Guilford B. Reed

Synthesis of radioactive glutamine from C14O2 in swiss chard leaves and its isolation by paper chromatography

Abstract 1. 1. Detached leaves of beet, swiss chard, and tobacco were found to produce large amounts of glutamine when supplied with NH 4 NO 3 in light. After an initial lag phase of 24 hr., the glutamine content of swiss chard leaves rose rapidly and continuously for at least 5 days, reaching a value of 5% of the fresh leaf weight. This glutamine was derived from photosynthetic carbon and was in dynamic equilibrium with the leaf carbohydrates. 2. 2. A method for the biosynthesis of radioactive glutamine was developed. A detached swiss chard leaf is supplied with 0.1 M NH 4 NO 3 for 48 hr. under continuous illumination. C 12 O 2 is fed during the first 24-hr, period, and C 14 O 2 during the second. In an experiment performed under these conditions, 28% of the fed C 14 was incorporated into glutamine. 3. 3. Since chemical isolation of glutamine from a leaf resulted in a product of low purity and specific activity, a Chromatographic technique has been evolved. Using this technique, 71.6% of the available glutamine was recovered from a leaf. This glutamine was over 99% pure. The final product from a 1 mc. feeding to one swiss chard leaf was 96.5 mg. of uniformly labeled glutamine with a specific activity over 4 × 10 6 counts/mm.

Labelling in the glucose deposited as starch during photosynthesis.

Summary It is shown that glucose recovered from starch deposited in photosynthesis in presence of C14O2 may be labelled primarily in 3 and 4 or 1 and 6 positions or evenly in all 6 positions, depending upon the periods of darkness and illumination to which the leaf is subjected.

Metabolism of radioactive asparagine in wheat leaves and Lupinus angustifolius seedlings.

Abstract 1. 1. Radioactive asparagine has been isolated from lupine seedlings, which were permitted to carry on photosynthesis for 6 days in the presence of C14O2. It was found to contain 55% of its activity in the α-carboxyl. 2. 2. Radioactive asparagine was vacuum infiltrated into wheat and lupine seedlings, and its subsequent metabolism was observed. 3. 3. Utilization of asparagine for both CO2 production and the synthesis of cells was observed in all cases. This was true even in young lupine seedlings before they develop their photosynthetic mechanism. 4. 4. Radioactive glucose, infiltrated into lupine seedlings, was utilized 2 1 2 –5 times faster than asparagine. Although glucose was used about equally for respiration and cell synthesis, asparagine was used mainly for the synthesis of cells.

Synthesis of glucose and starch by tobacco leaves from HC14OOH, CH3C14OOH, CH3CHOH · C14OOH, C14H3 · C14HOH · COOH, and C6H5 · -C14OOH☆

Abstract 1. 1. Detached tobacco leaves were placed on solutions of radioactive formate, acetate, lactate, and benzoate, and synthesis of free glucose and starch from these acids was studied. 2. 2. It was found that these acids were utilized for carbohydrate synthesis only in light, but not in darkness. Benzoic acid was not utilized even in light. 3. 3. The absence of CO 2 in the air or glucose in the acid solution had no effect on the utilization of formate or carboxyl-labeled lactate for the synthesis of glucose. On the other hand, carboxyl-labeled acetate was utilized for this purpose only in the presence of either CO 2 or added glucose. 4. 4. Both formate and carboxyl of lactate were converted to CO 2 and then entered glucose via 3,4 positions in the usual photosynthetic sequence. In addition, both contributed also directly to the 1,6 positions of glucose. 5. 5. The carboxyl carbon of acetate entered glucose both as photosynthetic CO 2 and also directly into the 2 and 5 carbon positions of glucose.

Gas Gangrene-Toxin Production in Gelatin-Thioglycollate Medium.

Conclusions The gas gangrene group of anaerobes grow luxuriantly in a medium consisting of a buffered salt-mixture with dextrose and gelatin. Highly purified gelatin is not suitable. In this medium all the toxigenic gas gangrene species produce a high and consistent yield of hemotoxin and lethal toxin. The toxins are actively antigenic and make effective toxoids.

Gas-Gangrene-Toxin Production

Since Bulls original demonstration that Cl. welchii is toxigenic it has been customary to cultivate this and other gas-gangrene anaerobes for toxin-production in chopped-meat media, rich infusion-broths or serum-broths. It seems evident from the literature that many workers have found these methods to give highly variable yields of toxin. In our experience different batches of such media made in an apparently similar manner, inoculated with the same strain of organism and handled in as nearly as possible an identical manner, give very different yields of toxin. The recent experience with the production of diphtheria toxin has indicated that more consistent yields may be obtained by substituting for complex infusions, media of known, or for the most part known, chemical composition. After several preliminary trials the following medium was found to give fairly high yields of toxin which are consistent from batch to batch with all the toxin-producing members of the gas-gangrene group of organisms: The most consistent results have been obtained with freshly prepared and autoclaved media. While small inocula give good growths, more uniform toxin-yields have followed the use of inocula consisting of 3% of a 5 to 6 hour culture in the same medium. Many modifications of this medium have been tried, particularly various proportions of gelatin and peptone. Variations of 1 to 10% of gelatin and 0.1 to 10% of peptone give fair yields of toxin but with a maximum at about 5% gelatin and 1% of peptone. Bacto peptone is slightly superior to several others tested. Fair yields of toxin are obtained with 5% of gelatin and no peptone. Larger concentrations of glucose and other sugars, fermentable by the organism in question, give somewhat more rapid growth but the yield of toxin is conspicuously lower probably due to the greater acid-production.

Body Temperature and Plasma Lipids in Rabbits.

Summary Plasma phospholipid and free cholesterol were estimated by oxidative micromethods in 30 normal young rabbits and their concentration found to bear no relation, individually or collectively, to variations in normal body temperature between 100 and 103°F.

Fibrinolytic Action of Gas Gangrene Anaerobes.

Conclusion 1. It is shown that most strains of the more important saccharolytic, toxin-forming gas gangrene species, Cl. welchii, Cl. novyi, Cl. septicum and Cl. sordellii, produce an active fibrinolytic substance. Certain other saccharolytic species, Cl. tertium, Cl. fallax, Cl. aerofoetidum fail to produce this substance. 2. The more common proteolytic gas gangrene species, Cl. histolyticum, Cl. sporogenes and Cl. tyrosinogenes produce an equally or more rapid destruction of coagulated plasma. 3. Most cultures which are active against plasma from one species will break coagulated human, guinea pig and rabbit plasma. Sheep plasma is less readily attacked and not attacked by any strains of Cl. welchii tested. 4. A few strains in both groups of gas gangrene anaerobes produce anticoagulation factors which prevent CaCl2 coagulation of plasma. It is shown that this is not a pH effect.