Combined Effects of Radiation and Inorganic Reagents during Irradiation on Radiosensitivity of Bacterial Cells

The combined effects of inorganic reagents and radiation on the inactivation of E. coli in the resting state were studied. Among these reagents halides such as NaCI, KCI, KBr and KI were found to have a considerable synergistic action to radiation. Temperature effect on the halide action during irradiation was not observed, but removal of oxygen from halide solutions increased the radiosensitivity of cells. Combined effects of radiation and some other inorganic reagents were also investigated. Heavy metal salts and hydrogen peroxide were synergistic, nitrates and sulfates having no influence or a slightly protective action. Barium chloride and calcium chloride were protective in lower concentrations and synergistic in higher concentrations. These synergistic actions of inorganic reagents except ferric salts were observed during irradiation, but not after the irradiation.     

Effects of Chemical Agents on Radiation Inactivation of Streptomyces Protease in Aqueous System

Inactivation of crystalline enzyme, Streptomyces protease G, by γ-ray irradiation in an aqueous system has been investigated. It is indicated that inactivation of the enzyme is attributable mainly to the indirect action of radiation. The inactivation curve is exponential and the G-value for enzyme inactivation is calculated as 0.1 at an enzyme concentration of 1×10^?5m, which is not influenced by varying pH. Effects of various other solutes on radiation inactivation have been also studied. Halogen ions, especially iodine ion, and nitrite ion are most protective among various inorganic anions examined, and alkali metal and alkali earth metal cations are ineffective. Among various organic compounds examined, sulfur-containg compounds and unsaturated compounds are generally effective for protection of enzyme activity against radiation damages. The protective effect of benzene is enhanced by the substitution of electron donating groups. Chloroform and chloral are found to act as a synergist for irradiation inactivation.     

Post-irradiation inactivation,protection, and repair of the sulfhydryl enzyme malate synthase

Malate synthase from baker's yeast, a trimeric sulfhydryl enzyme with one essential sulfhydryl group per subunit, was inactivated by 2 kGy X-irradiation in air-saturated aqueous solution (enzyme concentration: 0.5 mg/ml). The radiation induced changes of enzymic activity were registered at about 0, 30, 60 h after irradiation. To elucidate the role of OH., O-.2, and H2O2 in the X-ray inactivation of the enzyme, experiments were performed in the absence or presence of different concentrations of specific additives (formate, superoxide dismutase, catalase). These additives were added to malate synthase solutions before or after X-irradiation. Moreover, repairs of inactivated malate synthase were initiated at about 0 or 30 h after irradiation by means of the sulfhydryl agent dithiothreitol. Experiments yielded the following results: Irradiation of malate synthase in the absence of additives inactivated the enzyme immediately to a residual activity Ar = 3% (corresponding to a D37 = 0.6 kGy), and led to further slow inactivation in the post-irradiation phase. Repairs, initiated at different times after irradiation, restored enzymic activity considerably. The repair initiated at t = 0 led to Ar = 21%; repairs started later on resulted in somewhat lower activities. The decay of repairability, however, was found to progress more slowly than post-irradiation inactivation itself. After completion of repair the activities of repaired samples did not decrease significantly. The presence of specific additives during irradiation caused significant protective effects against primary inactivation. The protection by formate was very pronounced (e.g., Ar = 72% and D37 = 6 kGy for 100 mM formate). The presence of catalytic amounts of superoxide dismutase and/or catalase exhibited only minor effects, depending on the presence and concentration of formate. Both the presence of specific additives during irradiation and the addition of additives after irradiation may alter the post-irradiation inactivation. Catalase turned out to be the most potent inhibitor of post-irradiation inactivation; superoxide dismutase showed an ambivalent behaviour, it accelerated or impeded post-irradiation inactivation; formate, when added after irradiation, exhibited a moderate protective effect. The presence of specific additives, added before and/or after irradiation, influenced the repair behaviour to some extent. The highest activity achieved by repair amounted to about 90% of the activity of the corresponding unirradiated sample. The percentual gain of activity was found to be the greater the lower the residual activity of the enzyme was before initiation of repair.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interaction of Some Factors in the Mechanism of Inactivation of Bacteriophage MS2 in Aerosols

