Irradiated Suspensions Research Articles

The Relationship between the Phenomenon of Photoreactivation in Escherichia coli Following Ionizing Radiation and the Čerenkov Emission

The surviving fraction of the60 Co γ-irradiated Escherichia coli ${\rm B}_{{\rm s}-1}$ decreased, whereas the value of photoreactivation increased with increasing volume of irradiated suspension. A...

Chemical damage in gamma-irradiated human erythrocyte membranes.

In air saturated suspensions of erythrocyte ghost membranes gamma-irradiation causes formation of lipid peroxides, measured as malonaldehyde, and a loss of membrane protein sulphydryl groups. Addition of N-(p-amino-benzoyl)-1-glutamate prevented peroxidation up to doses of 2 x 10(3) Gy, due to scavenging of hydroxyl radicals. Another hydroxyl scavenger sodium formate, also prevented peroxidation at low doses, but lost its protective effect at higher doses probably because of secondary reactions of the resulting superoxide radical anion. Two sulphur containing radioprotectants also were able to reduce the extent of lipid peroxidation. The enzymes catalase and superoxide dismutase were added to the irradiated suspensions in order to determine the contribution from hydrogen peroxide and superoxide to peroxidation. The extents of peroxidation are compared with structural modification of the membrane under the same conditions of irradiation.

A model for oxygen-dependent damage from OH radicals in irradiated suspensions of bacterial spores.

A model for oxygen-dependent damage from OH radicals in irradiated suspensions of bacterial spores.

Photocatalytic oxidation of propan-2-ol in the liquid phase by rutile

The photocatalytic oxidation of liquid propan-2-ol to acetone has been investigated using irradiated suspensions of pure rutile. It is shown that ultraviolet radiation, rutile and a source of oxygen are all necessary for measurable reaction to occur at 310 K. The parameters controlling the photoactivity of pure rutile, together with effects of adding hydrogen peroxide, water or dissolved salts to the reaction mixture, have been investigated. Supporting measurements with doped rutile and less pure anatase have also been made.The experimental results, which include values of the quantum yield, do not support a chain mechanism for acetone formation. Mechanisms are proposed, in which reactive species are formed by the surface trapping of both photoelectrons and photoholes.

Direct evidence that pyrimidine dimers in DNA result in neoplastic transformation.

A specific test for the biological role of UV-induced pyrimidine dimers in DNA is photoreactivation (PR). Fish contain large amounts of the PR enzyme. Portions of cell suspensions of tissue from various organs of the fish Poecilia formosa were exposed to UV radiation (254 nm), then injected into isogenic recipients. An incident fluence of 20 J/m2 resulted in 10% of the fish with large granulomas and 100% with thyroid carcinomas. If the irradiated cell suspension was illuminated with PR light before injection, the yields of both types of lesion were reduced approximately 10-Fold. If the PR light was given before the UV exposure, there was no reduction in the numbers of growths. These experiments show that pyrimidine dimers in DNA can lead to neoplastic transformation.

Red light induced production of gibberellin-like substances in homogenates of etiolated wheat leaves and in suspensions of intact etioplasts

Homogenates of etiolated wheat leaves contain increased levels of acidic gibberellin (GA)-like substances following treatment with red light. Differential centrifugation of homogenates indicates that the response is confined to the 1000 g (or plastid) fraction. Irradiation of suspensions of intact etioplasts also increases the level of extractable GA-like activity. Phytochrome can be detected spectrophotometrically in preparations of etioplasts. The response in etioplasts can be inhibited by chloramphenicol, but not by cycloheximide, and partially by Amo 1618. The GA-like substances produced in etioplasts seem capable of passing into the surrounding medium within 20 min.

The photosensitizing action of carcinogens. I. The action of 2-naphthylamine on Escherichia coli K-12 and Paramecium caudatum.

Cultures of Paramecium caudatum incubated with 7 × 10−7 M 2-naphthylamine were rapidly killed when exposed to light of 366 nm. Cultures not exposed to the amine were unaffected by the light; cultures kept in the dark were unaffected by the amine. Escherichia coli K-12 populations were markedly reduced after irradiation of suspensions in water containing 3 × 10−4 M 2-naphthylamine with light simulating natural sunlight in intensity and wavelength distribution. Suspensions of E. coli in deionized water were unaffected by the light and E. coli suspended in solutions of the amine but kept in the dark were also unaffected. Since 2-naphthylamine is a known water pollutant these results may be of ecological importance.

