1. An unidentified orange-yellow colored compound was obtained from the cells of Pr. shermanii harvested from the medium containing cobinamide exogenously added. The compound exhibited the coenzyme activity in the propylene glycol-propionaldehyde conversion system of Abeles and Lee. Its K_m value was 6×10^-6M, 20 times as great as that of 5, 6-dimethylbenz imidazolyl cobamide coenzyme (DBCC).
2. The compound moved to the cathode 1.5 times faster than cobinamide cyanide, hydroxo- and cyanocobalamins, and DBCC at acidic pH levels in paper ionophoresis. After treatment with cyanide solution, the product showed the same mobility as cobinamide cyanide.
3. The shape of its absorption spectrum in a neutral solution was analogous to that of DBCC except the difference in the wave length of the absorption maximum at 450mμ.
The spectrum was replaced by a spectrum with double peaks at 350 and 500 to 550mμ by the exposure to light and changed to the same spectrum as that of cobinamide dicyanide after cyanide treatment.
4. The compound was converted to the violet dicyano form at acidic pH levels as well as under alkaline condition. This conversion is known to be specific for cobinamide analogues.
5. The microbiological activity of the compound was poor against E. coli No. 215 which is known to show a weak response to cobinamide and its derivatives.
6. From these results it would be concluded that the orange-yellow compound is the coenzyme form of cobinamide which has the structure as shown in Fig. 8. The authors tentatively designated it “cobinamide coenzyme”.

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1. Little has been known about the native forms of vitamin B₁₂-active substances and the natural occurence of Barker's cobamide coenzyme in the cells of methane bacteria, since most of non-cyano type vitamin B₁₂ were transformed to the cyano type by the extraction procedure using cyanide solution previously employed.
The authors employed an extraction method using hot ethanol instead of the cyanide treatment in order to avoid the transformation of cyanide-labile vitamin B₁₂ analogues.
2. A purified extract was obtained by the usual phenol treatment of the alcohol extract. By paper ionophresis performed in 0.5N acetic acid, five zones active to E. coli No. 215 were observed: three of them (tentatively named P1-P3 in the order of the mobilities) moved toward the cathode, one (P4) stayed near the start and the fifth (P5) migrated toward the anode. On the other hand, disappearance of P1 and P2 and concomitant increase in the area of P4 were observed in the extract with cyanide.
In the bioautogram using Ochromonas malhamensis which is known to have more specific responce to 5, 6-dimethylbenzimidazolyl cobamide than E. coli, the growth zones corresponding to P2 and P3 were not detected.
3. The partial purificate was fractionated by DEAE-cellulese column chromatography. A fraction eluted with water contained the vitamin B₁₂-active substance corresponding to the zone P1. The fraction exhibited the coenzyme activity in Abeles-Lee's propanediol-propionaldehyde conversion.
4. From these results it seems appropriate to conclude as follows: P1 corresponds to DBCC; P2, non-cyano type B₁₂ analogue lacking 5, 6-dimethylbenzimidazolyl cobamide structure; P3, pseudocobalamin; P4, cyanocobalamin and P5, an unidentified B₁₂ analogue with anionic character, the paper ionophoretic behavior of which is analogous to that of vitamin B₁₂ coenzyme M of Takeyama and Buchanan.

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1. The presence of a thiamine-sythesizing pathway from the pyrimidine and thiazole moieties in bakers' yeast was demonstrated by the washed cells and cell extracts by sonication of E. coli ATCC 9637, and the strains 70-17 and 26-43, the latter two of which were capable of growing in the presence of the pyrimidine (OMP_m) or the thiazole moiety (Th) alone.
2. The thiamine-synthesizing activity was higher in the mutants than the parent strain.
3. Only the strain 70-23 strictly requires thiamine for growth, failed to synthesize thiamine from OMP_m and Th, and no detectable OMP_m-kinase activity was found in the cell extract. It is therefore assumed that the thiamine requirement of this mutant is due to the decrease or lack of OMP_m-kinase activity.

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The formation of DBCC and unidentified vitamin B₁₂ analogues from exogenously added cyanocobalamin was demonstrated during thermophilic methane fermentation. Twenty-four hours after addition of cyanocobalamin the incubation mixture was centrifuged. The precipitate consisting of bacterial cells and sludges was extracted with hot ethanol. Vitamin B₁₂-active substances in the cultural filtrate were adsorbed on active charcoal and eluted with 80% ethanol. Each of the ethanol solutions obtained from the precipitate and from the filtrate was concentrated in vacuo, purified by usual phenol treatment and passed through DEAE-cellulose colume. Elution was carried out successively with water, 0.1N acetate buffer (pH 4.5) and 1M NaCl-acetone mixture as a developing solvent.
By column chromatography of the partial purificate obtained from the precipitate an orange-red effluent fraction was obtained (DC-1), which exhibited the same behaviors as DBCC in microbiological test, absorption spectroscopy and paper electrophoresis.
It also showed the coenzyme activity in Abeles-Lee's intramolecular oxidationreduction system. From these findings the identity of the orange-red substance with DBCC was concluded and the yield was calculated as ca. 20% of the cyanocobalamin added. Besides DBCC, the presence of an unidentified vitamin B₁₂-active substance was found in the fractions DC-2 (eluted with acetate buffer) and DC-3 (eluted with NaCl-acetone mixture).
Vitamin B₁₂-active substances in the cultural filtrate were fractionated in DS-1 (eluted with water), DS-2 (eluted with acetate buffer) and DS-3 (eluted with NaCl-acetone mixture).
In DS-1, the presence of a small amount of DBCC together with hydroxocobalamin, cyanocobalamin and an unidentified yellow-orange substance was detected.
In fraction DS-2, another yellow to yellow-orange substance was contained which moved slightly to the cathode. Though the lability in light was analogous to that of cobamide coenzyme (6), it did not show the coenzyme activity in Abeles-Lee's system. The significance of DBCC contained in methane bacteria was discussed.

