Maximal amounts of prodigiosin were synthesized in either minimal or complete medium after incubation of cultures at 27 C for 7 days. Biosynthesis of prodigiosin began earlier and the range of temperature for formation was greater in complete medium. No prodigiosin was formed in either medium when cultures were incubated at 38 C; however, after a shift to 27 C, pigmentation ensued, provided the period of incubation at 38 C was not longer than 36 hr for minimal medium or 48 hr for complete medium. Washed, nonpigmented cells grown in either medium at 38 C for 72 hr could synthesize prodigiosin when suspended in saline at 27 C when casein hydrolysate was added. These suspensions produced less prodigiosin at a slower rate than did cultures growing in casein hydrolysate at 27 C without prior incubation at 38 C. Optimal concentration of casein hydrolysate for pigment formation by suspensions was 0.4%; optimal temperature was 27 C. Anaerobic incubation, shift back to 38 C, killing cells by heating, or chloramphenicol (25 gg/ml) inhibited pigmentation. Suspensions of washed cells forming pigment reached pH 8.0 to 8.3 rapidly and maintained this pH throughout incubation for 7 days. Measurements of viable count and of protein, plus other data, indicated that cellular multiplication did not occur in suspensions of washed cells during pigment formation. By this procedure utilizing a shift down in temperature, biosynthesis of prodigiosin by washed cells could be separated from multiplication of bacteria.

Addition of casein hydrolysate to suspensions of washed, nonpigmented, nonproliferating Serratia marcescens incubating at 27 C induced biosynthesis of prodigiosin. Four amino acids of casein hydrolysate, DL-aspartic acid, L-glutamic acid, L-proline, and L-alanine caused formation of pigment when added individually. DL-Ornithine also was effective. Optimal concentrations for maximal pigmentation were 5 to 10 mg/ml; at these high concentrations, D-serine also induced biosynthesis of some prodigiosin. DL-Alanine and -ornithine were as effective as the Liosomers, but L-glutamic acid and L-proline gave better responses than their racemic mixtures. Kinetics of prodigiosin biosynthesis after addition of DL-alanine (20 mg/ml) were similar to those of cells suspended in 0.2% casein hydrolysate. The other amino acids were less effective. Addition of 5 mg of DL-alanine or casein hydrolysate per ml to minimal medium increased by 30% the amount of prodigiosin formed by growing cells after incubation for 7 days at 27 C. Cultures grown for 7 days at 27 C in 0.2% casein hydrolsate formed more prodigiosin than did suspensions of nonproliferating cells containing individual amino acids or casein hydrolysate. However, more pigment was produced by cells suspended in L-alanine (5 mg/ml) or L-proline (10 mg/ml) than when suspended in 0.4% natural or synthetic casein hydrolysate. Filtrates from suspensions of nonproliferating cells forming pigment in L-proline induced more rapid formation of prodigiosin, but filtrates from suspensions in DL-alanine did not. The data supported the hypothesis that pyrrole groups of prodigiosin may be synthesized from 5-carbon amino acids such as proline, ornithine, aspartic, and glutamic acids, but the role of alanine is unknown.on August 1, 2020 by guest

No abstract

Aggregation and adhesion of stored platelets were studied by a modified Wright technique. Comparisons of platelet rich plasma collected in ACD solution were made with and without aggregating agents, and at storage temperatures of 5"C, 24°C and 37°C. Platelets showed a loss of response t o 0.5 pg ADP/ml, collagen and agar after storage for 4-6 h a t the three temperatures with the greatest loss a t 37°C and the least change at 5°C. The response to 5 pg ADP/ml also was best maintained at 5°C. After 6-8 h of storage a t 5°C. there was some decrease in free (single) platelets after rotation in the absence of aggregating agents, with a marked decrease after 48 hours storage. With storage a t 24°C and 37"C, decrease in single platelets after rotation was not demonstrated during the first 24 h. A response to 5 pg ADP/ml was maintained for more than a week with the 5°C preparations. Acidification diminished the response to ADP for the first few days of storage and also depressed platelet aggregation and adhesion in the absence of aggregating agents on prolonged storage. The results of this irt uilro study would appear to correlate with the loss of hemostatic effect of stored platelets on transfusion. The sustained response to 5 pg ADP/ml a t 5°C suggests that this may be the best temperature for short term storage, although there is less spontaneous aggregation of platelets at the end of 24 h with 24°C storage. Also the 'storage lesion' appears to include loss of ADP or inability to release this substance from platelets.