Thesis (Ph. D.)--University of Wisconsin--Madison, 1955. / Typescript. Abstracted in Dissertation abstracts, v. 16 (1956) no. 2, p. 221. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 109-123).

Wuppertal, Universiẗat, Diss., 1999.

Some of the previous work on the mechanism of action of streptomycin has indicated the general area of nucleic acid metabolism as being a possible site of action. Specifically, a report has appeared that streptomycin inhibits the "exchange reaction" catalyzed by polynucleotide phosphorylase. Therefore, the possible action of streptomycin on polynucleotide phosphorylase from a streptomycin sensitive strain of Escherichia coli has been reinvestigated. In preparation for the study on polynucleotide phosphorylase, the nucleoside diphosphates of adenosine, guanosine, cytidine, and uridine were synthesized in sufficient yield and of a satisfactory purity by the recently developed method of Moffatt and Khorana. It was not possible to confirm the inhibition of the polynucleotide phosphorylase catalyzed "exchange reaction" reported by Kornberg, using enzyme from our streptomycin sensitive E. coli SA. Nor was it possible to demonstrate any inhibition of the polymerization reaction catalyzed by this enzyme, or any effect on the sedimentation properties of the polymer so formed. However, the diamines putrescine, cadaverine, spermine, and spermidine, while having no effect on the time course of the polymerization reaction, did lower the sedimentation coefficient of the polymer formed and cause a more heterogeneous polymer to be formed. Streptomycin has been suggested as an inhibitor of protein synthesis. Therefore, a study of the possible effects of streptomycin on ribosomes from E. coli was made. These subcellular particles have been shown to be a site of protein synthesis. Dihydrostreptomycin (DHSM) interacted strongly with the ribosomes. It was found that most of the ribonucleo-protein precipitated when ribosomes were dialyzed overnight at 4° against buffer containing about 500 μg./ml. of DHSM. A study of ultracentrifuge patterns of dialysates of ribosomes against lower levels of DHSM indicated that disruption, with a loss of discrete sedimentation coefficients, occurred when the level of DHSM was about 350 μg./ml. A study of elution patterns from DEAE-cellulose columns indicated only a partial change in the pattern after breakdown. Examination of sedimentation coefficients of ribosome dialysates at lower DHSM levels indicated no specific effect of DHSM. The autodegradation of ribosomes by polynucleotide phosphorylase was studied. DHSM, at low levels, had an effect on the time course of the auto-degradation. A study of the distribution of induced β-D-galactosidase associated with the ribosomes indicated that it was associated with that protein of the ribosomes not precipitated by DHSM. It can be concluded that streptomycin interacts strongly with ribosomes. This gives support to recent claims that streptomycin inhibits ‘in vitro' protein synthesis, and that the site of this inhibition is the ribosome. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Using three different streptomycin variants of Escherichia coli the mechanism of action of streptomycin, as dihydrostreptomycin, has been studied. The variants used were either susceptible to, resistant to or dependent upon, the presence of dihydrostreptomycin during growth. It is a well-known fact that streptomycin, in sufficient quantities, will form a precipitate with nucleic acids, thus the precipitate formed from dihydrostreptomycin and bacterial cell contents was studied. Streptomycin also inhibits the formation of proteins, as exemplified by adaptive enzyme production, when it is added, during induction, to susceptible organisms. Corresponding effects correlating growth and synthesis have been noted, also, with the dependent and resistant variants. Using these facts and the fact that adaptive enzymes can be removed by the precipitation of cell contents with dihydrostreptomycin, regeneration of enzyme activity from the precipitate was attempted with polymethacrylic acid. This compound will dissociate protamine-nucleic acid complexes but proved ineffective in regenerating enzyme activity. By using dihydrostreptomycin precipitates of disrupted susceptible E. coli the observation was confirmed that DNase is ineffective in depolymerizing these precipitates. Dihydrostreptomycin precipitates of susceptible E. coli are partially depolymerized by RNase, however. Analysis in the ultra-centrifuge was carried out on cell-free material from susceptible E. coli on the addition of RNase, DNase and low, biologically active concentrations of dihydrostreptomycin. RNase and dihydrostreptomycin both removed two of the three typical components shown to be present in susceptible E. coli cells. DNase had no effect on these components. Purine and pyrimidine analysis of these dihydrostreptomycin precipitates from E. coli showed an absence of uracil, as such, but a relatively unaffected thymine content which indicates that it is mainly DNA rather than RNA affected by the dihydrostreptomycin. From these and previously observed facts the following theory is advanced. Streptomycin reacts with DNA to form a complex which in susceptible E. coli is not depolymerized by DNase. This complex formation between dihydrostreptomycin and DNA prevents protein synthesis as exemplified by adaptive enzyme formation. Because depolymerization of DNA by DNase is prevented, cell division cannot take place in the presence of dihydrostreptomycin. And because the directing effect of DNA on enzyme production is blocked, this process cannot occur. It must then follow that in the streptomycin-dependent E. coli, the antibiotic is required for the depolymerization of DNA and for the directing effect of DNA on protein synthesis. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

The mode of action of streptomycin has been investigated with specific emphasis on the effect of the antibiotic on adaptive enzyme formation by Escherichia coli. The first phase of this study was to investigate the possibility that streptomycin interferes with the action of a metabolite required by E. coli for enzyme synthesis and growth. A resistant variant of E. coli, which exhibited partial dependence on the drug for enzyme formation, was used to test for the presence of a metabolite in other variants which did not exhibit this dependence. Subcellular fractions derived from susceptible and resistant cells were tested for the presence of a substance which would satisfy the streptomycin requirement of partially dependent organisms for enzyme induction. All attempts to demonstrate such a metabolite were unsuccessful. In the second phase of the study, the effect of streptomycin on the protein and nucleic acid content of partially dependent cells during enzyme induction was studied. By the standard techniques employed, it was shown that the antibiotic did not affect either the gross protein or nucleic acid content of the cells during the period in which the formation of a specific enzyme was demonstrated. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

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Thesis (Ph.D.)--Boston University / The investigation of the fate of bacteria which are normally intracellular parasites has interested workers for many years. or special interest have been the roles played by phagocytic cells and immune sera in the resistance of animals against infections produced by these organisms. The research whose results are presented here was undertaken to study the role of phagocytic cells (macrophages) in immunity to Salmonella typhosa. Experiments were performed with macrophages isolated from the peritoneal cavities of mice. Cultures of these cells were prepared, and infected with virulent and avirulent strains of S. typhosa and Salmonella typhimurium. Streptomycin was added to some cultures after the period of phagocytosis to prevent extracellular growth of the bacteria. The phagocytic and bactericidal abilities of the macrophages were determined by comparisons of viable counts of organisms released from the cells and the number of intracellular bacteria on stained cover slip preparations [TRUNCATED]

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Thesis (M.D.)--Boston University