Mixed lymphocyte reactions: Absence of stimulation by irradiated allogeneic cells

137Cs-irradiated mouse spleen cells, in contrast to mitomycin-blocked cells, do not stimulate a mixed lymphocyte reaction (MLR) when cocultured with normal allogeneic lymphocytes. Attempts to find an irradiation dose which blocks the DNA-synthetic capability of alloantigenic cells, but which does not also render them unstimulatory in mixed cultures, have not been successful. Low-level irradiation (100–500 rad) does not completely block mitogen (PHA) reactivity, hence the target cells may be capable of participating in a two-way MLR. High-level irradiation (> 1000 rad), however, thoroughly blocks PHA-stimulated DNA synthesis and eliminates the capacity of these cells to stimulate an MLR. Potentiation of MLR by irradiated cells syngeneic with the reacting cell population was not possible in these experiments. Cell death and lysis in irradiated suspensions occurred during the 72-hr culture period. It is believed that a combination of cell lysis and irradiation-blocked metabolic events normally necessary for the MLR sufficies to explain the poor or nonexistent MLR's obtained in these experiments.

Immunization with irradiated tumour cells and specific lymphocyte cytotoxicity in malignant melanoma.

A microculture technique has been adapted to assay the cytotoxic properties of the lymphocytes from patients with malignant melanoma when cultured with their autologous tumour cells. In a series of patients with established melanoma specific autologous cytotoxicity was extremely uncommon, being detectable in only 3 out of the 22 cases studied. This cytotoxicity did not correlate with clinical staging of the disease but may well have been related to tumour volume. By autoimmunization of patients with an irradiated suspension of their own tumour cells the appearance of cytotoxic lymphocytes could be provoked in 5 out of the 12 patients studied. This cytotoxicity was detectable at the end of the first week after the autograft and disappeared by the third week. Cytotoxic lymphocytes did not correlate in any obvious way with the appearance of specific antitumour antibodies detected by immunofluorescence. So far there has been no evidence of a serum factor capable of blocking the lymphocyte cytotoxicity in these patients.The presence and possible significance of cytotoxic lymphocytes in patients with malignant disease is discussed.

The Mechanism of Radiation Hemolysis in Human Erythrocytes

Human bank blood erythrocytes were given hemolyzing (16 to 50 kR) exposures of60 Co gamma rays, suspended in various media, and maintained at 5°C for 20 hours. Hemolysis occurred in isotonic NaCl but was prevented or reduced in isotonic choline chloride and in hypertonic NaCl or KCl. Prior to hemolysis in isotonic NaCl, the cells rapidly lost potassium and accumulated sodium. Although irradiated suspensions of cells in choline chloride did not hemolyze, they lost potassium. 2-Mercaptoethylguanidine (MEG) and reduced glutathione (GSH), which does not enter cells, reduced hemolysis and potassium loss when added to the suspensions before irradiation. Radiation caused a marked decrease in membrane sulfhydryl groups. Blockage of membrane sulfhydryl groups by p-chloromercuribenzoate (FCMB) and p-chloromercuribenzene sulfonate (PCMBS), which does not enter the cells, mimicked radiation by causing potassium loss and hemolysis. PCMBS-induced hemolysis was prevented by suspension of the cells in isotonic choline ch...

The Indirect Action of γ-rays on the Coat Protein of Bacteriophage Particles

SummarySuspensions of bacteriophage T4 in 25 mM phosphate buffer, pH 7·1, were irradiated with 60Co γ-rays. Measurements of the viscosity of irradiated samples before and after treatment with DNA-ase and trypsin showed that DNA is released from damaged phage particles. The phage debris acts as an effective radical scavenger and at higher doses protects the residual phage, thereby decreasing the inactivation rate. In irradiated suspensions of T4 and ΦX174 phage particles biologically-active subviral particles able to infect bacterial spheroplasts were found. Sedimentation in a sucrose gradient showed that the subviral particles of T4 have nearly the same sedimentation coefficient as unirradiated particles. Examination of irradiated samples with the electron microscope revealed that they contained many particles with the sheath contracted. The subviral particles produced by irradiation resemble those obtained with urea but do not show the tail clusters found in samples treated with urea.

Toxicity of Pasteurella tularensis killed by ionizing radiation.

