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Biomedical subjects

A R Stevens

Publications and source records attributed to A R Stevens.

At least 37 records · Page 2Linked to original sources
PubMed1975

DNA synthesis in growth and encystment of Acanthamoeba castellanii.

Differentiation of Acanthamoeba castellanii into dormant cysts occurs spontaneously in stationary phase cultures, or can be induced experimentally by starvation. Although no further increase in cell density occurred after induction in either case, incorporation of [H]thymidine into DNA continued at a reduced rate through the period when differentiated products (cyst wall components) were formed. No net accumulation of DNA occurred during differentiation, indicating that the DNA synthesis occurring at this time was balanced by breakdown. When either 5-fluorodeoxyuridine (FUdR) or hydroxyurea was added to exponentially growing cultures, growth was terminated and the subsequent spontaneous encystment was delayed in comparison with untreated stationary phase cultures. A similar delay was observed for experimentally induced encystment of FUdR-pretreated cells. In all cases, delay of encystment was correlated with inhibition of 32PO4 incorporation into DNA, and unexpectedly also into RNA. Addition of FUdR at zero-time of experimental induction of cells not previously exposed to FUdR, on the other hand, had no effect on encystment or on 32PO4 incorporation. The delay of encystment produced by FUdR and hydroxyurea, therefore, appeared to reflect a requirement for normal synthesis of DNA and/or RNA not only during encystment, but also during the period of exponential growth just before encystment induction.

Animals↗
PubMed1974

In vitro and in vivo activity of 5-fluorocytosine on Acanthamoeba.

The results of our studies indicated that the avirulent Neff strain of Acanthamoeba was more susceptible to the activity of the anti-metabolite 5-fluorocytosine (5-FC) than was the virulent A-1 strain or a mouse brain reisolate of this strain, designated A-3. Results of competition experiments in which cultures were exposed simultaneously to 5-FC and either uracil, thymidine, or both uracil and thymidine demonstrated that the drug was directed against both deoxyribonucleic acid and ribonucleic acid in the avirulent strain, whereas ribonucleic acid was mainly affected in the virulent amebas. Concentrations >10 mug of 5-FC per ml were amebicidal to the avirulent strain; lower concentrations of the drug, which only affected growth slightly, significantly impaired the capacity of the cells to spontaneously encyst in stationary-phase cultures. On the other hand, the virulent strains were capable of growing in the presence of 5-FC (40 mug/ml) after an initial period of susceptibility. After a few transfers in growth medium lacking the drug, 5-FC-treated virulent amebas exhibited growth parameters typical of untreated cells. However, after successive subcultures in drug-free medium, 5-FC-treated cells lost their resistance and were again susceptible to the drug. This result suggested that the capacity of the cells to develop resistance resulted from a drug-induced mechanism. Spontaneous encystment, which was normally minimal in stationary-phase A-1 or A-3 cultures, was enhanced in A-3 but not A-1 cultures treated with 5-FC (>30 mug/ml). Results obtained from experiments to determine the effectiveness of 5-FC in protecting mice experimentally infected with either A-1 or A-3 amebas indicated that the clinical usefulness of 5-FC may be limited by the capacity of the amebas to develop resistance.

Acanthamoeba↗
PubMed1973

RNA synthesis and turnover during density-inhibited growth and encystment of Acanthamoeba castellanii.

Alterations in transcription that precede and accompany encystment (E) of suspension grown A. castellanii have been investigated. Comparative studies were performed on cells undergoing spontaneous E in high density stationary phase cultures or after experimental induction of E at low cell densities by deprivation of nutrients in exponential growth. Onset of growth deceleration at high cell densities was accompanied by an increase in the cellular RNA. The maximum RNA content occurred in cells at stationary phase and subsequently declined with the appearance of cysts in the cultures. On the contrary, the RNA content in cells whose growth was immediately terminated by experimental E induction remained at a constant exponential level through 5 h postinduction and then began to decline shortly before the appearance of cysts. The mature cyst formed in stationary phase cultures and after experimental E induction contained an equivalent amount of RNA ( approximately 50% of the exponential value). Comparison of the kinetics of [(3)H]uridine incorporation demonstrated that there was an abrupt reduction in the rate of uridine incorporation into RNA with onset of growth deceleration or after growth termination in experimental E induction. The reduced incorporation of uridine into RNA could not be attributed to to a reduced uptake of the isotope by the cells or an altered capacity of the cells to phosphorylate uridine. Uridine continued to be incorporated into RNA at a reduced rate in cells throughout growth deceleration, in stationary phase, and up to 12 h postexperimental induction. Considered together, these results indicate that a buildup in RNA is not necessary for induction of encystment in acanthamoeba. The accumulated RNA in stationary phase cells appears to be due to the greater reduction in the growth rate than in transcription and the absence of RNA turnover in cells during growth deceleration. Initiation of RNA turnover appears to accompany growth termination and induction of E. The results further demonstrate that the regulation of the rate of transcription is closely coordinated with the control of growth and encystment in acanthamoeba.

Animals↗
PubMed1973

Quantitative growth of Naegleria in axenic culture.

A strain of Naegleria gruberi, isolated from a Vero cell culture and designated TS-1, was axenically cultivated in monolayer and mass aerating suspension culture. Cultural conditions for constant growth parameters and high-exponential cell densities were defined. Serum or other supplemented fractions were found essential in both Trypticase-yeast extract-glucose (TYG) and Casitone (CAS)-based media. Monolayer cultures grown in the CAS medium required lower levels of serum to reach maximum stationary densities of amoebae than cultures grown in the TYG medium. Heat-killed (121 C, 10 min) whole cell and cell lysate bacterial fractions were capable of replacing the serum in both the TYG and CAS media. Heat-killed bacterial fractions provided the same levels of growth as attained with serum in TYG medium, whereas the bacterial lysate supported only minimal growth in the same medium. In the CAS medium, both bacterial fractions resulted in the same level of growth which was equal to that obtained in reduced serum content. Strain TS-1 was established in suspension culture with the CAS medium used in monolayer culture. The addition of sheep red blood cells (RBC) or RBC lysate greatly enhanced growth responses. Further modifications resulted in a final medium for suspension culture consisting of Casitone-yeast extract-glucose-vitamin base, supplemented with serum and RBC lysate. This medium supported growth with a mean generation time of 9 h at 30 C and a stationary phase yield of greater than 5 x 10(6) amoebae per ml.

Air↗
PubMed1971

Reformation of nucleolus-like bodies in the absence of postmitotic RNA synthesis.

The dependence of nucleolar reformation on RNA synthesis that resumes in late anaphase or early telophase has been investigated in synchronously dividing Amoeba proteus. RNA synthesis was completely inhibited throughout all stages of mitosis and the early hours of interphase with high concentrations of actinomycin D. In such cells, nucleolus-like bodies that bind azure B and pyronin were apparent in the reformed nuclei. The bodies appear as dense, fibrous masses with loosely associated, finely fibrillar material. There are no characteristic granular regions in the reformed structures. It is suggested that the bodies probably represent mainly nucleolar protein and residual RNA which can bring about the reorganization of nucleoli in the absence of postmitotic RNA synthesis.

Amoeba↗