Physiological responses of the halophilic archaeon Halobacterium sp. strain NRC1 to desiccation and gamma irradiation

We report that the halophilic archaeon Halobacterium sp. strain NRC-1 is highly resistant to desiccation, high vacuum and ⁶⁰Co gamma irradiation. Halobacterium sp. was able to repair extensive double strand DNA breaks (DSBs) in its genomic DNA, produced both by desiccation and by gamma irradiation, within hours of damage induction. We propose that resistance to high vacuum and ⁶⁰Co gamma irradiation is a consequence of its adaptation to desiccating conditions. Gamma resistance in Halobacterium sp. was dependent on growth stage with cultures in earlier stages exhibiting higher resistance. Membrane pigments, specifically bacterioruberin, offered protection against cellular damages induced by high doses (5 kGy) of gamma irradiation. High-salt conditions were found to create a protective environment against gamma irradiation in vivo by comparing the amount of DSBs induced by ionizing radiation in the chromosomal DNA of Halobacterium sp. to that of the more radiation-sensitive Escherichia coli that grows in lower-salt conditions. No inducible response was observed after exposing Halobacterium sp. to a nonlethal dose (0.5 kGy) of gamma ray and subsequently exposing the cells to either a high dose (5 kGy) of gamma ray or desiccating conditions. We find that the hypersaline environment in which Halobacterium sp. flourishes is a fundamental factor for its resistance to desiccation, damaging radiation and high vacuum.     

The lethal effects of gamma irradiation on larvae of the Huhu beetle, Prionoplus reticularis: a potential quarantine treatment for New Zealand export pine trees

Gamma irradiation was investigated as a possible method for disinfestation of huhu beetle larvae, Prionoplus reticularis White, in Pinus radiataD. Don. Larvae of four representative size classes were irradiated at six doses, and the lethal dose (LD₉₉) calculated from mortality data 3 days and 10 days post treatment. All larval size classes showed a similar sensitivity to gamma irradiation and required 3677 Gray (Gy) and 2476 Gy for a LD₉₉ 3 and 10 days post-treatment, respectively. The penetration of gamma irradiation into pine wood was found to be lowest in freshly cut logs, and decreased linearly at a rate of 0.698 Gy mm^–1 of wood. The penetration was greatest in wood that had been stored for 2 years, and decreased 0.512 Gy mm^–1 of wood. These results are likely to be correlated with wood moisture content. Gamma irradiation appears to be a potential alternative method to fumigation for quarantine treatment of P. reticularis.     

Adaptation of Pseudomonas aeruginosa harbouring the multi-resistance plasmid Rip64 to high levels of mercury chloride,carbenicillin and gentamicin

In order to study phenotypic and genotypic alterations in response to selected conditions of stress, Pseudomonas aeruginosa PU21, harboring the multi-resistance plasmid, Rip64 (142.5 kb), was adapted by serial subcultivation on selective agar plates, amended with either Hg²⁺, carbenicillin, or gentamicin. Concentrations of the selective agents, to which P. aeruginosa PU21 Rip64 carried plasmid-encoded resistance genes, were close to or slightly above the respective minimal inhibitory concentration. Three different substrains were obtained: R64-M was adapted to Hg²⁺, R64-C to carbenicillin, and R64-G to geneamicin. All three substrains showed elevated levels of Hg²⁺ and carbenicillin resistance. Resistance to other antibiotics was either increased, decreased or unaffected. Increased Hg²⁺ resistance in all adapted substrains was accompanied by higher activity and less stringent induction of mercury reductase, as determined by Hg-volatilization assays with resting cells. No plasmid alterations compared to the non-adapted strain were detected in R64-C, but in R64-M and R64-G plasmid sizes increased by approx. 23 kb, as detected by pulsed field gel electrophoresis and the occurrence of additional DNA fragments after digestions with several restriction endonucleases. The plasmid localized gene rearrangements were accompanied by additional DNA fragments hybridizing with a merA gene probe.     