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1 Ascorbic acid continued to arrest the oxidation process of tyrosine-tyrosinase system until it undergoes oxidation.
2. The efficiency as expressed in molar concentrations for inhibiting the formation of melanin was, in a decreasing order, as follows: glutathione, ascorbic acid, thiamine, lipoic acid. The lowest molar concentration of ascorbic acid needed for inhibition was 0.1×10^-6M.
3. The skin ascorbic acid level is 1 in mormal persons and 5mg per 100g in rabbits. After intramuscular injection of 0.5g ascorbic acid the level was raised to 30 to 60mg per 100g in rabbits in 1 to 2 hours, consisting mostly of ascorbic acid rather than dehydroascorbic acid.
4. The highest level of blood ascorbic acid after repeated oral administration, 1g three times daily, was 2.6mg per 100ml.
5. Urinary exoretion of ascorbic acid was 10 to 20 per cent of the amounts administered. When 2g ascorbic acid was administered orally for 30 consecutive days, urinacy ascorbic acid in 24 hours on the 7th, 21st and 30th days did not show a marked increase.

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1. Degradation of Ψ-dihydrothiamine at 35° in aqueous solution was successively investigated. It was shown to be stable in alkaline solution, but it rapidly degraded in acid solution, being partially converted to thiamine.
2. Addition of hydrogen peroxide to either n- or Ψ-dihydrothiamine in acid solution resulted in 2-methyl-4-amino-5-aminomethylpyrimidine sulfate formation.
3. Dihydrothiamine was partially converted to thiamine by addition of cyanide in a weakly acid solution. Thiocyanate showed no effect. For the reaction a large amount of cyanide as compared with dihydrothiamine was required and the dissolved oxygen accelerated the reaction, whereas hydrogen or nitrogen inhibited the reaction. These findings suggest that the reaction is a kind of air oxidation catalyzed by cyanide.

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1. Distribution of S³⁵ in the organs of rats after oral and intravenous administration of thiamine propyl disulfide (TPD)-S³⁵ were compared with that of thiamine-S³⁵. The distribution of the radioactivity was much greater after administering TPD-S³⁵ than thiamine-S³⁵.
2. The percentage of S³⁵ found in the organs except for the stomach and small intestine 30 and 60 minutes after oral administration of TPD-S³⁵ were 35 and 36%, while those following oral administration of thiamine S³⁵ were 21.9 and 19.8 per cent.
3. Following intravenous injection of TPD-S³⁵, the S³⁵ found in the organs except for the stomach and small intestine were 52.7 and 68 per cent, while those following intravenous injection of thiamine-S³⁵ were 54 and 21 per cent.
4. There were differences in the amounts of S³⁵ found in various organs between the rats fed on a balanced diet and those fed on a thiamine-deficient diet after oral administration of TPD-S³⁵. The organs of the former had no radioactivity 7 days after administration, while the S³⁵ in the skin, muscles, testes and brain of the latter showed about the same levels 7 days after administration as those after 24 hours.

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1. When thiamine propyl disulfide (TPD) is added to erythrocyte suspension, much of it penetrates into the cells and thiamine level remain unchanged for 24 hours. Thiothiamine penetrates also into the cells under the same condition but not complete, the ratio of thiothiamine in the cells to that in plasma being roughly 1:1.
2. When the erythrocytes after above treatment were washed with a saline solution, thiothiamine was washed out but TPD could hardly be washed out of the cells.
3. Thiothiamine exists per se in erythrocytes but TPD does not. It is present as thiamine after reduction in the cells and it cannot be washed out with a saline solution.

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1. Thiamine propyl disulfide (TPD) was more effective than thiamine, and thiamine disulfide (TDS) was of the same activity as thiamine on the growth of L. ferementi in the Macias R medium containing cysteine, but in the medium without cysteine, both TPD and TDS were scarcely effective. In the medium containing sodium thiosulfate in place of cysteine, the same effects were observed.
2. TPD or TDS was easily converted to thiamine by preincubation with the cysteine contained in the Macias R medium. When the medium was inoculated with L. fermenti, it was showed that TPD was more effective than thiamine, but TDS was almost equally effective to thiamine. Cysteine in the medium can be replaced by thiosulfate.
3. The solution in which TPD or TDS had been previously reduced by cysteine to thiamine was added to the medium for L. fermenti and the growth-stimulating effects of TPD or TDS were found to be almost the same as thiamine.
4. It was confirmed using TPD-S³⁵ (outer) that the propylmercaptan moiety produced from TPD during thiamine formation by cysteine became S-propylmercapto-L-cysteine and that this compound had no effect on the growth of L. fermenti.
5. Taking these findings into consideration, it was discussed on the reason why TPD was more effective than thiamine alone in the cysteine (+) Macias R medium on the growth of L. fermenti.

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From the experiment presented in this paper it is evident that most strains of Cl. sporogenes and the organisms related closely to Cl. sporogenes produce extracellular thiaminase I. It will be worthy of note that the magnitude of the activity of the thiaminase of these organisms, the optimum temperature and pH were closely resembled to those of the enzyme of Cl. thiaminolyticum.

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