A rapid method for the purification of supercoiled PM2 DNA by affinity chromatography on H1 histone covalently coupled to agarose.

A simple and rapid method is described for the purification of supercoiled PM2 DNA by affinity chromatography on columns of H1 histone covalently coupled to agarose. The method does not require the use of intercalating agents or ultracentrifugation procedures. Under the conditions most appropriate for purification, elution is carried out in a single step with buffered 0.7 M NaCl after the sample has been loaded onto the column in buffered 0.2 M NaCl. The DNA eluted at the higher salt concentration consists of supercoiled closed circular DNA at greater than 90% purity independently of the ratio of supercoiled to nicked circular DNA in the input mixture.     

Directly observed 15N NMR spectra of uniformly enriched proteins

The proteins cytochrome c2, cytochrome c', and ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum were enriched in 15N by growth of the organism on 15NH4Cl. The proteins were purified to homogeneity and studied by 15N NMR. Longitudinal and transverse relaxation times as well as the nuclear Overhauser effects were determined for various groups of the proteins which vary in molecular weight from 13,000 to 114,000. The values of these parameters for the amide resonances or for groups thought to be rigid were consistent with the molecular weights of the proteins. Relaxation times of the amino-terminal alpha-amino groups and the side chain nitrogen atoms of arginine and lysine were consistent with much more rapid motion. Nitrogen atoms having bound protons were generally found to be decoupled from the protons by chemical exchange. Demonstrable 1H-15N coupling was taken as an indication that exchange was hindered, either by hydrogen bonding interactions or by inaccessibility of the group to solvent. Histidine side chain nitrogen atoms, which experience a large chemical shift upon protonation/deprotonation, were often found to be broadened beyond detectability by chemical exchange and tautomerization. Strategies for improving sensitivity and for obtaining specific peak assignments are also discussed.     

Oral behaviour following forebrain ablations in Gallus Domesticus

The oral behaviour of adult Brown Leghorn hens was recorded in response to oral stimulation with water, 2 M sodium chloride, 2 M acetic acid and 0.1 M quinine hydrochloride before and after surgical ablation of either the anterior telencephalon or the entire telencephalon and/or the diencephalon. It was found that beak wiping behaviour could be abolished by the removal of the anterior telencephalon. Head shaking behaviour was abolished only by the complete removal of the forebrain (telencephalon and diencephalon) whereas beak and tongue movements persisted after forebrain removal. Although these three behaviour patterns occur together in response to the stimulus, they appear to be controlled in different areas of the brain. The results are discussed in relation to the current work on the control of oral behaviour in mammals.     

Chemiluminescence associated with phagocytosis of foreign particles in rabbit alveolar macrophages

Rabbit alveolar macrophages exhibit a chemiluminescent response which is associated with phagocytosis of zymosan and polystyrene-butadiene particles. The chemiluminescence reaches a peak in 15 to 25 minutes and then gradually diminishes over the next 1 to 3 hours. During the time of maximal light emission there appears to be no actual uptake of particles, but the response is dependent upon the particle concentration. The metabolic inhibitor, DNP (2,4-dinitrophenol), causes a rapid inhibition of the chemiluminescent response. The addition of ATP to the medium prior to exposure of the cells to particles causes the chemiluminescent response to be greatly diminished, i.e., 0.3mM ATP virtually abolishes the response. These experiments suggest that some metabolic response of the cell to phagocytosis is responsible for the chemiluminescence.