The effect of electron and gamma irradiation on the induction of micronuclei in cytokinesis-blocked human blood lymphocytes

The effect of electrons and gamma irradiation on the induction of micronuclei in cytokinesis-blocked human peripheral blood lymphocytes was investigated to understand the relative biological effectiveness (RBE) of electrons compared with gamma rays. Blood samples were irradiated with an 8 MeV pulsed electron beam, at a mean instantaneous dose rate of 2.6 × 10⁵ Gy s⁻¹. Gamma irradiation was carried out at a dose rate of 1.98 Gy min⁻¹ using ⁶⁰Co gamma source. A dose-dependent increase in micronuclei yield was observed. The dose–response relationships for induction of micronuclei fitted well to a linear–quadratic relationship and the coefficients α and β of the dose–response curve were estimated by fitting the data using error-weighted minimum χ ² method. The RBE of 8 MeV electrons were found to be near unity as compared with gamma rays.     

Mutagenic DNA repair in Escherichia coli. VI. Gamma radiation mutagenesis in a tif-1 strain.

Summary In the non-filamenting tif-1 strain WP44_s NF trp a dramatic enhancement of both UV and gamma ray mutability to Trp⁺ was observed when irradiated bacteria were incubated on plates at 43°. This enhanced mutability was progressively suppressed when the initial plating density exceeded 10⁸ bacteria per plate and was not demonstrable in liquid media. Under optimal conditions more mutants were induced by gamma radiation than could reasonably be accounted for by the initial number of radiation-induced lesions in the DNA, implying the existence of some mechanism for amplifying the radiation effect. Moreover, the tif-enhanced mutation frequency could be obtained if incubation at restrictive temperature was delayed for up to 60 min in nutrient broth after irradiation, at a time when all known reparable DNA damage had been repaired and the number of viable bacteria had more than doubled. On plates the effect of high temperature was still fully demonstrable 120 min after irradiation. The results are hard to reconcile with the hypothesis that incubation of tif-1 bacteria at restrictive temperature causes the induction of a repair system acting on DNA damaged by gamma radiation. A more compatible interpretation would be that radiation causes a persisting physiological disturbance in the cell and that this enhances the spontaneous mutator effect occurring in tif-1 bacteria subjected to subsequent thermal shock.     

In vivo nicking and rejoining of nuclear DNA in ultraviolet-irradiated radiation-resistant and sensitive strains of Dictyostelium discoideum

Summary Some aspects of DNA repair in several radiation-resistant and radiation-sensitive strains of Dictyostelium discoideum were investigated by using alkaline sucrose gradients to analyze for the production and resealing of single-strand breaks following irradiation with 254 nm UV. All radiation-resistant strains and all mutants assayed that are sensitive to both UV and ⁶⁰Co gamma rays produced singlestrand breaks in their nuclear DNA after a UV fluence of 15 J/m². Mutants at the radC locus which are sensitive to UV but as resistant as their parental strains to ⁶⁰Co gamma rays produced many fewer single-strand breaks in their DNA after irradiation with UV. Thus, the radC mutations alter a repair pathway specific for UV-induced DNA damage and presumably affect the activity of a UV-damage-specific endonuclease involved in excision repair. All radiation-resistant strains and all of our mutants sensitive to gamma rays rejoined much of their DNA during a three-hour post-UV-irradiation incubation, suggesting that these strains have at least a partially intact excision repair system.Abbreviations used UV ultraviolet light - PBS phosphate buffered saline - cpm counts per minute     

Gamma Ray Irradiation of the Vasoactive Peptide Bradykinin Reveals a Residue- and Position-Dependent Structural Modification