The mechanisms involving inactivation of bacteriophage MS2 in aerosols and the effect of protective substances in the spray-medium were studied after spraying from various NaCl solutions. Results with aerosols generated from the salt solutions showed that with higher salt concentration in the spray-medium higher concentrations of protective substances were needed to protect phage MS2 against aerosol inactivation. Phenylalanine, which has a protective action at low concentration, produced less protection in aerosol droplets that were supersaturated solutions of this substance or in which crystals of phenylalanine can be expected to form. Our results suggested that protection by peptone and phenylalanine was related to the concentration in the aerosol droplet after evaporation to equilibrium, whereas protection by the surface active agent OED (a commercial mixture of oxyethylene docosylether and oxyethelene octadecylether) was related to the concentration at which a monolayer is formed around the aerosol particle. Inactivation of phage MS2 was maximal in the aerosol particle in fluid phase and became less at lower relative humidity where aerosol particles are expected to be in the solid state. It is suggested that inactivation of bacteriophage MS2 in aerosols could be explained by surface inactivation at the air-water interface.     

Modification of Combined Effect of Radiation and Sodium Chloride on Microorganisms by Chemical Agents during Irradiation

Effects of various chemical agents on the synergistic action of NaCl to the radiation inactivation of bacteria and yeast were studied. The remarkable modification of the radiation lethal effect by some reagents is considered to be a strong evidence for an indirect nature of NaCl synergistic action during irradiation. Most of these modification effects were restricted to the actions during irradiation, supporting the free radical hypothesis in which the short-life active species formed by radiation were considered to attack bacterial cells. Furthermore, pre-irradiation effects under various conditions suggest that the enhancement of radiation lethal effect by NaCl may involve the intracellular events.     

Use of Aqueous Silver To Enhance Inactivation of Coliphage MS-2 by UV Disinfection

A synergistic effect between silver and UV radiation has been observed that can appreciably enhance the effectiveness of UV radiation for inactivation of viruses. At a fluence of ca. 40 mJ/cm², the synergistic effect between silver and UV was observed at silver concentrations as low as 10 μg/liter (P < 0.0615). At the same fluence, an MS-2 inactivation of ca. 3.5 logs (99.97%) was achieved at a silver concentration of 0.1 mg/liter, a significant improvement (P < 0.0001) over the ca. 1.8-log (98.42%) inactivation of MS-2 at ca. 40 mJ/cm² in the absence of silver. Modified Chick-Watson kinetics were used to model the synergistic effect of silver and UV radiation. For an MS-2 inactivation of 4 logs (99.99%), the coefficient of dilution (n) was determined to be 0.31, which suggests that changes in fluence have a greater influence on inactivation than does a proportionate change in silver concentration.     

Effect of selenolipoic acid on peroxynitrite-dependent inactivation of NADPH-cytochrome P450 reductase

Seleno-organic compounds are known as efficient “scavengers” of peroxynitrite (PN). Here we studied the protective effect of selenolipoic acid (SeLA), the seleno-containing analogue of lipoic acid, on peroxynitrite-dependent inactivation of NADPH-cytochrome P450 reductase. 3-Morpholinosydnonimine hydrochloride (SIN-1) was used as a source of peroxynitrite. The reductase was irreversibly inactivated by PN generated from SIN-1. The inactivation occurred with the rate constant of about 3 × 10⁴M^-1s^-1. The presence of SeLA at low concentration (0.5 μM) led to synergistic increase of the reductase inactivation by PN. Our results suggest the formation of a reactive derivative of SeLA in the reaction of SeLA with PN, probably selenolseleninate, that mediates the aggravation of reductase inactivation. In the presence of SeLA, the inactivation was reversible under the action of thiols, allowing us to conclude that the observed action of SeLA may be considered as protective.     

小角X射线散射实验中蛋白质溶液的辐照损伤及防护方法

随着同步辐射光源（尤其是目前快速发展的第四代同步辐射光源）技术的进步,可用于实验的辐射通量越来越高,实验样品（特别是蛋白质等生物大分子样品）受到的辐照损伤也越来越严重。在全球现有的同步辐射装置上,蛋白质等生物大分子溶液专用小角X射线散射（SAXS）实验站的光子通量基本上都在1013cps量级。在如此高的通量下,蛋白质等生物大分子溶液样品在实验测量中受到的辐照损伤极其严重。如果没有有效的辐照防护措施,蛋白质溶液样品在毫秒级辐照时间内便会辐照损伤,导致不能获取有效的实验数据。辐照损伤严重制约了SAXS实验技术在蛋白质溶液样品方面的应用。因而,认识蛋白质溶液样品辐照损伤的产生机理、影响因素、判断标准,以及有效降低辐照损伤程度、延缓辐照损伤产生时间的方法,对于蛋白质等生物大分子溶液的散射实验具有重要的指导意义。本文在简要概述生物大分子溶液样品辐照损伤产生机理、影响因素、辐照剂量等基本概念的基础上,重点综述了同步辐射SAXS实验中辐照损伤的判断标准和防护措施。此外,本文还对比了各种防护措施的优缺点,讨论了在建HEPS新光源中SAXS束线可用的散射数据采集时间,指出辐照损伤防护剂是有价值的研究方向...     

