Alterations in Bacillus subtilis transforming DNA induced by beta-propiolactone and 1,3-propane sultone, two mutagenic and carcinogenic alkylating agents.
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Transforming DNA was exposed to either beta-propiolactone or 1,3-propane sultone and then used for transformation of competent bacteria to nutritional independence from tyrosine and tryptophan (linked markers) and leucine (an unlinked marker). The ability to transform was progressively lost by the DNA during incubation with either of these two chemicals. For all three markers the inactivation curve was biphasic, with a short period of rapid inactivation followed by one characterized by a much slower rate. The overall rate of inactivation was different for all three markers and presumably was related to the size of the marker. The decrease in the transforming activity was in part due to the slower rate of penetration of alkylated DNA through the cellular membrane and its inability to enter the recipient bacteria. This decrease in the rate of cellular uptake, even for DNA eventually destined to enter the cell, began almost immediately after its exposure to the chemical and ended up with an almost complete lack of recognition of the heavily alkylated DNA by the specific surface receptors of competent cells. Such DNA attached to sites on the surface of competent bacteria which were different from receptors specific for the untreated nucleic acid. This attachment was not followed by uptake of the altered DNA. Presence of albumin during the incubation with a carcinogen further increased the degree of inactivation, indicating that the artificial nucleoproteins produced under such conditions were less efficient in the transformation assay than was the naked DNA. Cotransfomration of close markers progressively decreased, beginning immediately after the start of incubation of DNA with the chemicals. Extensively alkylated DNA fractionated by sedimentation through sucrose density gradients showed a peculiar distribution of cotransforming activity for such markers; namely, molecules larger than the bulk of DNA ("megamolecules") showed less ability to transform the second marker than did some of the apparently smaller molecules which sedimented more slowly through the gradient. An increase in cotransformation of distant markers was evident in DNA molecules after a short exposure to an alkylating agent, but cotransformation of such markers was absent in DNA treated for longer periods. The observed changes in the transforming and cotransforming activities of the alkylated DNA can be explained by what is known about the physicochemistry of such DNA and in particular about the propensity of the alkylated and broken molecules to form complexes with themselves and with other macromolecules. (+info)
Detection of different types of damage in alkylated DNA by means of human corrective endonuclease (correndonuclease).
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Corrective endonuclease (correndunclease) activity of HeLa cells was assayed with alkylated DNA. Double-stranded, covalently closed DNA from phage PM II was treated with methyl methanesulfonate, N-methyl-N-nitrosourea, beta-propiolactone, or diepoxybutane to introduce alkylated bases and alkali-labile sites into the DNA. The damaged DNA was incubated with an extract of HeLa cells that catalyzes the formation of breaks at apurinic sites in double-stranded DNA. Methylated DNA was broken at every alkali-labile site by the HeLa correndonuclease, which indicated that these sites are similar to the apurinic sites produced by heating at acid pH. DNA alkylated with beta-propiolactone or diepoxybutane containing the same number of alkali-labile sites was broken to a far lesser extent. This indicates the presence of a second type of alkali-labile damage that is correndonuclease-insensitive. (+info)
Agents of transmissible spongiform encephalopathy (prion) are known to be extremely resistant to physicochemical inactivation procedures such as heat, radiation, chemical disinfectants such as detergents, alcohols, glutaraldehyde, formalin, and so on. Because of its remarkable resistance, it is difficult to inactivate prion. Chemical inactivation seems to be a practical method because it is applicable to large or fixed surfaces and complicated equipment. Here, three epoxides: beta-propiolactone, propylene oxide, and glycidol (GLD) were examined of their inactivation ability against scrapie-mouse prion protein (PrP(Sc)) under various conditions of chemical concentration, incubation time, and temperature. Among these chemicals, GLD worked most effectively and degraded PrP into small fragments. As a result of the bioassay, treatment with 3% GLD for 5 hr and 5% GLD for 2, 5 hr or 12 hr at room temperature prolonged the mean incubation time by 44, 30, 110 and 73 days, respectively. From dose-incubation time standard curve, the decrease in infectivity titers were estimated as 10(3) or more. Therefore, degradation of PrP(Sc) by GLD decreased the scrapie infectivity. It is also suggested that pH and salt concentrations influence the effect of GLD. Although further study is necessary to determine the optimal condition, GLD may be a potential prion disinfectant. (+info)
Effect of beta propiolactone viral inactivation on alpha1 antitrypsin values.