Approximately 2 x 10(11) viable Pasteurella tularensis cells per ml, contained in suspensions, were killed by exposure to 10(6) r of gamma-radiation. When injected intraperitoneally into mice, the irradiated suspensions initially contained about 10 ld(50) per ml, and immunized mice against challenge with fully virulent strains of P. tularensis. Toxicity and immunizing activity of the suspensions decreased significantly within a few days at 5 C. Mice were protected against the toxin by immune serum or by prior injection of endotoxin of Escherichia coli. Cortisone did not protect against the newly prepared suspension, but was effective against the aged suspension. Lethal doses of newly prepared suspension for guinea pigs and rabbits were approximately 0.5 ml and 2 ml, respectively. Cortisone protected rabbits, but not guinea pigs, against lethal challenge. Pyrogenic effects resembling those shown by endotoxin-containing suspensions were demonstrated in rabbits. The results suggested that two toxins are responsible for the toxicity of irradiated suspensions of P. tularensis: one labile and associated with the immunizing activity of the suspension, the other more stable and resembling classical endotoxin.

Sensitivity of stored melon fruit fungi to gamma irradiation

Direct effects of gamma rays on suspensions containing 10 5 spores/ml of the main fungi in Israel that cause the rot in stored melons, were tested by inoculating irradiated suspensions on Potato Dextrose Agar and into melon fruits. A 40 krad dose was lethal to Trichothecium roseum which was estremely sensitive. A 100 krad dose was lethal to Penicillium cyclopium, and P. viridicatum in vitro, while 150 krad was the lowest lethal dose tested in vivo. Lower doses (60 and 100 krad) caused some prolongation of the incubation period in melons stored at 17°C. The incubation period of Fusarium sp. inoculated into melons was 16–18 days for 100 krad doses, compared to ten days for controls. Its incubation was inhibited on melons by 200 krad for 24 days at 17°C, but there was a slow growth at 25°C in vitro between the 16th and 21st days. Alternaria tenuis was radiation resistance with 300 krad being sublethal both in vitro and in vivo.

Decomposition of phosphatidylethanolamine with phospholipase C from Clostridium perfringens

The optimum conditions for the enzymatic decomposition of phosphatidylethanolamine with the phospholipase C fromClostridium perfringens have been described. When a sonically irradiated aqueous suspension of lipid containing zinc ions is used, 70–80% decomposition is achieved.

X-irradiation of splenic lymphocytes. I. Thymidine pools and radiothymidine incorporation into DNA.

An immediate suppression of radiothymidine incorporation into DNA is found in splenic lymphocytes of the golden hamster when they are irradiated in vitro in the kilorad range (Manasek, Adelstein and Lyman 1966). Although the mechanism of this suppression has not been elucidated, Smets (1966 a) has put forth the hypothesis that at least part of the apparent depression in DNA synthesis is due to changes in the precursor-pool size. A number of investigators, working with a wide range of biological systems, have suggested that the decreased uptake of radiothymidine into DNA often observed after irradiation is due to an expansion of nucleoside and nucleotide pools consequent on DNA degradation and not to a net decrease in DNA synthesis (Hell, Berry and Lajtha 1960, Kelly 1961, Nygaard 1962, Smets 1966b). Several experimental approaches have been utilized to test for such amplification of intracellular pools. In one (Hell et al. 1960, Smets 1966b), the precursor is varied over a wide concentration range in the hope of eliminating pool effects by flooding. In the experiments to be described, the method is applied to x-irradiated splenic lymphocytes. Cell suspensions were prepared from the spleens of mature, male, golden hamsters (Manasek et al. 1966). From radioautographs we have determined that the predominant nucleated cell which is synthesizing DNA is a medium-sized lymphocyte. The minced tissue was passed through stainless-steel screens for dispersion, and the cells were suspended in Hanks' salt solution at pH 7-25. They were subsequently exposed to unfiltered 250kvp x-rays generated at a dose-rate of 300 rads per minute as determined with ferrous ammonium sulphate. The suspensions were maintained at a controlled temperature throughout their irradiation. Immediately thereafter, the cells were incubated with tritiated thymidine (specific activity 2 Ci/mmole) at 37°c for 30 min. DNA was isolated by a modification of Schmidt and Thannhauser's method (Munro and Fleck 1966). In this assay the incorporation of thymidine into DNA is linear for 4 hours in control and up to 1 hours in irradiated suspensions. In figure 1, the uptake of radiothymidine into the DNA of unirradiated splenic lymphocytes is shown for total thymidine concentrations varied from 1 x 10 - 7 M to 35 x 10- M. The 350-fold increase in concentration results in a ten-fold increase in radiothymidine incorporation. The ' S ' shape of the uptake curve suggests that the overall incorporation obeys Michaelis-Menten kinetics. Under

Nucleic acid synthesis in x-irradiated thymocytes.