Effect of salt solutions on radiosensitivity of mammalian cells. III. Treatment with hypertonic solutions.

V79 Chinese hamster cells were treated with hypertonic solutions of NaCl or KCl and irradiated rat various times before, during, or after exposure to the solution. In solutions of molarities between 0-2 and 0-5 M, the cellular radiosensitivity increases with the molarity of the bathing solution. At these molarities, the hypertonic solution need not be present during irradiation to sensitize cells. Furthermore, radiosensitivity of cells could be increased by exposing cells for longer times to the hypertonic solution before irradiation. At higher salt concentrations (at 1-5 to 1-8 M), significant radioprotection is observed. Survival curve data showed that this protection was characterized by an increase in DO and a decrease in n, while the survival curves of cells sensitized with 0-465 M NaCl or with lower concentrations exhibited mainly changes in DO. The 1-55 M NaCl solution must be present during radiation to give a protective effect. Prolonged exposure to the salt before irradiation reduced the amount of radioprotection afforded by the salt. The results are discussed in terms of the effects of ions on histones, cellular water structure and the cell-aging cycle.     

Radioprotective properties of ATP and modification of acid phosphatase response after a lethal dose of whole body p(66MeV)/Be neutron radiation to BALB/c mice.

The intraperitoneal administration of exogenous ATP prior to a lethal dose (7 Gy) of whole body neutron irradiation increased the radioresistance of BALB/c mice. This radiation used the beam from a neutron therapy facility produced by the reaction p(66 MeV)/Be. Survival of the mice, determined 7 days post-irradiation as the endpoint, was increased from 26% to 86% by the action of the exogenous ATP. Furthermore, the response of acid phosphatase activity as an indicator of the acute radiation effects showed a marked augmentation in both tissues studied, testes and small intestine. The activity of the enzyme after neutron irradiation with prior administration of ATP showed smaller increases when compared with the increases observed after neutron irradiation alone. This implies that exogenous ATP reduces the effect of the lytic enzyme and, hence, damage. Finally, changes were observed in the activity of acid phosphatase in the testes and intestine with different concentrations of exogenous ATP. In both tissues there was a monotonic decrease in the activity of the enzyme with increase of the concentration of exogenous ATP administrated before radiation. These results reflect the protective ability of exogenous ATP as an adaptive defence mechanism to reduce radiation damage in normal tissues after a lethal dose of neutron radiation.     

Use of aqueous silver to enhance inactivation of coliphage MS-2 by UV disinfection

A synergistic effect between silver and UV radiation has been observed that can appreciably enhance the effectiveness of UV radiation for inactivation of viruses. At a fluence of ca. 40 mJ/cm(2), the synergistic effect between silver and UV was observed at silver concentrations as low as 10 microg/liter (P < 0.0615). At the same fluence, an MS-2 inactivation of ca. 3.5 logs (99.97%) was achieved at a silver concentration of 0.1 mg/liter, a significant improvement (P < 0.0001) over the ca. 1.8-log (98.42%) inactivation of MS-2 at ca. 40 mJ/cm(2) in the absence of silver. Modified Chick-Watson kinetics were used to model the synergistic effect of silver and UV radiation. For an MS-2 inactivation of 4 logs (99.99%), the coefficient of dilution (n) was determined to be 0.31, which suggests that changes in fluence have a greater influence on inactivation than does a proportionate change in silver concentration.     

The action of air ions on bacteria. I. Protective and lethal effects on suspensions of Staphylococci in droplets

Techniques have been devised for studying quantitatively the effects of air ions on microorganisms suspended in small drops. In smog-contaminated atmospheres moderate concentrations of positive and negative air ions exerted a protective effect on staphylococci by delaying the drop in pH customarily observed and by diminishing the rate of evaporation. In clean air higher concentrations of positive and negative air ions accelerated the rate of death of staphylococci apparently by direct action on the cells and by increasing the rate of evaporation. Air ion action in these experiments did not involve cell agglutination or direct radiation from the radioactive isotopes employed.     

