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Biomedical subjects

D R Morris

Publications and source records attributed to D R Morris.

At least 73 records · Page 4Linked to original sources
PubMed1983

Positively supercoiled plasmid DNA is produced by treatment of Escherichia coli with DNA gyrase inhibitors.

A procedure has been developed whereby the relative amounts of the topoisomers of E. coli plasmid can be determined for cells grown under a variety of conditions. Several applications of the procedure are presented. Addition of either novobiocin or oxolinic acid, two inhibitors of DNA gyrase, gives rise to positively supercoiled plasmid. A likely model for the introduction of positive supercoils, involving DNA gyrase itself, is discussed. Oxolinic acid is also shown to induce linearization of plasmid in vivo. Starvation of cells for ATP is shown to cause relaxation of plasmid. The shift of a gyrB temperature-sensitive strain to the restrictive temperature is also shown to cause plasmid relaxation. Finally, it is noted that polyamine starvation of E. coli has no detectable effect on the distribution of topoisomers.

Aminocoumarins↗
PubMed1982

Purification, properties and regulation of the level of bovine S-adenosylmethionine decarboxylase during lymphocyte mitogenesis.

S-adenosylmethionine decarboxylase was purified from the livers of calves treated with methylglyoxal bis(guanylhydrazone) to elevate the level of the enzyme. Purified bovine S-adenosylmethionine decarboxylase was similar in specific activity and subunit molecular weight (32,000) to the enzymes previously isolated from rat and mouse. The bovine liver enzyme immunologically crossreacted with S-adenosylmethionine decarboxylase from resting and mitogenically activated bovine lymphocytes. The rate of enzyme synthesis in activated lymphocytes was determined by labeling the cells with [3H]leucine and isolating the radioactive decarboxylase by affinity chromatography and sodium dodecyl sulfate gel electrophoresis. The rate of enzyme synthesis was increased 10-fold by 9 h after mitogen treatment, which accounts for the initial increase in cellular enzymatic activity. There was no further increase in the rate of S-adenosylmethionine decarboxylase synthesis that correlated with a second elevation of activity occurring at approx. 24 h after mitogenic activation. It was concluded that the second increase in enzyme activity was due to lengthening the intracellular half-life of the enzyme by 2-fold.

Adenosylmethionine Decarboxylase↗
PubMed1981

Induction of polyamine limitation in Chinese hamster ovary cells by alpha-methylornithine.

Chinese hamster ovary (CHO) cells in culture were limited for polyamines through the use of alpha-methylornithine (alpha MO), a competitive inhibitor of ornithine decarboxylase. Initial exposure of the cells to the inhibitor caused growth rate and intracellular polyamine content to decline continuously. Reseeding the alpha MO-treated cells into medium containing the inhibitor resulted in steady-state (exponential) growth at cell densities below 5 x 10(3) cells/cm2, at a rate approximately twofold slower than untreated cells. Under these conditions, putrescine and spermidine were undetectable and spermine remained relatively constant at a level approximately half that found in untreated cells. Addition of exogenous putrescine elevated the polyamine content and stimulated the growth of alpha MO-treated cultures. Thus, growth rate correlated with polyamine content in the alpha MO-treated cells. The growth of reseeded, alpha MO-treated cells became nonexponential at a density (5 x 10(3) cells/cm2) far below that at which untreated cells departed from exponential growth (1 x 10(5) cells/cm2). Medium obtained from high density, alpha MO-treated cultures inhibited the growth of cells at low density in the presence of alpha MO. Doubling the concentration of the defined components of conditioned medium did not markedly affect its capacity to inhibit growth. However, dialysis completely not markedly affect its capacity to inhibit growth. However, dialysis completely removed the inhibitory activity from conditioned medium. The results imply that a low molecular weight inhibitor of growth is produced by polyamine-limited cells. This is a variable that must be controlled in studies with polyamine-limited animal cells. Morphological studies indicated that subcellular organelles, including mitochondria, were largely unaffected by treatment with alpha MO. The maintenance of mitochondrial integrity in the presence of alpha MO demonstrates that the swelling of mitochondria observed previously in cells treated with methylglyoxal bis(guanylhydrazone) was not due to polyamine limitation. alpha MO-treated cells did, however, accumulate numerous cytoplasmic vacuoles. The identity of these vacuoles and their relationship to cellular physiology is not yet understood.

Animals↗
PubMed1981

Cell cycle parameters of Chinese hamster ovary cells during exponential, polyamine-limited growth.

