An aerobic thiamine decomposing bacillus which produces thiaminase I was isolated by the author from human feces. One strain was first found from an adult of Pakistani. Later 12 simlar strains were isolated from patients and healthy subjects living near Taipei city, Taiwan. According to the results of bacteriological, fermentative and immunological investigations about these bacilli, it is obvious that all the strains is belonged to a new species of Genus Bacillus different from hitherto known thiamine decomposing bacilli, Thereafter the same strain was isolated also from feces of a patient in Niigata city. In this paper the experimental results about the morphological and cultural characteristics of this bacillus are mentioned.

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Experimental loading of γ-aminobutyric acid or β-hydroxy-γ-aminobutyric acid ar intravenous injection in rabbits following pretreatment with isonicotinic hydrazide resulted in the production of β-hydroxy-γ-aminobutyric acid in the case of γ-aminobutyric acid and glycine in the case of β-hydroxy-γ-aminobutyric acid. These products however were not obtained when pyridoxine was given concomitantly. A pathway from γ-aminobutyric acid to glycine via the β-hydroxy form was therefore shown to exist but in the normal state, it is believed that a more active metabolic pathway involving vitamin B_6 is present.

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The effects of vitamins B_6,thiamine and riboflavin on the active absorption of amino acid from the intestin were examined. Absorption of alanine was parallel to the vitamin B_6 content of the liver and was reduced in vitamin B_6 deficiency and was restored in the recovering stage. Inhibition of absorption was found in administration of a vitamin B_6 antagonist and it was prevented by administration of pyridoxine. In thiamine and riboflavin deficiency, active absorption of alanine from the intestine increased irrespective of the vitamin B_6 content of the organs. Administration of thiamine antagonist resulted in a decrease in oxygen consumption of the intestine as well as a decrease in absorption and this reduction was prevented only by administration of thiamine.

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A minute quantity of α-hydroxy-γ-aminobutyric acid is detected in the brain of normal rabbit but on experimental loading of γ-aminobutyric acid via the carotid artery an increase in α-hydroxy-γ-aminobutyric acid was observed. It was found that α-hydroxy-γ-aminobutyric acid undergoes transamination and vitamin B_6 is involved in the reaction. The optimal pH for the reaction is about 8 and the reaction is inhibited by carbonyl reagenst. The enzyme is proved to be specific in the brain.

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From the fact that pantothenic acid tends to be mobilized in the brain when it is given from the outside and it is also known to be widely distributed in the organs, it was assumed that if the β-alanine moiety was replaced with γ-aminobutyric acid, transfer to the brain would be greater compared to γ-aminobutyric acid. γ-Aminobutyric acid, α-and β-hydroxy-γ-aminobutyric acid and their pantoyl-bound forms were prepared and comparative studies were conducted on the effect on various types of seizures, inhibition of excitability and prolongation of sleep. It was found that compared to γ-aminobutyric acid or its hydroxyl derivatives, the pantoyl- or carbobenzoxy derivative had a stronger action. Homopantothenic acid especially, had a pronounced and stable action and moreover, it completely suppressed the running fit induced by hydroxymethylpyrimidine.

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In the previous communication (Vitamins 23,364,1961), it was demonstrated that ps-dihydrothiamine (DHB_1) was much more stable than n-DHB_1 in its aqueous solution but the both isomers showed nearly the same stability when they were first dissolved in HCl and then made alkaline. Now ps-DHB_1 was isolated and identified by the same HCl-alkaline treatment of n-DHB_1. The stability and its decomposition products of n- or ps-DHB_1 in various kinds of solutions were revealed by paper partition chromatogaphy (butanol・acetic acid・H_2O 4 : 1 : 5) : The alcoholic solutions indicated one spot of DHB_1 (Rf 0.56) in both isomers, while the aqueous solution of n-DHB_1 showed three spots ; DHB_1,tris-(2-methyl-4-amino-5-pyrimidinyl-methyl)-hexahydro-S-triazine (I), and 5-hydroxy-3-mercapto-2-pentanone (II) but the aqueous solution of ps-DHB_1 indicated two spots ; DHB_1 and (I). The neutralized solution of n- or ps-DHB_1 which was first dissolved in NHCl, showed the presence of thiamine distinctly beside DHB_1,whereas a small portion of DHB_1 converted into thiamine, was always detected as a tailing spot by the alkaline-ferricyanide reagent in all kinds of the solutions. Decomposition of DHB_1 into (I) and (II) in its aqueous solution was prevented by the addition of (I) or both (I) and (II) ; in the latter case DHB_1 was kept as high as 80-100% even after standing for 48 hours.