Protective effect of Na+ and K+ against inactivation of (Na+ + K+-ATPase by high concentrations of 2-mercaptoethanol at high temperatures

Purified dog kidney (Na⁺ + K⁺)-ATPase (EC 3.6.1.3) was inactivated with high concentrations of 2-mercaptoethanol at 50–55°C. The inactivation was prevented by NaCl or KCl, with KCl being more effective than NaCl (the former ion being about one order more efficient under a typical set of experimental conditions). A disulfide bond in the β-subunit of the enzyme protein was prevented from reductive cleavage by NaCl or KCl in accordance with protection of the enzyme activity. Choline chloride did not exert a significant protective effect over a similar concentration range. (Na⁺ + K⁺)-ATPase was also inactivated with high concentrations of 2-mercaptoethanol in the presence of low concentrations of dodecyl sulfate. This inactivation was also prevented by NaCl or KCl, with the latter being again more efficient than the former. These results indicate that Na⁺ and K⁺ bound to their respective ion-binding sites on the α-subunit exert a protective effect on a disulfide bond on the β-subunit. This suggests some sort of interaction between the α- and the β-subunits.     

Primary and post-irradiation inactivation of the sulfhydryl enzyme malate synthase: correlation of protective effects of additives

The presence of additives during X-irradiation of malate synthase led to radioprotective effects against primary and post-irradiation inactivation. Pronounced effects were provided by typical scavengers, sulfhydryl reagents and specific ligands (substrates, products, analogues). The results show that scavenging and specific protection are responsible for the protective efficiency of additives. Scavengers delete noxious species formed during irradiation or post-radiationem. Sulfhydryl reagents may act as repair substances. Specific ligands protect the active site of the enzyme and the essential sulfhydryls; specific protection is more pronounced post-radiationem. Ligands and sulfhydryl reagents may additionally act as scavengers. A cumulative index for the protective power of additives against both sorts of inactivation was established.     

Radiation inactivation of alpha-chymotrypsin in aqueous solutions. Effect of irradiation conditions

A comparative study was made of inactivation by gamma- and beta-radiation of alpha-chymotrypsin within a wide range of its initial concentrations (from 10(-4) to 10(-7) M). The regularities of gamma- and beta-inactivation are the same, and distinctions, if any, are due to a greater radiation effect of beta-rays on dilute enzyme solutions (less than or equal to 5 X 10(-6) M). The inactivation of alpha-chymotrypsin by radiation proceeds either via primary molecule unfolding followed by degradation of the most accessible and radiosensitive amino acid residues (pH 7.8) or, to a greater extent, via direct disruption of amino acid residues which can probably be random (pH 3.0). Calcium ions stabilize, on the whole, the enzyme molecule upon irradiation.     

Inactivation and unfolding of aminoacylase during denaturation in sodium dodecyl sulfate solutions

During denaturation by sodium dodecyl sulfate (SDS), aminoacylase shows a rapid decrease in activity with increasing concentration of the detergent to reach complete inactivation at 1.0 mM SDS. The denatured minus native-enzyme difference spectrum showed two negative peaks at 287 and 295 nm. With the increase of concentration of SDS, both negative peaks increased in magnitude to reach maximal values at 5.0 mM SDS. The fluorescence emission intensity of the enzyme decreased, whereas there was no red shift of emission maximum in SDS solutions of increasing concentration. In the SDS concentration regions employed in the present study, no marked changes of secondary structure of the enzyme have been observed by following the changes in far-ultraviolet CD spectra. The inactivation of this enzyme has been followed and compared with the unfolding observed during denaturation in SDS solutions. A marked inactivation is already evident at low SDS concentration before significant conformational changes can be detected by ultraviolet absorbance and fluorescence changes. The inactivation rate constants of free enzyme and substrate-enzyme complex were determined by the kinetics method of the substrate reaction in the presence of inactivator previously described by Tsou [Tsou (1988),Adv. Enzymol. Related Areas Mol. Biol. 61, 381–436]. It was found that substrate protects against inactivation and at the same SDS concentrations, the inactivation rate of the free enzyme is much higher than the unfolding rate. The above results show that the active sites of metal enzyme containing Zn²⁺ are also situated in a limited and flexible region of the enzyme molecule that is more fragile to denaturants than the protein as a whole.     

