Radiosensitization of GL261 glioma cells by tetraiodothyroacetic acid (tetrac)

We studied effects of tetrac (tetraiodothyroacetic acid) on survival of GL261, a murine brain tumor cell line, following single doses of 250 kVp x-rays and on repair of damage (sublethal and potentially lethal damage repair; SLDR, PLDR) in both exponential and plateau phase cells. Cells were exposed to 2 μM tetrac (1 h at 37oC) prior to x-irradiation. At varying times after irradiation, cells were re-plated in medium without tetrac. Two weeks later, colonies were counted and results analyzed using either the linear-quadratic (LQ) or single-hit, multitarget (SHMT) formalisms. Tetrac sensitized both exponential and plateau phase cells to x-irradiation, as shown by a decrease in the quasi-threshold dose (Dq), leading to an average tetrac enhancement factor (ratio of SF2 values) of 2.5. Tetrac reduced SLDR in exponential cells by a factor of 1.8. In plateau phase cells there was little expression of SLDR, but tetrac produced additional cell killing at 1-4 h after the first dose. For PLDR expression in exponential cells, tetrac inhibited PLDR by a factor of 1.9, and in plateau phase cells, tetrac decreased PLDR expression by a factor of 3.4. These data show that the decreased Dq value seen after single doses of x-rays with tetrac treatment is also accompanied by a significant decrease in recovery from sublethal and potentially lethal damage.     

Studies on the mechanism of action of ionizing radiations. VII. Cellular respiration,cell division,and ionizing radiations

On x-irradiation of the eggs and sperm of Arbacia punctulata there was inhibition of respiration with relatively large doses, whereas there was an increase with small doses. The dose required to produce an increase of respiration depended on the degree of sensitivity of the cell to the effect of ionizing radiation. Sperm cells were more sensitive; then came fertilized eggs; unfertilized eggs were the least sensitive. The inhibiting effect of x-rays on cell division was observed even on irradiation with x-ray doses which produced an increase of respiration. These results are compared to similar effects produced by thiol reagents and are attributed to oxidation of the thiol compounds in the cell.     

Quantum relations in photoreactivation of Colpidium

1. The amount of visible or long ultraviolet light (UV) required to photoreactivate Colpidium colpoda injured with known dosages of short UV (2654 A) was determined. 2. The effect of the short UV was tested by the delay in division of exposed animals compared to controls. Photoreactivation was tested by the effect of postillumination on the delay of division of treated colpidia compared to controls. 3. Colpidia were used in two physiological states: well fed and starved in balanced medium for 48 hours. The latter are much more sensitive to short UV although less susceptible of photoreactivation. 4. Photoreactivation occurred over the entire span from 3350 A to 4350 A for the well fed colpidia, from 3130 A to 5490 (green) for starved colpidia. 5. The photoreactivating effect of a single quantum of blue (4350 A) or long UV (3660 A) delivered per quantum of 2654 A used to injure colpidia was too slight to be considered significant. The effect of 10 quanta was usually more pronounced, but only after 100 quanta had been delivered was the photoreactivation nearly maximal for well fed colpidia. 6. The quantum requirement for maximal photoreactivation of the starved animals was greater at all wave lengths tried: 3660, 4050, 4350, and 5460 A being of the order of 800 incident quanta per incident quantum of 2654 A. 7. The transmission of UV(2654 A), blue, yellow, and red light by a suspension of colpidia was determined. 8. Large dosages of blue, violet, or long UV were slightly injurious to starved colpidia. In a few cases large dosages of 3660 A killed starved colpidia, especially after a non-lethal dose of short UV(2654 A). 9. Photoreactivation seems to be a balance between the slight injurious effect produced by the visible light or UV of long wave lengths and the injury produced by short wave length UV. 10. Possible reasons for the large number of quanta of photoreactivating light required per quantum of short UV are discussed.     

