Biomedical subjects
Publications and source records attributed to E L Powers.
The efficient export of galactose-binding protein to the periplasm of Escherichia coli is shown to be dependent on the presence of the cytosolic chaperone SecB.
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The original demonstration in the bacterial spore of the multiple actions of oxygen in modifying the responses of cells to ionizing radiation has now been verified in mammalian systems, pointing up the need for separate inquiry into each of the several components if the responses of mammalian cells are to be understood. We have provided physico-chemical explanations for only two of the four (at least) oxygen elements recognized in the spore: the reaction of diatomic oxygen with organic free radicals in dry spores; and an action of the hydroxy radical (OH) in spores at low [O2]. This paper will discuss newly recognized features of the oxygen responses that could reveal the nature of the other components. It will examine modulation of response by oxides of nitrogen, the similarities and differences among them and explanations for them that suggest further experiment, the importance of concentration of additive in determining both quantity and quality of response, and a general model that explains sensitizer action in terms of inhomogeneous chemistry.
The radiation sensitivity of spores of Bacillus megaterium is markedly increased by transplatin in both N2 and O2, and in a manner suggesting the involvement of hydroxyl radicals in both instances, thus differing mechanistically from its isomer cisplatin.
A rhodium compound, Rh(NH3)3Cl3, does not sensitize the spores of Bacillus megaterium to X-rays. However, it is a very effective sensitizer of vegetative cells of Staphylococcus aureus, raising the sensitivity four times in O2 and over 100 times in anoxia. The inhibition by oxygen of the sensitizing action of Rh(III), which operates over a wide range of [O2], is noteworthy. These experiments were performed in saline-phosphate buffer using 50 kVp X-rays. The results are discussed in terms of the known radiation chemistry of this compound.
As an extension of results obtained from radiation studies on caffeine both in other laboratories and more recently in this laboratory using the bacterial spore as the test system, six compounds with chemical structures closely resembling that of caffeine were tested as radiation modifiers. Of these compounds, purine, adenine and hypoxanthine resembled caffeine in sensitizing spores to radiation, while theobromine, xanthine and theophylline did not. These responses are discussed in relation to the electron sequestration hypothesis of cellular sensitization to high-energy radiation.
Studies were carried out on the effect of caffeine on the X-irradiation sensitivity of B. megaterium spores with the following results: Caffeine exerts a concentration-dependent modifying action on oxygen-dependent components of X-ray-induced damage in B. megaterium spore suspensions causing an 'over-O2 effect' at about 1 X 10(-4) mol dm-3, and as the concentration is increased to 1 X 10(-3) mol dm-3 or above, a small but consistent protection is seen. In the absence of O2, at a wide range of concentrations (8.5 X 10(-5) to 1 X 10(-1) mol dm-3), caffeine enhances the inactivation constant, k, from 1.17 to about 1.50 kGy-1. Both ethanol and t-butanol (5 X 10(-2) mol dm-3) remove the 'over O2-effect' produced by 1.10(-4) mol dm-3 caffeine in O2; such an effect, however, is not accompanied by reduction in the H2O2 concentrations in the spore suspensions. Ethanol prevents caffeine-induced anoxic sensitization, as well as H2O2 buildup. t-BuOH has no influence on either the low dose part of the log fraction survival curve or on the H2O2 yield in the spore suspensions. Caffeine reacts with radiation-induced eaq and .OH with rate constants of 1.5 X 10(10) and 6.9 X 10(9) dm3 mol-1s-1, respectively.
Several factors modifying radiation sensitivity in dry bacterial spores are described and discussed. Vacuum inducing the loss of critical structural water, very low dose rates of radiation from which the cell may recover, radiations of high linear energy transfer, and the action of temperature over long periods of time on previously irradiated cells are recognized from extensive laboratory work as important in determining survival of spores exposed to low radiation doses at low temperatures for long periods of time. Some extensions of laboratory work are proposed.
The effect of silver(I) and mercury(II) complexing upon the rate of reaction of both the hydrated electron and the OH radical with DNA has been investigated using the technique of pulse radiolysis. The results presented do not support the hypothesis that the mechanism of radiation sensitivity by these metals is mediated through increased rate of free radical attack upon DNA. Also, there is evidence presented that neither metal, when complexed with DNA, reacts at a significant rate with the free radical species e-aq and OH. The technique of pulse radiolysis was also used to study the complexation of both silver(I) and mercury(II) with DNA (calf-thymus, 40 per cent GC). Scatchard plots, derived from the experimental data, gave association constants of 1.85 x 10(5) dm3 mol-1 and 9.9 x 10(4) dm3 mol-1 and n values (number of complexing sites per nucleotide) of 0.71 and 0.31 for silver(I) and mercury(II) respectively.
