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Ultraviolet radiation effects on Ehrlich ascites tumor cells; observations using a flying spot ultraviolet microscope.

A flying spot ultraviolet microscope, employing a fast scan and pulsed operation of the raster, has been used to induce radiation damage in ascites tumor slide cultures, and to study by time-lapse cinematography the progressive stages of cell damage. The cells observed came from a strain (EF(7)) of the Ehrlich ascites carcinoma. Irradiated cells were found to show a characteristic syndrome of damage, involving blebbing at the cell surface, while control cells in the adjacent areas of the preparation remained unchanged. The end of the blebbing period is marked by swelling of the cells, and the time taken for this phenomenon to occur was used as a measure of the severity of the damage. It was found that the time required for swelling is dependent on the size of the dose employed, as well as on the sensitivity of the cells. This latter sensitivity was found to decline as the physiological age of the tumor increased. If ultraviolet illumination below 255 mmicro is excluded, no symptoms of damage occur, even when very large doses are used. These observations are discussed in relation to the nature of the system in the cell which is affected.

Animals↗

Studies on the mechanism of the photosensitized inactivation of E. coli and reactivation phenomenon.

In order to find a more satisfactory interpretation of the phenomenon of photosensitized inactivation of bacteria, studies were performed under various experimental conditions on methylene blue and E. coli. In summary the findings are as follow:- 1. The dye is absorbed by the bacteria according to the Langmuir isotherm and can be removed by ionic substitutions; the dye binding to the bacteria is predominantly ionic; the dye-bacteria complex produces a new absorption peak in the 610 mmicro wave length region, and the action spectrum corresponds to the spectral absorption of the dye-bacteria complex. 2. There is an optimum dye concentration range for the photosensitized inactivation. 3. Photosensitized inactivation of bacteria can take place both in the frozen and liquid states and the presence of oxygen is essential to the inactivation process. 4. Hydrogen peroxide, formed by reoxidation of the reduced methylene blue, does not inactivate bacteria. 5. Following the photosensitized inactivation, E. coli lose their ability to reduce the methylene blue in the presence of various hydrogen donors, suggesting that enzymes are involved in the inactivation process. 6. Bacteria inactivated by photosensitization can be reactivated by prolonged storage after irradiation; the recovery rate increases with increasing temperature (maximum 37 degrees ), and is also influenced by the presence of various hydrogen donors. In view of collected experimental data, the basic reaction mechanisms are analyzed in photosensitized inactivation. The first step of the reaction seems to be excitation of the dye-bacteria, or dye-bacteria oxygen complex, by a photon which produces an activated complex. In such a state, molecular oxygen is capable of producing an oxidizing reaction, which results in the inactivation of the bacteria. Some aspects of the detailed reactions taking place at the cell surface are discussed.

Escherichia coli↗

A comparison of five effects of ultraviolet light on the Arbacia egg.

The demonstration of five different effects of ultraviolet radiation on the sea urchin's egg indicates that more than one basic photochemical process goes on there. Photorecovery is observed in only one of these. The need for caution in interpreting such effects is obvious. Evidence for a different mechanism for the timing of cleavage in eggs activated by ultraviolet radiation as compared to normally fertilized eggs is presented. The bearing of these studies on survival curves for microorganisms is discussed.

Animals↗

Ultraviolet light and the mitotic cycle in the sea urchin's egg.

The effect of ultraviolet light in delaying certain events in the cell division cycle has been examined. The time to fusion of the egg and sperm nucleus is not affected by doses of ultraviolet that cause considerable delay in other parts of the cycle. The principal delay occurs before anaphase. Between anaphase and cleavage there is only slight delay. The "refractory period" during which the radiation does not delay the immediate cycle of cell division, does not seem to represent complete refractoriness of the mitotic cycle to interference during this period.

Animals↗

The effect of nutritional state on photoreversal of ultraviolet injuries in Didinium nasutum.

1. The effect of the nutritional state of Didinium nasutum on its resistance to short ultraviolet (UV) radiation (2654 A) and its recovery from the injury following illumination with visible light (4350 A, blue) was studied. 2. The resistance of a didinium to UV is considerably increased by feeding it a paramecium 15 to 60 minutes before exposure to UV. If fed just before exposure to UV, the resistance is less than that of an unfed control. 3. Photoreversal is only slightly greater in didinia fed after irradiation with UV but before exposure to visible light as compared to those fed after exposure to visible light. 4. Irradiated paramecia are eaten by didinia, provided they have not started to cytolyze. Didinia fed on irradiated paramecia divide at about the same rate as controls or slightly faster. 5. The available stock of Didinium declines in vigor with lapse of time after excystment, as measured by the time required for division. The sensitivity of Didinium to UV did not change essentially during the 5 month period over which tests were made. 6. The theoretical implications of the results are considered.

Ciliophora↗

The effect of chloramphenicol on deoxyribonucleic acid synthesis and the development of resistance to ultraviolet irradiation in E. coli infected with bacteriophage T2.

