Pyridazine-3-carboxylic acid (Pyd-3-CA), which is an analogue of nicotinic acid (NiA), inhibits competitively the growth of Lactobacillus arabinosus 17-5,and the inhibition index exhibits about 2200. On the other hand, it inhibits noncompetitively the growth of Escherichia coli B, which is able to synthesize NiA by itself. The later inhibition is, however, recovered only by NiA or nicotinamide (NAA), and is similar to the action of 6-aminonicotinamide (ANAA) known as the most potent antagonist of NAA. Pyd-3-CA is slightly less potent than ANAA for E. coli B, but it is interesting that, while the inhibition by ANAA is more effectively recovered by NAA than NiA, in the case of Pyd-3-CA the recovery of the inhibition by NiA is rather more than by NAA.

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When Escherichia coli B was grown in the salts-glucose medium in the presence of 5×10^<-5>M of pyridazine-3-carboxylic acid (Pyd-3-CA), it was found that quinolinic acid (QA) was accumulated in the medium. QA was isolated by absorption on charcoal from the medium, by ion exchange column chromatography, and in some cases, by thin layer chromatography, and it was identified by paper chromatography, paper electrophoresis, ultraviolet absorption spectra at various pHs, and infrared absorption spectra. The mechanism of the accumulation of QA was discussed in view of the inhibition of the biosynthesis of nicotinic acid derivatives by Pyd-3-CA in E. coli B.

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It was found that in the determination of thiamine of high lipid samples, such as egg yolk, the recovery of added thiamine is very low as about 40%. Therefore, the authors have attempted to improve the determination procedure of thiamine in egg yolk and it was proved that 98-102% recovery rates were obtained by removing the lipids by benzene extraction. By this improved procedure, the incorporation of thiamine supplemented in diet into egg was studied with thiamine hydrochloride and O, O'-isobutyrylthiamine disulfide administration.

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The utilization of hydrocarbons by microorganisms have recently been studied in rapid progress. In the vitamin formation from hydrocarbons, many investigations have also appeared in respect to vitamin B_<12> and B_6 by Fukui (1965-6), to biotin by Tsuboi (1966) and to vitamin B_2 with Pichia species by Matsubayashi (1966). In our laboratory, several attempts have already started prior to these reports to investigate the formation of riboflavin and its coenzymes by Eremothecium ashbyii using hydrocarbons or polyalcohols as carbon sources. Nitrogen sources seemed to be specially affected, and it was found that the utilization of the carbon sources may be recognized in the medium containing n-hexadecane plus NH_4H_2PO_4,liquid paraffin plus L-glutamic acid or polypeptone. Particularly, riboflavin formed when used n-hexadecane was estimated to be as free riboflavin and FAD in the strains whereas chiefly as free from in the broth. Further tests were repeated to elucidate the addition effect of nucleosides on the biogenesis, and it seemed that there may be affected some possible significance.

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A fat-soluble derivative of riboflavin, riboflavin-2', 3', 4', 5'-tetrabutyrate (B_2-But) was crystallized from methyl alcohol-water solvent system (19 : 5,v/v), and reddish-yellow, rhombic crystals were obtained. Melting point of this recrystallized sample was found at 147-148℃ which is a little higher than that previously reported. Infrared spectrum of this sample shows that 2', 3', 4', 5'-tetrahydroxyl groups of riboflavin disappeared completely and that newly formed ester linkage between alcoholic hydroxyl group and carboxyl group of butyric acid appeared. Spectroscopic properties of B_2-But in several solvents were studied, and the molar extinction coefficients were determined. It was also found that B_2-But dissolves in water at low concentration.

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The thiamine synthesis from hydroxymethylpyrimidine (OMP) analogues in the presence of thiazole was investigated by using the cell suspensions and cell free extracts of baker's yeast. 2-nor-OMP and 2-methylthio-OMP which are replaced the methyl group attached to 2C of the pyrimidine ring were effective while the 5-methyl derivatives of OMP had more effect as already reported. However, the thiamine synthesized from the both compounds could not be identified and the effect of the compounds was not detected in the case of cell free extract experiment. The conversion of the several analogues of OMP to OMP in the presence of methyl or amino-donors was tested using the extracts and suspensions, but no any positive results were obtained.

