Thiamine contents in pea seedlings and some fully grown plant leaves increased in vivo by administering both the pyrimidine derivative and the thiazole or the pyrimidine derivative only.
The enzyme system for the synthesis of thiamine, condensing both the pyrimidine and thiazole moieties in the presence of ATP and Mg²⁺, failed to be found in the homogenates of various plant leaves.

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1. It was observed that on the germination of higher plants, ω-methyl pantethine and bis (N-methyl pantoyl β-aminoethyl) disulfide, the compounds supposed to be the analogues of pantethine level, act similarly to ω-methyl pantothenic acid as a pantothenic acid antimetabolite. Inhibition by ω-methyl pantethine may be almost perfectly prevented by addition of pantothenic acid, but that by bis (N-methyl pantoyl β-aminoethyl) disulfide can be only partially prevented.
2. An experiment was conducted to quantitatively evaluate this inhibition on germination with the following results: In barley, 10 mM ω-methyl pantothenic acid was effective in fully inhibiting the germination and this inhibition was perfectly eliminated by addition of 0.1 mM pantothenic acid, the inhibition index being 100:1. Moreover, this inhibition could only partially be eliminated even by addition of pantoyl lactone in large quantities. However, the inhibition by ω-methyl pantoyl lactone was eliminated by 1/20 of pantoyl lactone. From this it was surmised that even in the process of germination pantothenic acid synthesis takes place with pantoic acid which acts as a precursor to this synthesis.
3. It was revealed that the inhibitory action of pantothenic acid analogues depend on the species of the plants on which they act.
Dicotyledoneous plants were all markedly inhibited by the analogues in general, and monocotyledoneous plants were classified into two groups according to the actions; one was affected and the other resistant to the action of the analogues. For instance, barley (belong to Festucoideae-subfamily) was markedly inhibited by ω-methyl pantothenic acid, whereas rice-plant (Panicoideae-subfamily) was resistant to the action. Thus, it was proved that the difference between two types of Gramineae plants in sensitivity to the effects of antimetabolites depends upon the difference in subfamily.
4. Difference in the effects cf pantothen is acid analogues depending on the species of plants was discussed in comparison with other physiological characteristics on germination. It ws suggested that the difference between species of plants in sensitivity may be concerned with its pattern of respiratory function.

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Using the purified tyrosinase from potato, the oxidation of normal-dihydrothiamine to thiamine was tested with the following results.
1. In pyrocatechol-tyrosinase system about 20% of the dihydrothiamine added was oxidized to thiamine. When either tyrosinase or pyrocatechol was omitted from the reaction mixture, the amount of thiamine formed decreased to less than 1%.
2. The amount of thiamine formed increased in proportion to the amount of the enzyme and of pyrocatechol used and the thiamine formation reached the saturation level in the presence of a definite amount of these components, no further increase being observed.
3. The optimal pH level for thiamine formation was 6 to 7, the activity being decreased rapidly in either acidic or alkaline side.
4. The thiamine formation was highest in pyrocatechol-tyrosinase system than in cresolase system.

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Using crystalline peroxidase 566 purified from wheat germ, the oxidation of normal-dihydrothiamine to thiamine was investigated and the following results were obtained.
1. In the oxidation of dihydrothiamine to thiamine by hydrogen peroxide-peroxidase system, 26 per cent of the dihydrothiamine added was oxidized to thiamine by adding hydrogen peroxide and peroxidase at an adequate time. This reaction was markedly inhibited by hydroxylamine and cyanide.
2. The optimal pH of thiamine formation was found to be 4.0, but the maximum was also found at near pH 7.0.
3. The amounts of thiamine formed were increased in proportion to the enzyme concentration.
4. When pyrocatechol was added to a hydrogen peroxide-peroxidase system, 23 to 24 per cent of dihydrothiamine added was oxidized to thiamine. Peroxidase, hydrogen peroxide and pyrocatechol are all required for this reaction and the amount of vitamin formed was decreased to less than 2 per cent, if any one of these components was omitted. This reaction was also strongly inhibited by hydroxylamine or cyanide.
5. Thiamine formation was highest at pH 6 to 7, in accordance with the optimal pH of Peroxidase using guaiacol as a hydrogen donor.
6. Thiamine formation was highest, when 0.10 μmole each of hydrogen peroxide and pyrocatechol were added to 0.0016 μmole of peroxidase.
7. The amount of thiamine formed was variable when the hydrogen donor was different. It was highest in the case of pyrocatechol and was very low in the case of guaiacol or p-hydroquinone.

