Inactivation of de novo DNA methyltransferase activity by high concentrations of double-stranded DNA

The activity of eukaryotic DNA methyltransferase diminishes with time when the enzyme is incubated with high concentrations (200–300 μg/ml) of unmethylated double-stranded Micrococcus luteus DNA. Under similar conditions, single-stranded DNA induces only a limited decrease of enzyme activity. The inactivation process is apparently due to a slowly progressive interaction of the enzyme with double-stranded DNA that is independent of the presence of S-adenosyl-l-methionine. The inhibited enzyme cannot be reactivated either by high salt dissociation of the DNA-enzyme complex or by extensive digestion of the DNA. Among synthetic polydeoxyribonucleotides both poly(dG-dC) · poly(dG-dC) and poly(dA-dT) · poly(dA-dT), but not poly(dI-dC) · poly(dI-dC), cause inactivation of DNA methyltransferase. This inactivation process may be of interest in regulating the ‘de novo’ activity of the enzyme.     

Inactivation of de novo DNA methyltransferase activity by high concentrations of double-stranded DNA

The activity of eukaryotic DNA methyltransferase diminishes with time when the enzyme is incubated with high concentrations (200-300 micrograms/ml) of unmethylated double-stranded Micrococcus luteus DNA. Under similar conditions, single-stranded DNA induces only a limited decrease of enzyme activity. The inactivation process is apparently due to a slowly progressive interaction of the enzyme with double-stranded DNA that is independent of the presence of S-adenosyl-L-methionine. The inhibited enzyme cannot be reactivated either by high salt dissociation of the DNA-enzyme complex or by extensive digestion of the DNA. Among synthetic polydeoxyribonucleotides both poly(dG-dC).poly(dG-dC) and poly(dA-dT).poly(dA-dT), but not poly(dI-dC).poly(dI-dC), cause inactivation of DNA methyltransferase. This inactivation process may be of interest in regulating the 'de novo' activity of the enzyme.     

Potentiometric titration of cytochrome-bo type quinol oxidase of Escherichia coli: evidence for heme-heme and copper-heme interaction

The cytochrome-bo quinol oxidase of Escherichia coli contains a high-spin b-type heme (cytochrome o), a low-spin b-type heme (cytochrome b) and copper. The EPR signal from cytochrome o is axial high spin and when titrated potentiometrically gives a bell-shaped curve. The low-potential side of this curve (Em7 approx. 160 mV) corresponds to the reduction/oxidation of the cytochrome. The high-potential side (Em7 approx. 350 mV) is proposed to be due to reduction/oxidation of a copper center; in the CuII form tight cytochrome o-copper spin coupling results in a net even spin system and loss of the EPR spectrum. Optical spectra of the alpha-bands of the reduced cytochromes at 77 K show that cytochrome b has its maxima at 564 nm when cytochrome o is oxidized but that this shifts to 561 nm when cytochrome o (max. 555 nm) is reduced. Both a heme-copper (cytochrome o-CuII) and a heme-heme (cytochrome o-cytochrome b) interaction are indicated in this quinol oxidase. These results indicate that cytochrome-bo quinol oxidase has a binuclear heme-copper catalytic site and suggest striking structural similarity to subunit I of the cytochrome aa3 system.     

Valinomycin-induced modulation of adriamycin resistance and cationic probe distribution in MCF-7 cell lines.

In vitro restoration of adriamycin sensitivity in a resistant human breast tumor cell line was obtained by continuous exposure to nanomolar nontoxic valinomycin concentrations. Seven-day treatment with nanomolar valinomycin concentrations caused a slight increase of the signal of the cationic fluorescent cyanine probe DiOC5(3) but did not appreciably affect adriamycin incorporation in the cells. A marked increase of the DiOC5(3) signal was obtained in the presence of micromolar valinomycin concentrations, which were incompatible with the in vitro cellular growth.     

Characterization of DNA sequences that mediate nuclear protein binding to the regulatory region of the Pisum sativum (pea) chlorophyl a/b binding protein gene AB80: identification of a repeated heptamer motif

Two protein factors binding to the regulatory region of the pea chlorophyl a/b binding protein gene AB80 have been identified. One of these factors is found only in green tissue but not in etiolated or root tissue. The second factor (denominated ABF-2) binds to a DNA sequence element that contains a direct heptamer repeat TCTCAAA. It was found that presence of both of the repeats is essential for binding. ABF-2 is present in both green and etiolated tissue and in roots and factors analogous to ABF-2 are present in several plant species. Computer analysis showed that the TCTCAAA motif is present in the regulatory region of several plant genes.     

Effects of protein-protein and protein-lipid interactions on heme site conformation in the mitochondrial b cytochromes

Removal of lipid from detergent-solubilized succinate cytochrome c reductase by a mild method leads to a series of changes in the optical and EPR spectra of the b cytochromes. This culminates in a state that resembles purified b cytochromes from the same source and bisimidazole ferriheme model complexes. Reconstitution of the lipid-depleted complex with phospholipid restores the native spectra in a significant fraction of the complexes in the early stages of lipid depletion. Once the final state has been reached, however, reconstitution has so far been incapable of restoring described in this communication can be related to a model for integral membrane cytochromes.     

Contribution of cardiogenic oscillations to gas exchange in constant-flow ventilation

The contribution of cardiogenic oscillations to gas exchange during constant-flow ventilation was examined in 11 dogs. With the use of two variations of cardiopulmonary bypass to maintain the systemic and pulmonary circulation, the influence of cardiogenic oscillations was removed by arresting the heart. Cardiac arrest by ventricular fibrillation was associated with a mean decrease in alveolar ventilation of 43% in five dogs on right and left heart bypass. However, successful defibrillation and return of the prearrest level of alveolar ventilation could not be achieved; thus we studied six dogs on left heart bypass. Alveolar ventilation decreased an average of 37% with cardiac arrest, and defibrillation resulted in a return of alveolar ventilation to 81% of the prearrest value. These results are consistent with previous predictions that cardiogenic oscillations are an important mechanism of gas transport during constant-flow ventilation.     

Spontaneous binding ofYersinia enterocolitica to murine leukocytes

Twelve strains ofYersinia enterocolitica were examined for their ability to bind spontaneously to murine leukocytes. Each of eight HeLa cell invasive strains exhibited nonselective binding to peritoneal leukocytes, lymph node leukocytes, and thymocytes, whereas four noninvasive strains lacked binding properties. Like the HeLa cell invasion, the binding ofY. enterocolitica to leukocytes was much less efficient for bacteria grown at 37°C than for bacteria grown at 22°C. The binding properties were not influenced by the virulence plasmid that codes for Vwa⁺ phenotype. This leukocyte binding test is proposed as a simple assay for invasive properties ofY. enterocolitica.