We have previously shown that Chinese hamster ovary cells made polyamine deficient by treatment with alpha-methylornithine, an inhibitor of ornithine decarboxylase, grow exponentially in culture at low densities at one-half the rate observed in untreated (control) cultures. In this study, the cell cycle of polyamine-limited cells was examined by using thymidine autoradiography, mitotic index analysis, and fraction labeled mitoses analysis. We found that the longer doubling time of inhibitor-treated cultures was a consequence of increases in the lengths of the G1 and S phases. The expansion of the S phase was proportional to the increase in doubling time (twofold), whereas the G1 phase was lengthened by slightly more than a factor of 2. The lengths of the G2 and M phases were essentially unchanged. Putrescine stimulated the growth of inhibitor-treated cultures and restored the cell cycle parameters to those of untreated cells.

Animals↗
PubMed1981

Regulation of the level of S-adenosylmethionine decarboxylase during lymphocyte mitogenesis.

Bovine lymphocytes were mitogenically activated by the lectin concanavalin A and the activity of the polyamine biosynthetic enzyme, S-adenosylmethionine decarboxylase (SDC), was followed (R. H. Fillingame and D. R. Morris, Biochem. Biophys. Res. Commun. 52: 1020-1025, 1973). Enzyme activity began to increase at approximately 3 hours after cellular activation, reaching a peak at 9-12 hours. A second elevation of SDC activity coincided with the entry of the cells into S phase at 24 hours. The early and late increases in SDC activity were regulated by different mechanisms. The initial elevation of activity was due to approximately a 10-fold enhancement of the rate of enzyme synthesis; the enhanced rate of synthesis was maintained throughout the remainder of the culture period. The second increase in SDC activity was due to a lengthening of the intracellular half-life of the enzyme from 80 to 170 min. These changes in the rates of SDC synthesis and degradation fully account for the observed biphasic enzyme induction curve. In vitro translation of the SDC messenger RNA has been achieved and this will be used in further studies of the mechanism of enhanced enzyme synthesis.

Adenosylmethionine Decarboxylase↗
PubMed1980

Specificity of the spermidine requirement for the replication of phi X174 DNA be cell-free extracts of Escherichia coli.

A new experimental approach for assessing the biological significance of spermidine interactions in isolated systems is applied to the stimulation by spermidine of the conversion of phi X174 virion DNA to its replicative form by cell-free extracts of Escherichia coli. At 2.5 mM Mg2+, spermidine activated the reaction 20-fold. Varying the spermidine concentration affected both the rate and extent of this DNA synthetic reaction without altering the nature of the reaction products. We evaluated the biological significance of the spermidine requirement by measuring reaction rates in the presence of a homologous series of spermidine analogs of known activity in vivo. There was a lack of specificity, in that all of these analogs were capable of efficiently substituting for spermidine in stimulating the reaction rate. The relevance of this in vitro spermidine stimulation to Escherichia coli chromosome replication in vivo is discussed in light of the results obtained with the spermidine analogs.

Bacteriophage phi X 174↗
PubMed1980

Activation of early enzyme production in small lymphocytes in response to high, nonmitogenic concentrations of concanavalin A.

Lymphocyte mitogenesis is generally assessed by measuring the incorporation of [(3)H]thymidine into DNA. By this criterion, small lymphocytes, which are activated by relatively low doses of concanavalin A, are either unresponsive to or inhibited by higher concentrations. Because lymphocytes begin to synthesize DNA about 24 hr after addition of mitogen, the response is far removed temporally from the initial stimulus. We have chosen to use the induction of S-adenosylmethionine decarboxylase (S-adenosyl-L-methionine carboxy-lyase, EC 4.1.1.50) to assess early activation events in bovine lymphocytes. Adenosylmethionine decarboxylase induction is bimodal, with an initial phase beginning 3 hr after addition of concanavalin A and a second wave coinciding with the onset of DNA synthesis. The initial accumulation of the decarboxylase (0-9 hr) in cultures treated with "nonmitogenic" levels of concanavalin A (108 mug/ml) was similar to that observed in cultures stimulated with optimally mitogenic doses (18 mug/ml). The early induction of ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) was also similar under these two culture conditions. However, the second phase of adenosylmethionine decarboxylase accumulation, the induction of thymidine kinase (ATP: thymidine 5'-phosphotransferase, EC 2.7.1.21), and DNA replication were blocked at the higher concentrations of concanavalin A. The inhibition of late events by high doses of concanavalin A was reversible. Cells treated with alpha-methyl-D-mannopyranoside 25 hr after addition of a high dose of lectin responded with a second period of adenosylmethionine decarboxylase accumulation, induction of thymidine kinase, and progression through S phase. These results suggest that initial lymphocyte activation occurs normally at high doses of concanavalin A, but that the cells are reversibly blocked prior to induction of "late" enzymes and progression through S phase.

Adenosylmethionine Decarboxylase↗
PubMed1980

The internal viscosity of the human erythrocyte may determine its lifespan in vivo.