In this work the effect of radical species generated by gamma ray irradiation of aqueous solution upon structure of vasoactive peptide bradykinin (BK, RPPGFSPFR) was investigated. Increasing doses of 1–15 kGy Co⁶⁰ gamma radiation were applied to BK solutions and a progressive degradation of its structure in a non-linear mode was observed. Two main peptide derivatives generated by these treatments were isolated and characterized through a combined amino acid analysis and daughter ion scanning mass spectrometry approach. Notably, it was observed that only the Phe residue located at position 8 and not 5 of BK was oxidized by reactive hydroxyl radical species given rise to Tyr⁸-BK and m-Tyr⁸-BK analogues. Comparative circular dichroism (CD) experiments of these peptides revealed that BK presents greater conformational similarity to Tyr⁸-BK than to m-Tyr⁸-BK. These results are in agreement with the biological potencies of these compounds measured in rat uterus and guinea pig ileum muscle contractile experiments. In summary, gamma irradiation of BK solutions revealed a residue- and surprisingly, position-structural modification effect of reactive radicals even in small peptides. Also of value for peptide chemistry field, the approach of applying controlled strong electromagnetic radiation in solution seems to be an alternative and unique strategy for generating, in some cases, peptides derivatives with uncommon structures and valuable for their further therapeutic potential evaluations.     

Gamma radiation effects on growth and yield attributes of <Emphasis Type="Italic">Psoralea corylifolia</Emphasis> L. with reference to enhanced production of psoralen

The present work describes radiation-induced effects on vegetative, reproductive traits and psoralen content in Psoralea corylifolia L. The effects of gamma radiation on Psoralea seeds were investigated by exposing seeds with doses of 2.5, 5, 10, 15 and 20 kGy at dose rate of 1.65 kGyh⁻¹ and studying the plant growth at three developmental stages: preflowering, flowering and post flowering (seed to seed) after irradiation. Irradiation with lower doses of gamma rays significantly improved vegetative traits while higher doses proved depressing for same parameters. Similar trend was followed in reproductive traits. Psoralen, showed highest concentration in seeds (7.56%) at 20 kGy and lowest in control roots (0.23%). Increment in psoralen was striking for higher gamma doses applied. These long-term changes in plant development may be attributed to alteration in plant genome induced by irradiation. The results show in depth development stimulation and enhancement of secondary metabolite in Psoralea corylifolia L. following low and high dose treatment respectively depicting the potential of gamma rays in plant biotechnology and metabolomics.     

Gamma irradiation alters the phenotype and function of CD4CD25 regulatory T cells

To examine the effects of gamma irradiation on Tregs, changes in phenotype and suppression function in Tregs treated with or without gamma ray were analyzed. Purified CD4⁺CD25⁺ regulatory T cells were irradiated at different dosages with a ¹³⁷Cs source gamma ray at 4.8 Gy/min. After culture, the phenotype and function changes were determined by flow cytometry and [³H]-thymidine incorporation, respectively. A dose-dependent reduction of Tregs proliferation in response to gamma irradiation was noted, which paralleled the apoptosis induction of Tregs. Gamma irradiation downregulated the Tregs expression of CD45RO, CD62L, FOXP3, membrane TGF-β, but upregulated Bax and GITR. High dose gamma irradiation (30 Gy) significantly abolished the suppression of Tregs on CD4⁺CD25⁻ T cells proliferation. Thus Tregs not only influences the phenotype but also alters their suppressive capacities. Our findings suggest that radiotherapy may be an important strategy to alter the immunologic balance of Tregs and effector cells in cancer therapy.     

The effect of continuous gamma irradiation on formation of sister chromatid exchanges (SCEs) and chromosomal aberrations in meristematic cells ofVicia faba

Germinated seeds ofVicia faba were continuously irradiated at low dose rate of gamma rays (0.05 Gy h-¹) up to a total accumulated dose of 2 Gy. The FPG (fluorescence plus Giemsa) technique of differential chromatid staining was used to monitor the frequency of sister chromatid exchanges (SCEs) in irradiated root tip meristem cells. The results of the experiments have demonstrated that SCE frequency is raised by continuous gamma irradiation only in plant cells containing BrdU in the chromosomal DNA. No effect concerning SCE formation was recorded at continuous irradiation of meristematic cells of Vicia faba with native, i. e. BrdU-nonsubstituted, DNA. In contrast to SCEs, a significant increase was found in the yield of chromosomal aberrations in all variants of irradiation.     

