Diethyl-α-benzyl-α-(3-(3-methylphthalidyl))-malonate and diethyl-α-benzoyl-α-(3-(3-methylphthalidyl))-succinate as intermediates of aureomycinic acid analogues, and 2, 10-dibromo-l. 4-dioxo-1. 4. 5. 8. 9. 10-hexahydronaphthalene as intermediate of tetracyclin A. B ring analogue have been prepared.

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The effect of riboflavin on hypertenstion induced by high salt diets in rats was studied and the following results were obtained.
1. Riboflavin was found effective to inhibit the elevation of blood pressure in rats fed on high salt diets.
2. Riboflavin slightly increased the activity of liver monoamine oxidase.
3. Riboflavin reduced serum cholesterol levels, significantly in rats fed on high salt diets.

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(1) In order to investigate the fitness of water-dispersible nitrogen of heat-treated soy-bean flour as a criterion of the degree of denaturation, a method for determining the amount of native protein in aqueous solution which was based on the increase of suscepti-bility to protease action of Asp. sojae prior and after heat treatment was established.
(2) In the autoclaving of flour, there existed a minimum of water-dispersed nitrogen with regard to the heating time or the water con-tent of flour. The higher the temperature of treatment or the water content of flour, the larger was the increase of dispersed nitrogen beyond the minimum.
(3) The higher the temperature or the water content of flour, the easier denaturation oc-curred. The native protein in the water ex-tract of heated flour disappeared at the minimum point of water dispersibility.
(4) The water dispersibility method served as a criterion of denaturation for the flour heated within the range where a minimum point of water dispersibility was attained. Within this range amounts of water-dispersed nitrogen ran paralell to the amount of the native protein content. However, this water dispersibility method is not suitable for com-parison of the degree of denaturation of flours treated under different conditions.

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(1) The majority of soybean proteins were hardly hydrolyzed in native state by the pro-tease of Asp. sojae, but became susceptible to the enzyme upon heating in a solubilized state. From this fact the method for deter-mining the native protein in the soybean flour was investigated.
(2) The insolubilization of the protein, which occurred by heating under low water contents, was not attributed to its denaturation. The native protein in these flour was not extracted by water or buffer solution, but could be solubilized after the protease action. There-fore the measurement of the native protein in the water extract by the method previously described was inadequate for the purpose of measuring a degree of denaturation of heat treated flours.
(3) A new method was described, which was quantitative and applicable to the flours heated under various conditions. In this method the native protein in the flours was determined by applying the turbidimetric method to proteins which were extracted but unhydrolyzed after a twenty-hour digestion (30°C, pH 7.2) of flours by the protease of Asp. sojae with an activity of 65 [PU]^{cas. 30° FR} _{m. eq. tyr.} at pH 7.2.

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The extracellular proteinase and peptidase of Pediococcus soyae¹⁾ is not found in its growing culture solution and culture filtrate. In the cell extract, weak activities of proteinase and pe-ptidase are observed.
Weak transaminase and reductive amination activities of producing glutamic acid from α-keto-glutaric acid are estimated in the resting cell suspension and also in the crude cell extract. The potent amino group doners are; alanine, leucine, lysine, aspartic acid and ammonium chloride.
Starch fermentation by Ped. soyae strain d 6 is confirmed.
The numbers of lactic acid bacteria in soy koji are about 0.1-3 billion per gram and are much numerous than aerobic spore forming bacteria. The majority of them are salt sensitive Streptococci and Lactobacilli, but fewer salt tolerant Pediococcus soyae also exist. In the soy mash, salt sensitive lactic acid bacteria decrease rapidly within a period of two months. On the contrary, Ped. soyae grows well in the 18%, salted mash, starting at 10²-10⁸/ml, reaching to 10⁸-I0⁹/ml at the 4th month of brewing. Thereafter the viable counts of Ped. soyae gradually decreased and settled down to about 10⁴/ml after 9 months of brewing. The pH value in soy mash was lowered while Ped. soyae was 10⁶/ml or more.

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The amino acid composition of hydrolysates of crystalline Japanese-radish peroxidase a has been determined mainly by chromatography on columns of Dowex 50-X8. The recoveries of the residues and the nitrogen were 93.52 and 100.90 per cent respectively, both including those of the carbohydrate residues and protohematin. The analyses indicate the presence of the following 384 amino acid residues, 65 carbohydrate residues and I protohematin in the molecule of peroxidase a (mol. wt. 54500): Asp₅₁Glu₂₆Gly₆Ala₆₄Val₂₀Leu₃₆Ileu₁₅Ser₅₁Thr₂₇(Cys-)₁₀Met₄Pro₁₇-Hypro₁₂Phe₁₈Tyr₃Try₂ His₄Lys₇Arg11(-CONH₂)₅₀Mannose₃₅Xylose₁₁Arabinose₄Hexosamine₁₅Protohe-matin₁.