To elucidate the role of protein synthesis in DNA formation, E. coli R2 infected with phage T2 was studied as a model, employing chloramphenicol to inhibit protein synthesis. The following results were obtained. 1. Chloramphenicol inhibited protein synthesis but not synthesis of nucleic acids in uninfected bacteria. 2. Studies of the effect of chloramphenicol on phage maturation indicated a delay of 2 minutes between time of addition and cessation of phage growth. 3. The increase of DNA in phage-infected bacteria was completely suppressed by the addition of chloramphenicol within 2 minutes following infection. Addition at later times showed progressively less inhibitory action depending upon the time interval, and addition after the 10th or 12th minute showed no appreciable effect on DNA synthesis despite the cessation of intracellular phage formation and protein synthesis. 4. When chloramphenicol was added to infected cells the increase of resistance to UV stopped within 2 minutes, whether or not DNA synthesis continued. Thus evolution of resistance paralleled the rate of DNA synthesis achieved, but not the amount of DNA accumulated. 5. We conclude that in infected bacteria, protein synthesis is necessary to initiate DNA synthesis but is not essential for its continuation. The resistance to UV that characterizes infected cells near the midpoint of the latent period is not due to accumulation of DNA, but depends on some chloramphenicol-sensitive process (probably protein synthesis) completed at about the time the rate of DNA synthesis becomes maximal.

Bacteriophage T4↗

Photoreversal of nuclear and cytoplasmic effects of short ultraviolet radiation on Paramecium caudatum.

1. Irradiation with three short ultraviolet (UV) wave lengths, 226, 233, and 239 mmicro rapidly immobilizes Paramecium caudatum, the dosage required being smaller the shorter the wave length. 85 per cent of paramecia immobilized with wave length 226 mmicro recover completely. Recovery from immobilizing doses is less the longer the wave length. 2. Irradiation continued after immobilization kills the paramecia in a manner which is markedly different for very short (226, 233, and 239 mmicro) and longer (267 mmicro) wave lengths. 3. An action spectrum for immobilization in P. caudatum was determined for the wave lengths 226, 233, 239, 248, and 267 mmicro, and found to resemble the absorption of protein and lipide in the wave length region below 248 mmicro. Addition of these data to those of Giese (1945 b) gives an action spectrum resembling the absorption by albumin-like protein. 4. Division of P. caudatum is delayed by doses of wave lengths 226, 233, and 239 mmicro which cause immobilization, the longest wave length being most effective. 5. Immobilization at any of the wave lengths tested (226, 233, 239, 248, 267 mmicro) is not photoreversible when UV-treated paramecia are concurrently illuminated. 6. Division delay resulting from immobilizing doses of 226, 233, and 239 mmicro is photoreversible by exposure to visible light concurrently with the UV. 7. Division delay induced by exposure to wave length 267 mmicro is reduced by exposure to visible light applied concurrently with UV or immediately afterwards. 8. The data suggest that the shortest UV wave length tested (226 mmicro) affects the cytoplasm selectively, because it is absorbed superficially as indicated by unilateral fluorescence in UV. Consequently it immobilizes paramecia rapidly but has little effect on the division rate because little radiation reaches the nucleus. 9. The data support the view that nuclear effects of UV are readily photoreversed but cytoplasmic effects are not.

Ciliophora↗

The action of ultraviolet radiation on yeast catalase.

The effect of prolonged UV irradiation (mostly 2537 A) on the catalase activity of an aqueous yeast suspension was divisible into 4 periods. First, the period during which the cells lost their ability to form colonies, but during which no change in catalase activity was noted. Second, the period during which a considerable rise in catalase activity (Euler effect) occurred. The Euler effect was accompanied by enzyme alteration as shown by the simultaneous decrease in the activation energy of the enzyme-substrate system. However, during the initial phase of this period, as the catalase activity of the suspension began to increase, the activation energy rose to a transient level higher even than that characterizing the unaltered enzyme. Heat accelerated the rate of alteration when applied either during or after the irradiation; the activation energy for the over-all alteration reaction was 24 kcal., a value close to that recorded previously for alteration induced by chemical agents. Nevertheless, the rate-limiting step appeared to be different in the two cases. A model of these events was presented in which the primary photochemical action was on the site at which catalase is located within the cell. Third, a rather long period during which irradiation led to no diminution in the catalase activity of the maximally active suspension. This protection effect was duplicated in intro by a model crystalline catalase-KNA system, or by adding either ribonuclease digestion products of RNA or adenine to a catalase solution prior to irradiation. Evidence was adduced that the protection effect was not a simple screening, but involved some sort of interaction between the enzyme and the nitrogenous components of RNA, an interaction which must likewise occur within the cell. Alteration induced by CHCl(3) did not eliminate the protection effect, but that by butanol did. The onset of photoinactivation was due to modification of protein structure, not of RNA. Fourth, the period of photoinactivation of the intracellular enzyme, which was quite similar to that of the crystalline enzyme in vitro.

Catalase↗

Evidence for thermal reactions following exposure of Didinium to intermittent ultraviolet radiations.

1. The nature of ultraviolet injury and its variation with the same dose given at different intensities and wave lengths have been investigated in the protozoan Didinium nasutum, using time to the fourth division as a measure of injury. 2. The injury has been found to consist of a "slowdown" of division rate, which always occurs, and a "stasis," usually at the second division after irradiation, which appears in varying degrees among more severely injured samples. 3. Injury was found to be almost independent of intensity at three wave lengths out of four studied over a wide range of intermediate and high intensities, but was found to rise sharply with lower intensity at all except the longest wave length. 4. Flashed UV of high intensity is much more effective than the same dose of continuous radiation at high intensity and shorter total time of treatment. It is also more effective than the same dose at low intensity and equal time of treatment, though only slightly so. 5. An increase of injury with rise of temperature and with increase of dark period clearly indicates that injury depends on thermochemical reactions following the absorption of UV in Didinium. 6. The most reasonable assumption is that a similar conclusion applies to other organisms as well, and that its general application may be useful in the investigation of UV effects on protoplasm.

Temperature↗