Separation and Determination of cis- and trans- dl-Chrysanthemumic Acid by Gas Liquid Chromatography

Macromolecular synthesis in M. radiodurans after irradiation with the wide range of doses has been investigated in connection with the mechanism for cell killing in radioresistant bacteria. Incorporations of ¹⁴C-amino acids into protein and ³²P_i into RNA were considerably inhibited by gamma irradiation at higher doses as well as synthesis of DNA. From the results obtained, it is possible to consider that inhibition of RNA and protein synthesis may have an important role in primary events leading to radiation lethality of this radioresistant bacterium.     

Gamma-ray-induced changes in hypodermal mesocarp tissue plasma membrane of pre- and post-storage muskmelon

Gamma irradiation (1.0 kGy) of intact, newly harvested, mature muskmelon fruit (Cucumis melo L. var. reticulatus Naud.) appears to have an immediate deleterious effect, but also a long-term beneficial effect, on the integrity and function of the plasma membrane (PM) of hypodermal mesocarp tissue. The initial consequences of gamma irradiation included an increase in the free sterol:phospholipid ratio, resulting at least in part from deglycosylation of steryl glycosides, a decrease in the spinasterol:7-stigmastenol ratio in each of the PM steryl lipids (free sterols, steryl glycosides, and acylated steryl glycosides), and a decrease in H⁺-ATPase activity. Irradiation did not increase protein loss, suggesting that the decrease in H⁺-ATPase activity resulted from either direct inactivation of the enzyme or altered PM ordering caused by the steryl lipid modifications. The long-term beneficial effects of irradiation, observed following 10 days of commercial storage, included greater retention of total PM protein, a diminished decline in total PM phospholipids (PL) and in the PL:protein ratio, and maintenance of greater overall H⁺-ATPase activity (activity was the same as in controls on a per mg protein basis, but there was > 30% more protein in the PM of stored irradiated fruit). These results indicate that 1 kGy gamma irradiation administered prior to storage slowes the progression of two key parameters of senescence, PM protein loss and PL catabolism.     

Gamma irradiation‐induced disease resistance of pear (Pyrus pyrifolia “Niitaka”) against Penicillium expansum

In this study, the effects of gamma irradiation on the resistance of pear fruit against Penicillium expansum, the causal agent of blue mould disease, were investigated. A low dose of gamma irradiation for 14 days increased the disease resistance and firmness of pear fruits. Remarkably, exposure to 200 Gy of gamma irradiation significantly maintained fruit firmness, markedly reduced disease incidence and enhanced the activity of defence‐related enzymes (e.g., β‐1,3‐glucanase, phenylalanine ammonia lyase, peroxidase and polyphenol oxidase) and expression of pathogenesis‐related (PR) genes (e.g., PR‐1, PR‐3 and PR‐4). Therefore, the gamma irradiation‐induced resistance against P. expansum involves both metabolic changes and the induction of expression of defence‐related genes. In addition, scanning electron microscopic analysis revealed that gamma irradiation significantly inhibits the growth of P. expansum. These results suggest that exposure of mature harvested pear fruits to artificial gamma irradiation confers fungal disease resistance; therefore, gamma irradiation represents an important strategy for controlling postharvest diseases in pear fruit.     

Effects of Gamma Stress and Carbon Dioxide on Eight Bioactive Flavonoids and Photosynthetic Efficiency in <Emphasis Type="Italic">Centella asiatica</Emphasis>