酵母、大肠杆菌和枯草杆菌基因组中短ORF的分布与形成原因

用终止密码方法计算了酵母、大肠杆菌和枯草杆菌基因组中所有的第一类开阅读框架（记为理论ORF）,给出了理论ORF和已知ORF随长度的分布,发现长度大于150个氨基酸后,理论ORF与已知ORF分布基本趋于一致,小于150个氨基酸的理论ORF数目的对数随长度线性变化,并提出这些短ORF是随机产生的猜想;研究了组分约束下的随机DNA序列中ORF数目、ORF的长度与随机序列总长度和GC含量之间的关系,证明了本文猜想的正确性;给出了短的理论ORF中可能的编码序列所占比例的分布曲线,这对识别短的编码序列有参考价值。     

Different photodynamic action of proflavine and methylene blue on bacteriophage

Summary The irradiation with visible light (Li) of temperate Serratiaphage that is maximally sensitized with either proflavine (PF) or methylene blue (MB) induces—apart from lethal lesions—mutations which are phenotypically expressed as clear (c) or lightly turbid (l) plaques. The mutagenicity of the MB+Li and PF+Li treatment differs in several respects: (i) Up to an inactivation of 6 to 8 lethal hits MB+Li is a much more potent mutagen than PF+Li. (ii) at low levels of survival the dose curve of mutation frequency with MB+Li reaches a peak and then decreases drastically while the mutation frequency after PF+Li continues to increase in proportion to lethal hits induced. (iii) Mapping of 100 MB+Li and 77 PF+Li induced c or l mutants indicates significant difference in the electiveness of the four genomic regions of c or l mutants.     

Restoration induced by catalase in irradiated microorganisms

1. E. coli, strain K-12, and B. megatherium 899, irradiated in strict but still undefined physiological conditions with certain heavy doses of ultraviolet light, are efficiently restored by catalase, which acts on or fixes itself upon the bacteria in a few minutes. This restoration (C. R.), different from photorestoration, is aided by a little visible light. 2. At 37° the restorability lasts for about 2 hours after UV irradiation; the restored cells begin to divide at the same time as the normal survivors. 3. C. R. is not produced after x-irradiation. 4. B. megatherium Mox and E. coli, strain B/r show little C. R.; E. coli strain B shows none. None of these three strains is lysogenic, whereas the two preceding catalase-restorable strains are. 5. Phage production in the system "K-12 infected with T2 phage" is restored by catalase after UV irradiation, whereas phage production in the system "infected B" is not. 6. With K-12, catalase does not prevent the growth of phage and the lysis induced by UV irradiation (Lwoff's phenomenon). 7. Hypotheses are discussed concerning: (a) the chemical nature of this action of catalase; (b) a possible relation between C. R. and lysogenicity of the sensitive bacteria; (c) the consequences of such chemical restorations on the general problem of cell radiosensitivity.     

RESULTS OF IRRADIATING SACCHAROMYCES WITH MONOCHROMATIC ULTRA-VIOLET LIGHT : IV. RELATION OF ENERGY TO OBSERVED INHIBITORY EFFECTS

Data obtained on yeast irradiated with monochromatic ultra-violet radiation has been analyzed for the number of quantum hits involved in the production of different degrees of inhibition of cell division, according to the method proposed by Mme. Curie (1929). Sufficient data are not available for a rigorous determination, but the calculated results tend to indicate that a multiple hit to kill relation is followed, that different numbers of hits are involved in the production of different degrees of inhibition, and that this number increases with increase in the degree of damage sustained.     

Saccharomyces cerevisiae mutants selected for increased production of Trichoderma reesei cellulases

 Trichoderma reesei endoglucanase I (EGI) was used as a reporter enzyme for screening mutagenized yeast strains for increased ability to produce protein. Sixteen haploid Saccharomyces cerevisiae strains, transformed with a yeast multicopy vector pALK222, containing the EGI cDNA under the ADH1 promoter, produced EGI activity of 10^-5–10^-4 g/l. On the average 93% of the total activity was secreted into the culture medium. Two strains with opposite mating types were mutagenized, and several mutants were isolated possessing up to 45-fold higher EGI activity. The best mutants were remutagenized and a second-generation mutant, strain 2804, with an additional twofold increase in EGI activity was selected. The mutant strain 2804 grew more slowly and reached a lower final cell density than the parental strain. In the selective minimal medium, the 2804 strain produced 40 mg/l immunoreactive EGI protein, but only 2% was active enzyme. In the rich medium the secreted EGI enzyme stayed active, but without selection pressure the EGI production ceased after 2 days of cultivation, when the strain 2804 had produced 10 mg/l of EGI. A sevenfold difference was found between the parental and the 2804 strain in their total EGI production relative to cell density. The difference in favour of the mutant strain was also detected on the mRNA level. The 2804 mutant was found to be more active than the parental strain also in the production of T. reesei cellulases, cellobiohydrolase I, and cellobiohydrolase II. Received: 22 December 1995/Received revision: 26 February 1996/Accepted: 17 March 1996     

