Sulfhydryl Modification of Two Nicotinic Binding Sites in Mouse Brain

Two distinct binding sites with properties corresponding to those expected for nicotinic cholinergic receptors can be identified in brain by the specific binding of nicotine (or acetylcholine) and alpha-bungarotoxin. The effects of modification of these binding sites by treatment with the disulfide-reducing agent dithiothreitol were examined in tissue prepared from DBA mouse brains. Treatment with dithiothreitol reduced the binding measured with either ligand, and reoxidization of the disulfides fully restored binding. The effects of dithiothreitol treatment appeared to be due to a reduction in the maximal binding of nicotine and to a decrease in the binding affinity for alpha-bungarotoxin. Agonist affinity for the alpha-bungarotoxin binding site was reduced by treatment with low concentrations of dithiothreitol. The nicotine binding sites remaining after disulfide treatment displayed rates of ligand association and dissociation similar to those of unmodified tissue, but treatment of previously unmodified tissue with dithiothreitol accelerated the rate of nicotine dissociation. After reduction, both binding sites could be selectively alkylated with bromoacetylcholine. The results suggest that both putative nicotinic receptors in brain respond similarly to disulfide reduction and that their responses resemble those known for the nicotinic receptor of electric tissue.     

A protocol based on gas chromatography--flow counting for monitoring intermediary metabolism.

A method is described which allows the user to track the influence which changes in nutritional state have on specific areas of intermediary metabolism. The approach relies on combined gas chromatography-gas flow proportional counting; data processing programs are used. To illustrate the operation and potential of the method, the effect of serum supplementation of the growth medium on overall fatty acid metabolism in HeLa cells is reported.     

Spiral Plate Method for Bacterial Determination

A method is described for determining the number of bacteria in a solution by the use of a machine which deposits a known volume of sample on a rotating agar plate in an ever decreasing amount in the form of an Archimedes spiral. After the sample is incubated, different colony densities are apparent on the surface of the plate. A modified counting grid is described which relates area of the plate of volume of sample. By counting an appropriate area of the plate, the number of bacteria in the sample is estimated. This method was compared to the pour plate procedure with the use of pure and mixed cultures in water and milk. The results did not demonstrate a significant difference in variance between duplicates at the α = 0.01 level when concentrations of 600 to 12 × 10⁵ bacteria per ml were used, but the spiral plate method gave counts that were higher than counts obtained by the pour plate method. The time and materials required for this method are substantially less than those required for the conventional aerobic pour plate procedure.     

Repeated use of Bacillus subtilis cell walls for copper binding

Purified cell walls from Bacillus subtilis were repeatedly suspended in 5 mm CuCl₂ and, after removing unbound Cu, were suspended in 1% (v/v) HNO₃ to release bound Cu. The walls were then regenerated by washing in H₂O. After five cycles, copper binding actually increased slightly, probably due to enhanced exposure of binding sites in the walls. Thus bacterial walls may be used repeatedly for metal removal during bioremediation of heavy metal pollution.R.J.C. McLean is with the Department of Biology, Southwest Texas State University, 601 University Drive, San Marcos, TX 78666-4616, USA A.M. Campbell is with the Department of Chemical Engineering, Queen's University, Kingston, Ontario, K7L 3N6, Canada. P.T. Khu is with the Department of Mining Engineering, Queen's University, Kingston, Ontario, K7L 3N6, Canada. A.T. Persaud, L.E. Bickerton and D. Beauchemin are with the Department of Chemistry, Queen's University, Kingston, Ontario, K7L 3N6, Canada.