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A mutant strain of Escherichia coli. D-30, requiring the pyrimidine moiety of thiamine (OMP) for both aerobic and anaerobic growth in a minimal medium supplemented with the thiazole moiety of thiamine (Th) was isolated from mutant 26-43, requiring Th. The requirement for OMP could be replaced by methionine and lysine, but not by succinate. The mutant could not grow in the minimal medium supplemented with 1 nM OMP, but the rapid growth was observed by the addition of methionine. The growth in the minimal medium Supplemented with methionine and lysine was strongly inhibited by adenosine. The growth of mutant 26-43 in the minimal medium supplemented with Th was also considerably inhibited by adenosine. This inhibition was restored by the addition of both methionine and lysine. A mutant strain, 6-60, isolated from a Th-requiring mutant (26-43), required OMP and adenosine for growth. The requirement for OMP in mutant 6-60 was substituted by methionine and lysine as found in mutant D-30. The formation of OMP by cell suspension of mutant D-30 was shown to be dependent on the presence of methioninc. This formation by cell suspensions of mutants D-30, 26-43 and 6-60 was shown to be activated by the presence of methionine and lysine. These results suggest that the OMP synthesis in mutant D-30 was not completely blocked. Possible mechanisms for the activation of the OMP biosynthesis by methionine and lysine are discussed.

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A cup-plate technique for the microbioassay of folate using Lactobacillus casei as a test organism was attempted. The folate content of powdered agar added to basal medium affected the clearness and sharpness of the growth zones. There was a linear relation between the diameters of growth zones and logarithms of the concentrations within the range of 10〜400 ng/ml of pure folate solution. In the statistical evaluation of the assay results the maximum errors of. thc estimate were below 10%. The total folate contents in some natural products obtained by cup-plate method were a little high to those obtained with turbidimetric one and the recovery experiments were successful enough. These resuls suggest that the cup-plate method may be available for the microbiological assay of folate in natural products.

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The antiscorbutic activity of 5,6-O-isopropylidene ascorbic acid (IAsA) and 5,6-O-isopropylidene ascorbic acid 2-sulfate (IAsS) in Hartley guinea pig fed a scorbutigenic diet has been investigated. Oral administration (0.1 mmol/animal) of IAsA completely prevented development of scurvy and continued to increase in body weight throughout the feeding experiment in guinea pig. An equimolar dose of IAsS did not prevent scurvy, and total vitamin C levels in tissues of IAsS-administrated animals were similar to those in C-free animals. The vitamin C Levels (μmol/ml or g) of IAsA, ascorbic acid (ASA), IAsS, and vitamin C deficient groups after the feeding test (IAsA, ASA group : 40 days, IAsS, C deficient group: 20 days) were 0.14, 0.11, 0.01, and 0.01 in blood, 4.90, 3.97, 0.07, and 0.05 in liver, 1.89, 1.47, 0.08, and 0.02 in kidney, respectively.

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It has been proposed by several groups of investigators that the pyrimidine moiety of thiamin is synthesized via 5-aminoimidazole ribotide, an intermediate of purine biosynthetic route in bacteria. To decide whether the methyl group attached to C-2 atom of the pyrimidine is introduced after the recyclization of the imidazole ring of 5-amino-imidazole ribotide (AIR) to the pyrimidine, the competition of radioactive formate incorporation into the pyrimidine moiety by 4-amino-5-hydroxy-methylpyrimidine was investigated. The results showed that this compound was not used as a biosynthetic intermediate by Escherichia coli and Saccharomyces cerevisiae. It remains unsolved, at which step methyl group is introduced and which compound is methyl origin of the pyrimidine moiety of thiamin.

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