Several well-known transforms of the widely-used Michaelis-Menten function linearize the kinetics of many competition reactions such as enzymatic processes. These transforms allow easy and accurate evaluation of the mathematical constants of the system, as well as giving clues to the various mechanisms involved in these competitions. In this paper these linearization techniques are applied to several sets of data from several authors that describe the radiation sensitivity determined by varied concentrations of two sensitizers--O2 and misonidazole. It is shown that, when the increment in sensitivity determined at the various concentrations of sensitizer is used as the dependent variable, straight lines are obtained from various sets of data when either the so-called Lineweaver-Burk or the Eadie-Hofstee transform is used. The E-H transform results in a better distribution of data points and, accordingly, is preferred. The transform allows recognition of two oxygen-dependent processes (one at low [O2] and one at high [O2]) in data apparently demonstrating but one; and, as well, two processes determined at two levels of misonidazole from data that appeared to describe one. These results support the evidence given earlier for two oxygen effects in other cells. Also, the transform reveals that in different cell systems two inhibitors of the oxygen effect appear to act in the same manner on one oxygen effect and in a different way on the other. In discussion the value of the transform in analysing mechanisms of sensitization is examined, and its further potential use in understanding the action of chemical protective agents is pointed out.
A transforming DNA system has been used to study the effect of silver ion on the X-ray-induced inactivation of the functional ability to DNA. In N2 transforming DNA is sensitized to radiation by Ag+, with the maximum sensitization occurring at a ratio of one Ag+ added per DNA base. This response can be partially prevented by addition of .OH scavengers. In O2 the transforming DNA is only slightly sensitized by Ag+ except at very high Ag+ concentrations. These results are interpreted in terms of the eaq- sequestration theory of radiation sensitization. Studies on the binding of Ag+ to DNA show that at the maximum sensitization in N2, there is one Ag+ bound per DNA base pair and that this binding is unaffected by .OH scavengers, although radiation may change these binding properties. Steady-state radiation chemistry studies indicate a difference in the response of DNA-Ag+ complexes to irradiation in N2 and in O2. As with the transforming DNA studies, these can be interpreted in terms of the eaq- sequestration theory of radiation sensitization.
The X-ray-induced inactivation of the biological activity of Bacillus subtilis transforming DNA in dilute aqueous solution has been studied over a wide range of O2 concentrations in an attempt to elucidate the mechanisms involved in O2 action. When the DNA is irradiated in the presence of 100 per cent O2 there is a protection of the transforming DNA compared to the sensitivity in N2-saturated or in N2O-saturated solutions. When the equilibrating gas contains intermediate concentrations of O2 (1 per cent--90 per cent) in N2 or N2O, the DNA sensitivity is equivalent to that in pure N2 or N2O respectively. At low O2 concentrations (approximately 0.14 per cent O2 in N2 or in N2O) there is a sensitization of the DNA and this sensitization can be prevented by .OH scavengers. Possible mechanisms for these actions of O2 on the radiation sensitivity of transforming DNA are discussed.
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Biologically active DNA isolated from Bacillus subtilis was exposed in vitro to X-rays at a concentration of 10 microgram/ml in 29 mM phosphate buffer. Radiation-induced damage to the DNA was quantitatively determined by measuring the decrease in its transforming activity (try2 locus) using B. subtilis 168M (try-) as recipient. In O2, which removes .H and eaq-, the radiation sensitivity of the DNA is less than that in N2-saturated water. In N2O, which has been shown to increase yields of .OH in irardiated aqueous solutions, the radiation sensitivity of Transforming DNA is twice that observed in O2 and 1.5 times that in N2. Addition of 5 X 10(-2) M ethanol or 1.7 X 10(-1) M t-butanol, both .OH scavengers, causes large (about tenfold) reduction in the radiation sensitivity in all three saturating gases. These results suggest the importance of the .OH radical in the loss of biological activity of DNA.
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Studies of irradiated bacterial spores in aqueous suspension indicate that the sensitization of spores by oxygen can depend on three chemical processes. One of these processes involves reactions of hydroxyl radicals; the other two apparently do not.
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