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The cell suspensions of baker's yeast and a thiamine requiring mutant of E. coli (OMP-less, 70-17) are able to synthesize a thiamine analogue, 2-nor-thiamine from 2-nor-OMP and thiazole. This is determined fluorometrically and bioautographically by paper partition chromatography after treatment of the extract with Takadiastase. The formation of 2-nor-thiamine by yeast was limited and showed substrate inhibition differing from thiamine synthesis from OMP. The OMP-less mutant of E. coli had response for growth in 1×10^<-9>-10×10^<-9>M 2-nor-OMP or 2-nor-thiamine, but the activity was 70% of OMP or thiamine. The growing cells of the mutant in the presence of 2-nor-OMP produced 2-nor-thiamine, probably its esterified form, not thiamine. In addition, they pyroduced a larger amount of 2-nor-thiamine than they can produce thiamine in the presence of same molar OMP. This indicates that the mutants grow by utilizing 2-nor-thiamine formed and can not convert it to thiamine. The concentrates obtained from the extracts of the growing cells supplemented with 2-nor-OMP or OMP were subjected to estimation of the cocarboxylase activity manometrically using partially purified apocarboxylase from baker's yeast. The cell extracts supplemented with 2-nor-OMP showed much lower activity than that of the cell extracts supplemented with OMP, though the former contained much more esterified compound as determined by the thiochrome method than the latter.

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The effect of hydroxymethylpyrimidine analogues on the thiamine biosynthesis from hydroxymethylpyrimidine and thiazole by baker's yeast was examined. Among various pyrimidine compounds including vitamin B_6,2-nor-4-amino-5-hydroxymethylpyimidine (2-nor-OMP), 2-methyl-4-amino-5-methylpyrimidine (5-CH_3-OMP), 2-methyl-4-amino-5-nor-pyrimidine (5-nor-OMP) were inhibitory in this order in the thiamine synthesizing system containing ATP and Mg^<2+> in the extract, while they were less inhibitory in the suspension of the cells. The growth inhibition of these compounds for a thiamine requiring mutant of E. coli (OMP-less strain. 70-17) was not observed. The structural analogy of these compounds were discussed comparing with the toxopyrimidine groups which were inhibitory on pyridoxal kinase of rat brain investigated by K. Makino, et al.

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The author found an enzyme which produces FMN-like substance from riboflavin in a salting out fraction by 70-80% saturation of ammonium sulfate of cell-free extract of dried bakers' yeast. The enzyme requires guanosine-, uridine or inosine-5'-triphosphate, whereas adenosine- or cytidine-5'-triphosphate is ineffective. The nucleotide specificity of the enzyme entirely resembled the novel enzyme which he had found from Rhizopus oryzae.

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The manometric assay methods for lipoic acid by Stokstad, et al. and Gunsalus, et al. were examined and simplified as follows : Time required for preparing lipoic acid-deficient cells of Streptococcus faecalis 10 C1 was remarkably reduced. Namely, after cultivating for 8 hours in AC broth at 37℃, washed cells were directly transferred to the lipoic acid-deficient medium and highly active apo-cells were obtained within 15 hours at 37℃. For the manometric assay, reduced glutathione, adenosine and MgSO_4 were necessary for the maximum activity. Thiamine, riboflavine, glutamate and MnSO_4 could be omitted. By this simplified procedure a sufficient response of O_2 uptake was obtained to 0-10 mμg of DL-lipoic acid per vessel. The lipoic acid contents in acid digests of several natural substances were succesfully determined. Discussion was also made on an unidentified active substance(s), presumably a complex form of lipoic acid contained in yeast extract.

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Bioactivities of DBCC were investigated on 2 kinds of experiments ; replicate culture of glia cell-line, established from newborn rat cerebellum and regeneration of sciatic nerve amputated on adult rabbits. The growth curves of glia cell after treatment with DBCC were found to show a more upward than CN- or OH-cobalamin. Accompanying a process of the regeneration of amputated sciatic nerve in adult rabbits, electromyogram pattern drived from M. gastrocnemius become to indicate the recovered nerve function. With daily intravenous injection of DBCC 125μg/kg, the occurances of these patterns were all accerelated and further the amplitude of each patterns was obviously. higher than that of control. RNA value of motor neurons innervated sciatic nerve in spinal cord treated with DBCC was also increased than CN-or OH-cobalamin. 34 patients of neurological disturbances were selected for the clinical application of DBCC. These patients had subjective and/or objective neurological signs, such as neuralgia, paresthesia, hypesthesia, absence of knee jerk, abnormal electromyogram patterns, etc. Most of the cases received 2 or 3 times per week of 25Oμg of DBCC for 11-90 days. By the treatment, an improvement or a disappearance of neurological signs was obtained in 21 of the cases. A marked therapeutic effect of DBCC was observed in a case of toxic neuritis, in that CN- and OH-cobalamin was found ineffective despite massive doses of total 116 mg.

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Enrichment of polished barley with riboflavin tetrabutylate was studied. Polished barley was mixed with riboflavin-tetrabutylate, then water (50% volume of the polished barley) was added, mixed and heated at 130℃ for an appropriate time. By this treatment, riboflavin tetrabutylate was easily fixed on the surface layer of the barley and very small amount of the vitamin was extracted by washing with water.

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