Detecting ultraviolet damage in single DNA molecules by atomic force microscopy

We report detection and quantification of ultraviolet (UV) damage in DNA at a single molecule level by atomic force microscopy (AFM). By combining the supercoiled plasmid relaxation assay with AFM imaging, we find that high doses of medium wave ultraviolet (UVB) and short wave ultraviolet (UVC) light not only produce cyclobutane pyrimidine dimers (CPDs) as reported but also cause significant DNA degradation. Specifically, 12.5 kJ/m(2) of UVC and 165 kJ/m(2) of UVB directly relax 95% and 78% of pUC18 supercoiled plasmids, respectively. We also use a novel combination of the supercoiled plasmid assay with T4 Endonuclease V treatment of irradiated plasmids and AFM imaging of their relaxation to detect damage caused by low UVB doses, which on average produced approximately 0.5 CPD per single plasmid. We find that at very low UVB doses, the relationship between the number of CPDs and UVB dose is almost linear, with 4.4 CPDs produced per Mbp per J/m(2) of UVB radiation. We verified these AFM results by agarose gel electrophoresis separation of UV-irradiated and T4 Endonuclease V treated plasmids. Our AFM and gel electrophoresis results are consistent with the previous result obtained using other traditional DNA damage detection methods. We also show that damage detection assay sensitivity increases with plasmid size. In addition, we used photolyase to mark the sites of UV lesions in supercoiled plasmids for detection and quantification by AFM, and these results were found to be consistent with the results obtained by the plasmid relaxation assay. Our results suggest that AFM can supplement traditional methods for high resolution measurements of UV damage to DNA.     

Recovery in the survival capacity of ultraviolet-irradiated 3T3 mouse cells at G0 cannot be solely dependent on the excision of pyrimidine dimers

Mouse cells (3T3 line) excised at most 20% of the pyrimidine dimers introduced into their DNA by a dose of short-wavelength ultraviolet radiation that allows a significant fraction of the cells to survive. When irradiation was delivered at the pre-replicative stage, a significant repair of lethal events was observed, as the cells progressed toward S phase. The recovery in survival cannot be accounted for solely by excision of pyrimidine dimers. Therefore, either another lesion produced by ultraviolet radiation is critical in terms of lethality, or the dimer, which may trigger the lethal event, becomes no longer an obstacle for the replication system after a certain period of time.     

Linear dose--response relationships after prolonged expression times in V-79 Chinese hamster cells.

The expression time for induced mutants resistant to 6-thioguanine, in V-79 Chinese hamster cells, was determined by respreading the cells in the selective medium, at various times after treatment. The length of the expression time for mutants induced by X-rays, ethyl methane sulphonate and ultraviolet irradiation was dose dependent. For the highest dose used this was 7 to 8 days, beyond which there was no further changes in mutant frequency. The dose-response relationship of these agents does not appear to deviate from linearity; this permits the calculation of mutation rate per unit dose. For X-rays this value was 1.35 - 10(-7) per rad per locus, for ethyl methane sulphonate, 2.2 - 10(-2) per mole per locus and for ultraviolet irradiation, 6.3 - 10(-6) per erg per mm2 per locus. The effectiveness of the 3 different mutagens for the induction of mutations was compared by calculating the increase in mutant frequency per unit of decrease in survival (Do). These increments in frequency were: 5.6 - 10(-5) for X-rays, 69.5 - 10(-5) for ethyl methane sulphonate and 16.1 - 10(-5) for ultraviolet irradiation.     

Properties of Haemophilus influenzae mutants that are slightly recombination deficient and carry a mutation in the rec-1 gene region.

The highly recombination-deficient rec-1 mutants of Haemophilus influenzae are, as far as tested, equivalent to recA mutants of Escherichia coli. By selection for mutations in the rec-1 gene of H. influenzae, mutants designated ird (intermediary recombination-deficient) mutants were isolated; these mutants were much less recombination deficient (degree of transformability, 0.2 to 30% of wild-type value) than previously isolated rec-1 mutants (degree of transformability, 0.0001% of wild-type value). The ird mutants were more sensitive to ultraviolet irradiation and mytomycin C treatment than the wild type, but less sensitive than rec-1 mutants. Spontaneous production of phage HP1c1 by lysogenic MC11 cells and prophage induction by mitomycin C or ultraviolet irradiation were the same as in the wild type. In the ird mutants endogenous deoxyribonucleic acid was degraded both spontaneously and after ultraviolet irradiation to the same extent as in the wild type. Examination of one of the ird mutants revealed that recombination could be enhanced by ultraviolet irradiation, possibly because of an increased synthesis of the rec-1 gene product induced by ultraviolet irradiation.     