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AIMS: alpha1 Antitrypsin was undetectable in several patient samples treated with 0.5% beta propiolactone, which was used as a virucidal agent. This study was designed to confirm beta propiolactone as the cause and determine why it might have such an effect. METHODS: Volumes of 0, 5, 10, and 20 micro l of beta propiolactone were added to 2 ml aliquots of serum to make final concentrations of 0%, 0.25%, 0.5%, and 1% of beta propiolactone. alpha1 Antitrypsin concentrations and the pH were measured at different time intervals. The effects of adding buffer before the addition of beta propiolactone, NaOH after beta propiolactone, and 6M HCl instead of beta propiolactone were also measured. RESULTS: The addition of beta propiolactone to a volunteer's serum showed a fall in both alpha1 antitrypsin values and pH with increasing time and concentration of beta propiolactone. This effect was also seen when adding HCl, but was partially prevented by buffering the serum or adding NaOH. CONCLUSIONS: These results suggest that it is the acidity of the degradation products of beta propiolactone that is responsible for the fall in alpha1 antitrypsin values. This fall in alpha1 antitrypsin values was dependent on the concentration of beta propiolactone used and the length of time before the test was performed. The effect of beta propiolactone on laboratory tests should be re-evaluated, with attention being paid to sample pH, storage time, and storage temperature. (+info)
Ethylenimine-inactivated rabies vaccine of tissue culture origin.
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The replication of seven rabies virus strains (CVS, HEP, PV, ERA, WIRAB, CPZ and BOLIVAR) in BHK cells and the inactivation dynamics of these strains by beta-propiolactone, acetylethylenimine, and ethylenimine were studied to find the most immunogenic strain and the most economic and stable inactivating agent for the production of an inactivated tissue culture rabies vaccine for animal use. The seven strains reached the peak of virus production 3 to 5 days after inoculation of the cell culture; PV yielded the highest virus titer (10(9) plaque-forming units/ml). The infectivity of virus suspensions containing 10(7) to 10(8) plaque-forming units/0.1 ml was inactivated by beta-propiolactone in 0.5 h, acetylethylenimine in 3.0 h, and ethylenimine in 1.0 h. Most of the vaccine lots prepared with the different strains and inactivating agents passed a modified National Institutes of Health potency test. The vaccines prepared with the PV strain had consistently higher antigenic values (equal or better than four) than the other six strains. This difference was highly significant (F6,12=59.8), whereas there were no statistically significant differences among the antigenic values of the vaccine lots prepared with the three inactivating agents. Batches of lyophilized and liquid vaccine stored at 4 C maintained potency for over 1 year. Ten dogs vaccinated with a vaccine prepared with the PV strain and inactivated with ethylenimine developed a good antibody response and resisted challenge 60 days after vaccination, while seven of eight nonvaccinated controls died of rabies. This information indicates that an inactivated, stable, economic, and easy-to-prepare rabies vaccine can be produced in BHK cells by using the PV strain and ethylenimine as an inactivating agent. (+info)
Restriction of carcinogen-induced error incorporation during in vitro DNA synthesis.
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The in vitro accuracy of DNA replication has been investigated through the measurement of the frequency with which noncomplementary nucleotides were incorporated during polynucleotide replication. The effect of beta-propiolactone treatment of deoxynucleotide templates, ribopolynucleotide templates, and the DNA polymerase from avian myeloblastosis virus was determined. Treatment of the deoxynucleotide template, poly(dA) (see article) oligo(dT) 12-18, by beta-propiolactone resulted in an increased frequency of noncomplementary nucleotide incorporation during DNA polymerization. Carcinogen treatment of the ribonucleotide templates, poly(rA) (see article) oligo(dT) 12-18, and poly(rC) (see article) oligo(dG) 12-18, and carcinogen treatment of avian myeloblastosis virus DNA polymerase did not alter the frequency of noncomplementary nucleotide incorporation. This suggested that carcinogen-induced error incorporation during DNA synthesis was restricted solely to the treatment of a deoxynucleotide template. (+info)
Immunopathogenicity and oncogenicity of murine leukemia viruses. II. Infection of mice and rats with Scripps leukemia virus.
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The pathologic consequences of infection of newborn mice and rats with MuLV (Scripps leukemia virus--SLV) were observed. Serum MuLV p30 concentrations of most strains were elevated 20 to 100 times that of controls while MuLV gp70 levels were elevated only 1.1 to 14.8 times, probably reflecting in part the higher concentrations of gp70 in control sera but also indicating the lack of parallelism in regulation of synthesis of these two viral antigens. Infected mice of most strains developed immunologic diseases, including antinuclear antibody and glomerulonephritis, but not Coombs' antibodies. The nature and severity of the immunologic disease varied considerably, depending upon the genetic character of the host. Most infected animals developed lymphatic leukemias, but four strains showed partial to complete resistance to SLV oncogenesis: BALB/c (nude); C57 Bl/6; (NZB times NZW) F1, and (NZW times BALB/c) F1. (+info)
Development of a potent inhibitor of 2-arachidonoylglycerol hydrolysis with antinociceptive activity in vivo.
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