Wild-type cells of Escherichia coli growing at 37 C in mineral salts-glucose medium with vigorous aeration were lysed at maximal exponential rates by 10-4 to 102M D-cycloserine. At concentrations above 2 X 10-2M, D-cycloserine was bacteriostatic. Low levels of D-cycloserine (10-6 M) and pencillin G (10 units per ml) interacted synergistically to cause a rapid exponential rate of lysis. Spontaneous mutations to D-cycloserine resistance occurred in discrete steps at frequencies of 106 to 107 for each step. First-, second-, and third-step D-cycloserine-resistant mutants were lysed at maximal exponential rates by D-cycloserine concentrations of 10-3, 3 X 10-3, and 5 X 103 M, respectively. D-Alanine, L-alanine, and DL-alanyl-DL-alanine reversed D-cycloserine-induced lysis, in that order of effectiveness. On the basis of these observations, a D-cycloserine-enrichment cycling technique was developed for isolation of auxotrophic mutants. D-Cycloserine at 2 X 10-3 M was as efficient as penicillin G (1,000 units per ml) for mutant enrichment in E. coli and should be useful for isolation of mutants in penicillin-resistant microorganisms. Bacterial conjugation experiments indicated that all three mutations conferring D-cycloserine resistance were linked to the met, locus. Transduction experiments showed that the mutation conferring first-step resistance was at least 0.5 min away from the mutations conferring secondand thirdstep resistance. The latter two mutations possibly occurred in the same gene, since they were sometimes carried in the same transducing phage. Studies on expression of D-cycloserine resistance indicated that these mutations were neither dominant nor recessive to each other nor to the D-cycloserine-sensitivity allele. Each allelic state exerted its influence on the phenotype independently of the others. These results are discussed in terms of the known inhibition of alanine racemase and D-alanyl-D-alanine synthetase by D-cycloserine.

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Large-scale adaptation of a recently reported glycine precipitation method for the production of factor VIII (FVIII) concentrate is described. Scaling up of the method required some modification including the addition of aluminum hydroxide to the glycine buffer to reduce the level of contaminating proteins in the final preparation and the use of centrifugation to replace filtration by glass beads. Furthermore, the resultant product was virus inactivated by incorporation of the organic solvent and detergent technique. At industrial level, the modified method gave a good recovery of FVIII activity (230 IU/l plasma) with high purity (4 IU/mg protein). The final product, after virus inactivation and lyophilization, yielded 185 IU of FVIII activity per liter of starting plasma and was considered to be suitable for clinical evaluation.