Increased atmospheric CO₂ and gamma irradiation have a significant impact on the plant photosynthetic apparatus and organic compound production. In this study, we evaluated the effect of elevated CO₂ on the photosynthetic efficiency and production of defensive secondary metabolites (flavonoids) induced by gamma irradiation as a physical elicitor in Centella asiatica. Irradiated and non-irradiated 10-week-old plants of C. asiatica were exposed to 400 and 800 μmol mol^?1 of atmospheric CO₂ in growth chambers for 2 h every day until six weeks. A CO₂-enriched atmosphere initially improved the photosynthetic efficiency and ameliorated the detrimental impact of gamma irradiation on the photosynthetic apparatus, increasing carbon allocation into the flavonoid pathway. Elevated CO₂ combined with gamma irradiation resulted in the highest concentration of flavonoids in C. asiatica tissues compared with the other treatments. There was an enhancement in rutin (2.49 fold), naringin (2.15 fold), fisetin (4.07 fold), and morin (4.62 fold) with rising CO₂ concentrations from 400 to 800 μmol mol^?1 in the irradiated plants. With increasing CO₂ concentration, the compensation point and the respiration declined, whereas the apparent quantum yield and the maximum net photosynthesis (A _max) rate increased. The efficiency of photosystem II (PSII) was improved in the irradiated plants grown under high concentrations of CO₂. The total carbohydrate concentration reached the maximum value at the highest level of CO₂, followed by gamma irradiation combined with the highest level of CO₂. Irradiated plants of C. asiatica grown under elevated CO₂ could be superior to non-irradiated plants due to increased carbon availability both for the flavonoid biosynthesis and for the photosynthetic pathway.     

Effect of gamma irradiation on microbial load,aflatoxins and phytochemicals present in Trigonella foenum-graecum

The effects of gamma irradiation on microbial load, total aflatoxins and phytoconstituents content of Trigonella foenum-graecum have been studied. Gamma irradiation at a dose of 2.5 kGy resulted in 2 log reduction of the total aerobic microbial count. A complete sterilization was, however, observed at 10 kGy. The total aflatoxin level decreased gradually with increase in gamma irradiation dose as compared to its un-irradiated counterparts, whereas the high performance liquid chromatography (HPLC) profile showed no change in the levels of phytochemicals up to the gamma irradiation dose of 10 kGy. HPLC profiles, however, differed in peak areas, and retention times of the components. These results suggest that gamma irradiation at a dose of 5.0 kGy was very effective for microbial decontamination because it did not adversely affect the active components of T. foenum-graecum.     

Double-strand Scissions in DNA of Gamma-irradiated Micrococcus radiodurans and their Repair during Postirradiation Incubation

Strand scissions in DNA of M, radiodurans after in vivo irradiation with ⁶⁰Co gamma rays were investigated by the sedimentation analysis using neutral sucrose gradients. Double-strand scission in DNA was estimated to occur at the rate of one double cut per 800 eV. This rate is in a good agreement of the value reported for mammalian cells. The rejoining of these double-strand scissions was observed during the repair process of the post-irradiation incubation and the mean rejoining time, i.e., the time reducing the remaining fraction of the double-strand scission to 0.37, was found to be 52 min. This rejoining repair was inhibited by adding chloramphenicol, tetracycline or actinomycin D to the postirradiation incubation medium. It is suggested that the high resistance character of M. radiodurans to gamma rays may be due to the efficient capacity of this rejoining repair.     

Radiosensitivity and Biological Effects of Gamma and X-Rays on Germination and Seedling Vigour of Three Coffea arabica Varieties

Effects of gamma and X-ray treatments were studied on three varieties of Coffea arabica (Kent, Mundo Novo and Geisha) to determine their radiosensitivity and relative biological effects. The coffee varieties seeds were subjected to 0, 50, 100, 150, 200 and 400 Gy of gamma and X-rays from Cobalt 60 (⁶⁰Co) source irradiation. The irradiated seeds were pre-germinated in Petri dishes placed in a germination chamber, whilst some were sown in the greenhouse for germination studies. Data were collected on germination date and rate, root and hypocotyl length to determine the relative biological effectiveness of treatments and the optimum dose. The results showed varieties responding differently to the irradiations and doses. There was a decrease in germination with increasing doses of the irradiation. The X-ray-treated seeds had less germination percentage and seedling vigour measured at 28 days after treatment compared to the gamma-irradiated seeds. The irradiation effects on germination suggest that lower doses of X-rays give the same Relative Biological Effects as higher gamma doses for both growth chamber and greenhouse germination for Geisha at LD₅₀, where the effects were similar for the two irradiations. Whereas 50–100 Gy stimulated germination and seedling vigour, 150 Gy adversely affected germination and no germination occurred at 200–400 Gy. The study concluded that all the coffee varieties evaluated are sensitive to gamma and X-ray irradiation in terms of germination, seedling vigour and biological effects with an optimum dose of 50–100 Gy. Therefore, both gamma and X-rays could be utilized in a future mutational breeding programme for coffee seedlings.

