations. The photoinactivation rate was enhanced either with a decrease in the multiplicity of infection or with the use of spheroplasts of recA mutants strains. The results are discussed in terms of the nature of and possible repair mechanisms of photodynamically induced lesions in φ times 174 phage DNA.     

DOES LOW-INTENSITY He-Ne LASER RADIATION PRODUCE A PHOTOBIOLOGICAL GROWTH RESPONSE IN Escherichia coli?

Abstract A photobiological study was camed out on the bacterium Escherichia coli in order to determine whether stimulation of growth occurred after irradiation of an inoculum with coherent red light. No enhancement or inhibition of growth was observed for cultures of the bacterium following irradiation of inocula with a Helium-neon laser (continuous wave, λ= 632.8 nm) at irradiances of 7.7 × 10¹⁵ and 1.8 × 10¹⁶ photons cm⁻² s⁻¹ using fluences of 4.5 × 10^−-1 and 4.5 J cm⁻² at each irradiance. Bacterial growth in irradiated and control cultures was monitored during a growth period of ca 2 h using a viable count technique after inocula in the early exponential phase had been diluted with fresh growth medium. These results do not provide support for the work of Kam et al . (1983, Nuov. Cim . 2D, 1138–1144), and Tiphlova and Karu (1988, Photochem. Photobiol . 48 , 467–471), which appear to show substantial enhancement of E. coli growth under these conditions.     

EFFECTS OF DOSE FRACTIONATION ON ULTRAVIOLET SURVIVAL OF ESCHERICHIA COLI

Abstract— Exposure of E. coli B/r and B at low average dose rates of u.v. radiation (2537 Å), produced either by fractionated doses or by continuous irradiation at a very low dose rate (80 ergs/mm²/hr), results in much increased survival compared to single exposure at high dose rate. This increase is attributed to repair taking place during the irradiation period. The effect is small in the repair-deficient strains E. coli B_8-1_, and C syn- , and is absent in phage T1 and T4, which cannot undergo repair in the extracellular state. However, the prolonged time available for repair in these experiments accounts for only a very minor part of the increase in survival. The principal factor apparently is that the number of lesions present at any time remains relatively low. Presumably complete repair, not only the excision step, can occur in buffer during the irradiation period. This interpretation is supported by experiments in which cells were exposed to combinations of highly fractionated irradiation and single-dose irradiation. We therefore propose that mutual interference in repair, possibly by overlapping of repair regions in complementary DNA strands, reduces considerably the repair efficiency if many lesions are present. This hypothesis explains the 'shouldered' survival curves of B/r and possibly other E. coli strains as due to decreasing repair efficiency with increasing u.v. dose     

THE ROLE OF HOST-CELL REPAIR IN LIQUID-HOLDING RECOVERY OF U.V.-IRRADIATED ESCHERICHIA COLI

Abstract— The experiments reported give evidence that liquid-holding recovery (LHR) of u.v. irradiated E. coli cells involves basically the same type of dark repair which causes reactivation of phage and which results in much increased survival of the cells themselves [host-cell reactivation (HCR)]. LHR is very small in the two HCR(-) strains B syn^- and B_s-1, but occurs to larger but different extents in the three HCR(+) strains B, B/r, and B/r (Λ). LHR is inhibited if the liquid contains caffeine or acriflavine, both of which are known to inhibit HCR. The results indicate that most of the LHR effect, if not all, occurs during the liquid holding, rather than under growth conditions after liquid holding. It is assumed that the holding itself allows a prolonged time for, and therefore an enhancement of, HCR. It is thus implicit that LHR can be observed only where otherwise HCR of repairable u.v. damage would be incomplete, and that different extents of LHR, as observed in the three HCR(+) strains, reflect different extents of incompleteness of HCR. It is concluded that the repairable u.v. hits which are not fully repaired by HCR are predominantly those concerned with the extra u.v. sensitivity of the strains B and B/r (Λ), relative to B/r.     

UBER DIE FEHLENDE PHOTO— UND WIRTSZELLREAKTWIERBARKEIT DES UV-INAKTIVIERTEN RNS-PHAGEN FR

Abstract. The plaque forming ability of the E. coli phage fr, containing single stranded RNA, is highly UV-resistant. One lethal hit corresponds to approximately 1'2 × 10⁵ erg cm^-2. photoreactivation of fr due to illumination of infected bacteria has not been found after UV-inactivation of either extracellular or intracellular phage. This shows that the already known PR-enzyme of E. coli K12 is probably specific only for some of the UV-lesions in DNA but not in RNA. Furthermore, it may indicate that the photoreactivation of some RNA-containing plant viruse^(1,2) is due to a PR-enzyme which is not present in K12.
UV-inactivated fr is also not host-cell-reactivable. The plaque forming ability of fr infected bacteria decreases rapidly with increasing doses of PR-light wherea: the colony forming ability of uninfected host cells is very resistant to PR-light.     

ON THE MECHANISM OF RADIOSENSITIZATION BY 5-BROMOURACIL. THE OCCURRENCE OF DNA STRAND BREAKS IN U.V.-IRRADIATED PHAGE T4 AS INFLUENCED BY CYSTEAMINE

Abstract— U.V.-irradiation of phage T 4Bo^r results in a decrease in sedimentation rate of BU-DNA which is attributed to single- and double-strand breaks. No breaks could be observed in unsubstituted DNA. Cysteamine present during u.v.-irradiation is able to prevent double-strand breaks but does not influence the production of single-strand breaks measured by alkaline sucrose gradient centrifugation. The biological importance and nature of DNA strand breaks due to BU-incorporation as well as the action of the protective agent on these breaks and on the biological activity are discussed.     

HOST-CELL REACTIVATION OF NON-LETHAL ULTRAVIOLET-EFFECTS

Abstract— Delay of intracellular growth of u.v.-irradiated bacteriophage T1 and Λ was compared in host-cell reactivating [HCR(+)] and non-host-cell reactivating [HCR(—)] bacterial strains. At a given phage survival level, intracellular growth delay occurs to the same extent in HCR (+) and HCR (-) strains; at a given absolute u.v.-dose, this delay is considerably more expressed in HCR (-) than in HCR (+) strains. Therefore, it does not reflect the time required for the HCR repair of otherwise lethal U.V. lesions. The results rather suggest that U.V. causes, besides lethal lesions, stable photoproducts in the DNA, which are a priori non-lethal, and which are recognized and efficiently eliminated by the HCR repair system. The HCR enzymes likewise act on (non-lethal) u.v.-photoproducts causing prophage induction in lysogenic cells. Consequently, one obtains the maximum induction effect in a lysogenic HCR (-) strain at a much lower u.v.-dose than in the corresponding lysogenic HCR (+) strain. In contrast, u.v.-damage causing loss of the host cell's capacity to support growth of unirradiated phage is not affected by HCR.