Substrate Inhibition Competes with Halide Inhibition in Polyphenol Oxidase

Polyphenol oxidase (PPO) is a ubiquitous enzyme important in the food industry. Although PPO activity followed Michaelis?CMenten kinetics at catechol concentrations of up to 1?mM, it slowly decreased at catechol concentrations above 2?mM. This result indicated that in addition to the active site (site A), the enzyme possesses a second catechol-binding site (site B) that exerts an inhibitory effect on PPO activity. Halides inhibit PPO activity in such a way that substrate inhibition is lessened when halide concentration is increased. Furthermore, elevated concentrations of catechol diminished the degree of inhibition by halides. These findings suggest that halides also bind to site B to inhibit PPO activity. A steady-state kinetic analysis demonstrated that the dissociation constant between catechol and PPO depended on the binding of halides to site B. The dissociation constants were greatest when chloride bound to the site. Bromide and iodide yielded lower dissociation constants, in that order. These data indicate that the binding of halide to site B modulated the structure of site A, thereby exerting an inhibitory effect.     

Optical manipulation of Saccharomyces cerevisiae cells reveals that green light protection against UV irradiation is favored by low Ca and requires intact UPR pathway

Optical manipulation of Saccharomyces cerevisiae cells with high density green photons conferred protection against the deleterious effects of UV radiation. Combining chemical screening with UV irradiation of yeast cells, it was noted that the high density green photons relied on the presence of intact unfolded protein response (UPR) pathway to exert their protective effect and that the low Ca²⁺ conditions boosted the effect. UPR chemical inducers tunicamycin, dithiotreitol and calcium chelators augmented the green light effect in a synergic action against UV-induced damage. Photo-manipulation of cells was a critical factor since the maximum protection was achieved only when cells were pre-exposed to green light.     

Stimulation of the salt receptor of the blowfly. III. The alkali halides

Application of solutions of each of the alkali halides to the tip of a labellar sensillum of the blowfly elicited a repetitive neural discharge from the salt receptor. The records were qualitatively similar to those for NaCl. For each of the alkali chlorides and sodium halides, the shapes of the curves of the response of the salt receptor as a function of concentration were similar to that for NaCl. The alkali halides exhibited a regular pattern of relative stimulating effectiveness for the salt receptor. The effectiveness of the anions increased monotonically with atomic number. The effectiveness of the cations was greatest for potassium and declined as the atomic number was increased or decreased. This hierarchy for stimulating effectiveness was maintained at all tested molarities. The response to a mixture of two salts appeared to be an average of those to the single salts at concentrations equal to the total concentration of the mixture. Cross-adaptation was observed between the alkali halides. The results indicate that an explanation of stimulation of the salt receptor must apply to all the salts tested and that both the anion and the cation affect a salt's stimulating effectiveness.     

Ectomycorrhizal fungi: A new source of atmospheric methyl halides?

Incomplete source budgets for methyl halides – compounds that release inorganic chlorine and bromine radicals which, in turn, catalyze atmospheric ozone depletion – limit our ability to predict the fate of the stratospheric ozone layer. We report here the first measured emissions of methyl chloride, methyl bromide, and methyl iodide from ectomycorrhizal fungi. We grew nine fungal isolates on growth media containing halide concentrations similar to those found in soils and plant tissues. The observed range of emissions was 0.003–65 μg methyl chloride, 0.001–3 μg methyl bromide, and 0.02–12 μg methyl iodide g^?1 dry weight fungi day^?1. Species varied in production rates of methyl chloride vs. methyl bromide vs. methyl iodide. Cenococcum geophilum, a widespread ectomycorrhizal fungus, was further tested to investigate the effects of halide substrate concentration in growth media. Emissions from this species increased linearly with increasing concentrations of both bromide and iodide. In addition, a subset of four fungi was studied with two media concentrations each of chloride, bromide, and iodide (0.2 or 20 mm ). These fungi had similar responses to halide concentration, despite 1000‐fold differences in baseline emission rates between isolates. Finally, high chloride concentrations (20 mm ) in media did not appear to inhibit emissions of methyl bromide or methyl iodide. Overall, ectomycorrhizal fungi might be an important source of methyl halides to the atmosphere, and substrate concentrations and community composition may influence production levels in ecosystems.