The rate of incorporation of thymidine into the deoxyribonucleic acid (DNA) of rat thymocytes in vitro was not immediately affected by low doses of X-radiation, but became progressively more inhibited as the irradiated cells were incubated at 37 °C for periods of up to 6 hours. Ribonucleic acid (RNA) synthesis deteriorated almost at the same rate as DNA synthesis after X-irradiation in vitro, but protein synthesis was slightly more resistant. Addition of 10–50 mM nicotinamide to the irradiated cell suspensions tended to retard the development of this inhibition, particularly at low temperatures, but high concentrations of nicotinamide were also toxic to these cells. The inhibition of DNA synthesis by X-irradiation appeared to follow a qualitatively similar pattern in thymus, spleen, and regenerating liver in vivo.In addition to the inhibition resulting from degenerative processes in the irradiated cells, DNA synthesis in vitro was directly inhibited by 20–30 kr X-radiation. The effects of radiation on DNA synthesis paralleled to some extent its effects on the gel-forming capacity of the deoxyribonucleoprotein from the thymus cells. It is suggested that the normal synthesis of both RNA and DNA in thymocytes depends on the integrity of the deoxyribonucleoprotein.

Mitotic stimulation in normal and irradiated suspension cultures of rat bone marrow by an elevated salt concentration

Increasing the sodium concentration of the medium of suspension cultures of rat bone marrow accelerated the entry of normal cells into mitosis. A similar acceleration was obtained in irradiated (75 and 100 r) suspensions and the duration of postirradiation mitotic delay was strongly reduced.

The dewaxing of oils using ultrasonics

1. It has been established that the ultrasonic irradiation of distillate oils subjected to dewaxing, using controlled dilution and MEK as solvent, enables this process to be intensified to a considerable extent. 2. In the two-stage dewaxing of irradiated suspensions a highly deoiled wax is obtained besides the dewaxed oil. The irradiation of a suspension before the first stage enables filtration to be intensified in both stages. 3. Narrowing of the feedstock fractional composition enables the dewaxing properties to be greatly improved. The use of ultrasonics raises the dewaxing quality still further.

VIABILITY AND METABOLISM OF STAPHYLOCOCCUS AUREUS AFTER FREEZING, LYOPHILIZATION, AND IRRADIATION.

Ahn, T. H. (University of California, Los Angeles), H. Nishihara, Charles M. Carpenter, and G. V. Taplin. Viability and metabolism of Staphylococcus aureus after freezing, lyophilization, and gamma irradiation. J. Bacteriol. 88:545-552. 1964.-Reproductive viability and oxidative activity on various substrates (alanine, glutamic acid, glucose, lactic acid) were studied in Staphylococcus aureus cells subjected to (i) freezing and thawing, (ii) lyophilization, (iii) irradiation in suspension, (iv) irradiation in lyophilized state, and (v) lyophilization and irradiation in the presence of various substrates. Freezing and thawing in dilute albumin solution had no effect on viable cell count and on lactic acid oxidation, but the oxidation rates of alanine, glutamic acid, and glucose were decreased. Lyophilization reduced the viable cell count to 25 to 30% of the initial value, and decreased the oxidation rates of glutamic acid and alanine proportionally, whereas decrease in glucose oxidation was less than proportional. The lactic acid oxidation rate was not affected by lyophilization. An irradiation dosage of about 40,000 r was required to reduce the viable count of cells irradiated in suspension by one log(10) unit; lyophilized cells required 140,000 to 170,000 r. Cells irradiated in suspension with dosages sufficient to render them completely nonviable on culture continued to respire on lactic acid at about 60% the rate of nonirradiated cells, but the residual activities on the other substrates tested were less than 10 to 12%. Lyophilized cells irradiated with sufficient dosages for cultural nonviability retained 40 to 80% of the oxidative capacity of nonirradiated cells on the test substrates. Cells lyophilized and irradiated in the presence of albumin generally retained a greater portion of their oxidative activities compared with cells lyophilized and irradiated in buffer. Cells irradiated in the presence of various oxidizable substrates gave varying results, depending both on the substrate present during irradiation and the substrate added for the oxidative studies.

Reduction of mitotic delay in irradiated suspension cultures of rat thymocytes by an elevated salt concentration

Increasing the concentration of NaCl in the medium of thymocyte cultures from 0.12 M to 0.15 M after irradiation with 225 and 300 r reduces the duration of the initial mitotic delay. This procedure had no effect on cultures irradiated with 150 r.