Young red cells obtained by high-speed centrifugation of a normal blood sample had an MCHC of 31.7 g/dl which corresponds to an internal viscocity of about 9 cP. Old red cells from the same blood sample had an average MCHC of 37.5 g/dl which is equivalent to a mean internal viscosity of about 54 cP. This increased internal viscocity of old cells must result in a prolonged transit time through the narrow channels of the splenic microvasculature and consequently an increased probability of contact with a phagocytic macrophage. This probability of contact increases as the internal viscosity of the cell is further increased and may represent one of the major factors associated with the detection and subsequent elimination of senescent cells by the spleen.

Erythrocyte Aging↗
PubMed1980

Structural specificity of the spermidine requirement of an Escherichia coli auxotroph.

A homologous series of spermidine analogs was synthesized with the general structure NH3+ (CH2)nNH2+(CH2)3NH3+, where spermidine has n = 4. The influence of these compounds on growth and on the syntheses of protein and messenger ribonucleic acid was examined in a spermidine auxotroph of Escherichia coli. All of the homologs tested were taken up by the cells to an intracellular level equivalent to the level of spermidine which gives optimal growth. With increasing chain length of the homologs, there was reduced ability to stimulate growth. The homologs with n = 7 and n = 8 were essentially inactive. A similar specificity was observed when the ability of the homologs to restore the rates of protein and messenger ribonucleic acid chain elongation was compared to that of spermidine. These results suggest that a definite spatial arrangement of the amino groups of spermidine is required for productive interaction at its intracellular site(s) of action.

Bacterial Proteins↗
PubMed1980

Stimulation of deoxyribonucleic acid replication fork movement by spermidine analogs in polyamine-deficient Escherichia coli.

We examined the rate of deoxyribonucleic acid (DNA) replication fork movement in polyamine-deficient cells of Escherichia coli by two independent techniques. DNA autoradiography was used to directly visualize the length of DNA produced during a given time interval, and replication rates were calculated. The amount of DNA synthesized after blocking protein synthesis also allowed calculation of replication rates. We found that the DNA chain elongation rate in polyamine-deficient cells was about half that of putrescine- or spermidine-supplemented cells. We also found that spermidine homologs of increasing chain length, when present at equal intracellular concentrations, exhibited a decreasing ability to support growth and the rate of DNA replication fork movement. The kinetics of recovery of DNA synthesis from the polyamine-deficient state were also investigated. A new rate of DNA synthesis was reached about 20 min after addition of spermidine to polyamine-limited cells. The rise in the rate of DNA synthesis was preceded by a rise in the intracellular concentration of spermidine.

DNA Replication↗
PubMed1979

The effects of suspending medium viscosity on erythrocyte deformation and haemolysis in vitro.

Fresh adult human erythrocytes were suspended in isotonic pH adjusted solutions containing various concentrations of Dextran T.500. The cells were subjected to uniform hydrodynamic shear stress in a Ferranti Shirley Cone and Plate Viscosimeter. The amount of lysis incurred at any given combination of exposure parameters was markedly affected by the viscosity of the suspending medium. Optical diffraction patterns obtained whilst the cells were undergoing shear demonstrated that cellular deformation was also a function of viscosity. Consequently, the distorted shape of the stressed cell may play a crucial role in the haemolytic process.

Adult↗
PubMed1978

Cellular polyamine depletion reduces DNA synthesis in isolated lymphocyte nuclei.

The accumulation of putrescine, spermidine and spermine in activated bovine lymphocytes was blocked by the combined action of two inhibitors of polyamine biosynthesis, methylglyoxal bis(guanylhydrazone) (MGBG) and alpha-methylornithine. Lymphocytes were cultured under three conditions: (1) alpha-methylornithine alone, (2) MGBG alone, or (3) alpha-methylornithine plus MGBG. DNA synthesis in nuclei isolated from these cells was reduced from control rates by approx. 10, 55 and 75%, respectively. In each case, the degree of inhibition was similar to that observed with the intact cells. Stimulation of nuclear DNA synthesis with the postnuclear supernatant fraction was not affected by polyamine depletion of the cells. Several experiments indicate that the reduced rate of in vitro DNA synthesis was caused by the lack of polyamines and not by alternate effects of the drugs. No inhibition was observed (1) when spermidine was added to inhibited cultures 12 h before harvest and nuclear isolation, (2) when the drugs were added after polyamines had accumulated, and (3) when the drugs were added directly to the in vitro assay. In addition, the degree of inhibition of in vitro DNA synthesis correlated with the degree of polyamine deficiency. These in vitro studies confirm the results obtained with whole cells and support the hypothesis that DNA synthesis is one cellular site of action of the naturally occurring polyamines.

Animals↗