Studies on the mechanism of action of ionizing radiations. X. Effect of x-rays on some physico-chemical properties of proteins

On x-irradiation of aqueous solutions of serum albumin, serum globulin, and egg albumin, there was an increase in the absorption of ultraviolet light, the increase being more marked around 2400 A. The increase in optical density was proportional to the x-ray dose and inversely proportional to protein concentration. Addition of salts protected greatly the protein solutions against these changes. The increase in optical density was more marked in alkaline solutions. Irradiation of oxygenated solutions showed a greater increase in optical density around 2400 A. The absorption spectrum changes seem to be due to oxidation tyrosine residues and of other oxidizable groups.     

Influence of confluent holding time on UV light mutagenesis in human diploid fibroblasts

We have investigated the induction of mutants resistant to 6-thioguanine (6TG) following 254 nm ultraviolet light exposure of density-inhibited cultures of human diploid fibroblasts. Phenotypic expression of 6TG resistance was maximal within 9 days and remained stable through 19 days after irradiation. In reconstruction studies, complete recovery of 6TG-resistant mutants occurred at cell densities of up to 35 000 cells per 100-mm petri dish. The induced mutation frequency increased linearly with dose over the range of 3–9 J/m²; the D₀ of the survival curve was 4.2 J/m². Delaying subculture to low density for 1.5–24 h after irradiation produced unexpected alterations in induced mutation frequencies. An increase in UV-induced mutations of approximately 3-fold was observed in cultures maintained in confluence for 3 h. This trend was reversed with longer holding times: the mutation frequency declined sharply in cultures held for 6 h compared to the 3-h value, and thereafter showed a steady and gradual diminution to background levels.

These data suggest that the repair of potentíally mutagenic damage is a complex phenomenon which can lead to an increase or decrease in mutation frequency as a function of holding time. Although the decline in mutation frequency observed following longer holding intervals is consistent with the notion of an error-free process, we hypothesize that the increased mutation frequency produced by a short holding period reflects the existence of a cell-mediated process which enhances the mutagenic potential of at least some UV-induced DNA photoproducts.     

Pedigree Analyses of Yeast Cells Recovering from DNA Damage Allow Assignment of Lethal Events to Individual Post-Treatment Generations

Haploid cells of Saccharomyces cerevisiae were treated with different DNA damaging agents at various doses. A study of the progeny of individual such cells (by pedigree analyses up to the third generation) allowed the assignment of lethal events to distinct post treatment generations. By microscopically inspecting those cells which were not able to form visible colonies we could discriminate between cells dying from immediately effective lethal hits and those generating microcolonies (three to several hundred cells) probably as a consequence of lethal mutation(s). The experimentally obtained numbers of lethal events (which we call apparent lethal fixations) were mathematically transformed into mean probabilities of lethal fixations as taking place in cells of certain post treatment generations. Such analyses give detailed insight into the kinetics of lethality as a consequence of different kinds of DNA damage. For example, X-irradiated cells lost viability mainly by lethal hits (which we call 00-fixations); only at a higher dose also lethal mutations fixed in the cells that were in direct contact with the mutagen (which we call 0-fixations), but not in later generations, occurred. Ethyl methanesulfonate (EMS)-treated cells were hit by 00-fixations in a dose dependent manner; 0-fixations were not detected for any dose of EMS applied; the probability for fixation of lethal mutations was found equally high for cells of the first and second post treatment generation and, unexpectedly, was well above control in the third post-treatment generation. The distribution of all sorts of lethal fixations taken together, which occurred in the EMS-damaged cell families, was not random.(ABSTRACT TRUNCATED AT 250 WORDS)     

Response of potassium retentivity and survival of yeast to farultraviolet,near-ultraviolet and visible,and x-radiation