     

Induction of c-mutations in extracellular phage lambda by gamma-rays

Summary Mutagenic action of ⁶⁰Co -rays on extracellular phages red ⁺ and red1 13 after irradiation in 4% nutrient broth in the absence or in the presence of 0.1 M cysteamine or in dried samples was studied. The yield of c mutations was almost independent of the repair genotype of the host cells (uvrA6, polA1, recA13, lexA102, uvrE502, uvrD3 or xthA9), of the phage Red function and of the conditions of -irradiation and was 1·10^-12 per base pair and 1 rad. When the SOS-repair system of the host cells was induced by moderate UV irradiation, the yield of c-mutations was drastically enhanced in phage irradiated in broth, but not in phage irradiated in the dried state. These data allow us to suppose that the direct action of -rays induces, in phage DNA, premutational lesions that are fixed into mutations by replication. On the other hand after -irradiation in broth, when indirect radiation effects are only partially suppressed, about 85% of premutational lesions are converted into mutations by means of the inducible, errorprone SOS-repair system.     

Association of genetic polymorphisms with embryonic mortality in the chicken

Summary Association of the egg-white genotypes of dams with the mortality of their embryos was studied in 1966 in the pure and crossbred embryos of dams of two relatively noninbred Light Sussex substrains, 6D and 6F. These had been derived two generations earlier, in 1964, by equal division of strain 6, which we previously studied in 1962; while the sires of the crosses came from two Rhode Island Red/New Hampshire substrains, 5D and 5F, similarly derived. The effects were estimated parametrically for the three loci Tf, II and III singly and in two- and three-way interaction.In the first week of incubation, Tf had an additive effect in all matings, while II (and perhaps III) had a near-additive effect in the pure matings only. There was an additive X additive interaction effect of II X III in the last six days of incubation, which was consistent over all matings except 5F X 6F. The only effect to influence the total mortality over the whole of incubation was the three-way additive X additive X additive interaction, and there was substantial agreement in this over the matings. Some triple homozygotes were superior and others inferior to the genotypes with one or more loci heterozygous.This evidence of multiple interaction between loci affecting fitness is discussed in relation to hypotheses for the maintenance of genetic variability in populations.     

Accumulation of small heat shock proteins, including mitochondrial HSP22, induced by oxidative stress and adaptive response in tomato cells

Changes in gene expression, by application of H₂O₂, O₂°^–generating agents (methyl viologen, digitonin) and gamma irradiation to tomato suspension cultures, were investigated and compared to the well-described heat shock response. Two-dimensional gel protein mapping analyses gave the first indication that at least small heat shock proteins (smHSP) accumulated in response to application of H₂O₂ and gamma irradiation, but not to O₂°^– generating agents. While some proteins seemed to be induced specifically by each treatment, only part of the heat shock response was observed. On the basis of Northern hybridization experiments performed with four heterologous cDNA, corresponding to classes I–IV of pea smHSP, it could be concluded that significant amounts of class I and II smHSP mRNA are induced by H₂O₂ and by irradiation. Taken together, these results demonstrate that in plants some HSP genes are inducible by oxidative stresses, as in micro-organisms and other eukaryotic cells. HSP22, the main stress protein that accumulates following H₂O₂ action or gamma irradiation, was also purified. Sequence homology of amino terminal and internal sequences, and immunoreactivity with Chenopodium rubrum mitochondrial smHSP antibody, indicated that the protein belongs to the recently discovered class of plant mitochondrial smHSP. Heat shock or a mild H₂O₂ pretreatment was also shown to lead to plant cell protection against oxidative injury. Therefore, the synthesis of these stress proteins can be considered as an adaptive mechanism in which mitochondrial protection could be essential.