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Hydroxocobalamin was found to be as effective as cyanocobalamin on the growth of Ochromonas as well as on Lactobacillus leichmannii. When it is autoclaved with the Ford medium at 120° for 10 minutes, it is decomposed and the activity was reduced to as low as 20 per cent, and it is not reproducible. In order to determine hydroxocobalamin it is necessary to add it aseptically to the sterilized medium or to autoclave with stabilizing agents such as ascorbic acid.

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The bacillus producing thiaminase discovered by Chang should be recognized as a new species and the necessity of the establishment of the taxonomical system of Bacillus thiaminolyticus has been stressed, taking into consideration the various facts appearing in many papers concerning the bacillus.

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Thiamine-protein complex is formed by the reaction of O-benzoyl thiamine disulfide or other symmetrical thiamine disulfides with proteins such as heat-denatured egg albumin. The complex is not separated into the components by means of solvent extraction or zone electrophoresis, but thiamine is liberated through reduction by sodium thiosulfate. This complex formation does not occur when the denatured protein is treated with iodine or monoiodoacetic acid, showing evidently the participation of protein-SH. The complex formation seems to be produced by the interchange reaction between protein-SH and disulfide type thiamine.

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1. Symmetrical disulfides, such as O-benzoylthiamine disulfide (BTDS) or thiamine disulfide (TDS) are reduced to free thiamine when it is allowed to react with heat- or urea-denatured egg or serum albumin under certain conditions, whereby a protein-thiamine complex is partially formed. The complex can be separated by deproteinization with metaphosphoric acid, which can also stop the reducing reaction of protein-SH. The complex thus separated can easily be converted into free thiamine by reduction with thiosulfate, followed by determination by thiochrome method. Applying this method, reactions of BTDS or its related compounds with protein-SH were examined and the following results were obtained.
2. Egg albumin in native state scarcely reacts with the thiamine derivatves. Further, denatured protein, whose SH group had been oxidized by iodine or blocked by P-chloromercuribenzoate, loses its reactivity, scarcely producing the complex.
3. Serum albumin reacts both in native and denatured states, producing a complex.
4. The complex formation with denaturedprotein takes place only with symmetrical disulfides. Asymmetric thiamine derivatives such as thiamine propyl disulfide, produce only free thiamine. It was also suggested that the same complex was formed by a reaction of thiamine or O-benzoylthiamine (OBT) with oxidized protein.
5. In the reaction of BTDS with heat-denatured protein, the optimun pH of the reaction, the effects of the protein amount, the concentration of added BTDS, temperature and time were investigated and the relationship between complex formation and production of free thiamine was clarified. In any case, complex formation was proportional to the amount of free thiamine formation, increasing with the progress of the reaction.
6. The protein-thiamine complex is, therefore, considered to be due to chemical binding of the thiamine derivatives with protein-SH as protein-S-S-thiamine.

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Folic acid absorption studies were made on twenty patients, fourteen normal and six abnormal. The sera were assayed microbiologically using Lactobacillus casei and Streptococcus faecalis as the test organisms.
No significant difference was observed between the results. L. casei may therefore replace S. faecalis as the test organism in assays associated with folic acid absorption studies.

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1. The chicks were given S³⁵-sulfate orally and intramuscularly and the radioactivities of the left tibia, keel and skin were tested. The deposition of S³⁵-sulfate in the tissues of the birds receiving vitamin D₃ was found to be higher than those receiving vitamin D₂ or vitamin D-deficient group.
2. Carrier-free P³²-phosphate solution was supplied orally or intramuscularly, and the radioactivities of the left tibia, keel and skin were estimated. Increased levels of P³²-phosphate were always seen in the groups receiving vitamin D₃.
3. The activity of citrate oxidase increased significantly in the group receiving vitamin D₃, while no influence was observed on the ability of oxidative phosphorylation of citrate in the liver.
4. No influence was observed on the pyruvate oxidative phosphorylating ability and pyruvate oxidase activity in the liver homogenate by the administration of vitamin D₃.
5. The glutathione level in the blood was estimated, and the increase was statistically significant in the group receiving vitamin D₃. But no influence of glutathione in the liver was observed. As for the oxidized glutathione levels, no effects were seen in both blood and liver.
6. From these findings, there seems to be some intimate relationship in chicks between vitamin D₃ metabolism and active sulfate metabolism postulated by Lipmann.

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