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The growth of Lactobacillus fermenti 36 and Kloeckera apiculata which both require thiamine for their growth, was checked on the broth aseptically added with dihydrothiamine. It showed 1/100 growth-stimulating activity on Lactobacillus and 1/10 activity on Kloeckera compared to that of thiamine. Both n- and ps-dihydrothiamine indicated nearly the same activity but at the high concentration such as 10^<-2>M (x1/4) especially on Kloeckera, inhibition of growth was markedly observed, while thiamine had no inhibitory effect at higher concentrations. Two spots of Rf 0 (thiamine) and Rf 0.75 (dihydrothiamine) were detected by bioautography of pure sample of dihydrothiamine developed on a paper-strip with isoamyl-alcohol・pyridine・H_2O (5 : 1 : 3) and it was also demonstrated by the two-dimensional bioautography that dihydrothiamine was easily oxidized to thiamine on the paper-strip during the process of development. It was concluded, therfore, that the apparent growth stimulation by dihydrothiamine was based on its conversion into thiamine.

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Mycelium of Aspergillus oryzae (Fugu) cultured for 48 hours under aeration in a medium containing 1% potato starch, 3% corn steep liquor and 0.2% NaNO_3 and its aqueous extract, deproteined extract, charcoal and resin treated extracts were administered to albino rats and their effects on the growth of the animal were examined in order to know in which fraction the growth factor was concentrated. However, not only any fraction, but also the mycelium itself did not exert the growth stimulating effect. Since the mycelium which has been found to be effective in previous experiments was that cultured for 24 hours, it was supposed that formation and accumulation of the effective factor may be dependent on culture conditions. Then, an examination was made on effects of mycelia obtained under such different conditions as surface culture for 48-72 hours or aerating culture for 24 hours and showed that only the mycelium used in previous experiments was effective.

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In our previous papers it has been reported that charactaristic glutamic pyruvic transaminase (GPTM) and glutamic oxalacetic transaminase (GOTM) located at mitochondoria have quite different natures from those in nonparticulate fraction. It was elucidated the significance of their localization in mitochondoria from the view point of the coupling reaction with TCA-cycle in this paper. The data presented in this paper indicate that C^<14>-glutamic acid, when added to mitochondrial preparations, is rapidly converted into C^<14>-aspartic acid attended with C^<14>O_2 release stoichiometricaly. In this system malonate inhibited the formation of C^<14>-aspartate and hydroxylamine at a very low concentration, at which it did not inhibit the oxidation of α-ketoglutaric acid and glutamic dehydrogenase activity, also strongly inhibited the production of C^<14>-aspartic acid. These data indicated that the convertion of glutamate to aspartate in mitochondria was an altanative coupling reaction between GOTM and a half of TCA cycle as Krebs's suggestion. In the same way aspartate and acetate added was converted into glutamate. No detectable amount of C^<14>-glutamate was found from C^<14>-α-ketoglutarate which was supplied from TCA-cycle, when ammonia, C^<14>-acetate and succinate were added to mitochondrial preparations, though much amount of glutamate was detected, when ammonia, acetate, succinate and α-ketoglutarate were added as substrates. These data suggested that glutamic dehydrogenese could not react with α-ketoglutarate formed in TCA-cycle but could react with it added from outside of mitochondria. The following unit reaction of GOTM would be expected to occur between oxalacetate located at TCA cycle and aspartate added from outside of mitochondria. Aspartate + C^<14>-Oxalacetate &rlarr; Oxalacetate + C^<14>-Aspartate. Cold aspartate, when added C^<14>-succinate only to mitochondria was converted into C^<14>-aspartate strongly. The same kind of reaction of GOTM at glutamate and α-ketoglutarate could be recognized.

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