Potassium retentivity and survival of yeast were studied after exposure to various kinds and conditions of irradiation. The radiations used were: 2537 A ultraviolet, 3500 to 4900 A long-ultraviolet and short visible, and 250 kvp¹ x-rays. Both potassium retentivity and survival are decreased by these radiations. The dose-response of survival is about 16 times as sensitive as is potassium retentivity after 2537 A irradiation. Potassium retentivity is about twice as sensitive as survival after irradiation of 3500 to 4900 A. Survival after x-irradiation under aerobic conditions is five times as sensitive as potassium retentivity. Survival of cells irradiated with x-rays under anaerobic conditions was about half as sensitive as under aerobic conditions. The response of potassium retentivity to x-radiation at 25°C. under anaerobic conditions is only slightly affected below 160 kr, at which dose the slope abruptly increases to that obtained under aerobic conditions; lowering the temperature to 0°C. moves this point to about 300 kr. These differential effects are indicative of interaction of radiations with the yeast cell at sites that independently control survival and the retention of potassium.     

Role of the competitive microbial flora in the radiation-induced enhancement of ochratoxin production by Aspergillus alutaceus var. alutaceus NRRL 3174.

The radiation sensitivity and the toxigenic potential of conidiospores of the fungus Aspergillus alutaceus var. alutaceus were determined after irradiation with 60Co gamma rays and high-energy electrons. Over the pH range of 3.6 to 8.8, the doses required for a 1 log10 reduction in viability based on the exponential portion of the survival curve ranged from 0.21 to 0.22 kGy, with extrapolation numbers (extrapolation of the exponential portion of the survival curve to zero dose) of 1.01 to 1.33, for electron irradiation, and from 0.24 to 0.27 kGy, with extrapolation numbers of 2.26 to 5.13, for gamma irradiation. Nonsterile barley that was inoculated with conidia of the fungus and then irradiated with either electrons or gamma rays and incubated for prolonged periods at 28 degrees C and at a moisture content of 25% produced less ochratoxin A with increasing doses of radiation. Inoculation of barley following irradiation resulted in enhanced ochratoxin levels compared with unirradiated controls. In these experiments, inoculation with 10(2) spores per g produced greater radiation-induced enhancement than inoculation with 10(5) spores per g. There was no radiation-induced enhancement when the barley was surface sterilized by chemical means prior to irradiation. These results are consistent with the hypothesis that a reduction in the competing microbial flora by irradiation is responsible for the enhanced mycotoxin production observed when nonsterile barley is inoculated with the toxigenic fungus A. alutaceus var. alutaceus after irradiation.     

Calculation of boron neutron capture cell inactivation in vitro based on particle track structure and x-ray sensitivity

The Monte-Carlo technique was used to perform quantitative microdosimetric model calculations of cell survival after boron neutron capture irradiations in vitro. The high energy ⁷Li and alpha-particles resulting from the neutron capture reaction ¹⁰B (n,α)⁷Li are of short range and are highly damaging to cells. The biophysical model of the Monte-Carlo calculations is based on the track structure of these α-particles and ⁷Li-ions and the x-ray sensitivity of the irradiated cells. The biological effect of these particles can be determined if the lethal effect of local doses deposited in very small fractional volumes of the cell nucleus is known. This lethal effect can be deduced from experimental data of cell survival after x-ray irradiation assuming a Poisson distribution for lethal events. The input data used in a PC-based computer program are the radial dose distribution inside the track of the released particles, cell survival after x-ray irradiation, geometry of the tumor cells, subcellular ¹⁰B concentration, and thermal neutron fluence. The basic concept of this Monte-Carlo computer model is demonstrated. Validations of computer calculations are presented by comparing them with experimental data on cell survival.     

Estimation of mutation rates induced by large doses of gamma, proton and neutron irradiation of the X-chromosome of the nematode Panagrellus redivivus

The radiation-resistant free-living nematode Panagrellus redivivus was used to study mutation rates in oocytes, following gamma, proton and neutron irradiation in the dose range 45-225 grays. gamma-Radiation produced approximately 0.001 lethal X-chromosomes per gray over the range tested. Proton or neutron irradiation produced approximately 0.003 lethal X-chromosomes per gray at lower doses, with the mutation rate dropping to 0.001 lethal X-chromosome per gray at the higher doses. These results suggest a dose-dependent mutation-repair system. Cell lethality was also examined. gamma-Radiation produced the greatest amount of cell lethality at all doses, while neutron irradiation had no cell lethal effect at any of the doses examined.