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An amperometric modification of the potentiometric determination of starch-iodine affinity by Bates et al. has been developed. The procedure is essentially the same as that of the poten-tiometric method, but free iodine is determined amperometrically by using a rotating platinum electrode as an indicating electrode without the application of an external e. m. f. This method is of special advantage for routine work, since a sharp and exact end point is obtained. The amperometric titration curve can also easily be converted into the iodine affinity curve, which has been recommended as an exact method to evaluate the iodine-binding capacity of starchy materials. Using this technique, the iodine affinities of some starchy materials and their change during β-amylolysis are determined, successfully.

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1. Polarography of each fraction isolated from sarkomycin by the countercurrent distri-bution method revealed the existence of two components, showing polarographic waves. One of them shows biological activity, while the other shows no biological activity.
2. The pure sarkomycin obtained by the countercurrent distribution method, showed two reduction waves in the acid region and one wave in neutral and alkaline regions, resulting in the approach of both waves.
3. Analysis of the slope of the reducing waves of sarkomycin gives values of 0.063 v. (for the first wave) and 0.076 v. (for the second).
4. Crystals Z and D, which are considered as degradation or polymerization products, were polarographed and showed one polaro-graphic wave in the acid region.
5. 3-Methyl-2-oxocyclopentane-l-carboxylic acid was polarographed and showed one wave below pH 4.0.
6. The correlation between reducing cur-rents and concentrations of sarkomycin solu-tion is linear, restrictive to the same sample. However, polarographic method can not be applied for determination, because the decrease rate of biological activity is not proportional to that of the reducing current.

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The quaternary system KCl-MgC1₂-Mg(OH)₂-H₂O and the associated systems, (a) KCI-Mg(OH)₂-H₂O and (b) MgC1₂-Mg(OH)₂-H₂0 were investigated at 100° Though the isotherm (a) exhibited nothing new, isotherm (b) exhibited a new basic magnesium chloride, MgC1₂•2Mg(OH)₂•6H₂O, as a solid phase in such a solution having a higher concentration of magnesium chloride than 51.5 gram per 100 gram water. The solid phases of quaternary isotherm were the new basic magnesium chloride, bischofite, carnallite, potassium chloride and magnesium hydroxide.

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Factors affecting the proteinase production in cell suspensions of Ps. myxogenes sp. were studied and the following facts are shown:
1) The production was observed in cell suspensions in a phosphate buffer of pH 7.0, containing a carbon source and calcium ion under aerobic conditions, without accompany-ing growth. The calcium ion could be replaced with strontium but not by others, and inhibi-tion was observed in the presence of calcium-removing agents such as EDTA, Oxalate, Citrate and NaF. The carbon sources which were to be utilized by the cells were all found to be effective, and production was inhibited by various metabolic inhibitors.
2) The optimum pH was about 7.0, and the promoting effect of phosphate was observed remarkably at pH 6 and slightly at 7 but not at 8.
3) The enzyme producing abilities by nutritionally differentiated types of cells pre-pared from synthetic media (of which the components were differentiated) in various concentrations of glucose (1-7%), in the presence or absence of CaCO₃ or excess addition of a growth promoting agent, were almost identical with one other. Furthermore ability was not altered throughout the stationary phase when the pH of the cultural medium was kept at neutrality.
4) The production was more inhibited along with the increasing of the concentrations of carbon sources such as organic acids and glucose or ethanol in which were observed high acid formation, but not in fructose or glycerol in which low acid formation was seen. The inhibition, however, was recovered to some extent by the further addition of calcium. In other words, it could be said that the optimum concentration of calcium was differentiated by the sorts of carbon sources or by their concentrations.
5) The balance between the concentrations of carbon source and cells was important for the production, and when kept the balance of both factors suitably the enzymatic activity produced was parallel to the concentrations of cells.
6) The addition of a nitrogen source in the reaction mixture promoted production about three times. However, in the presence of amino acid analogues, which did not affect the production in the reaction mixture with-out a nitrogen source, the promoting effect of the nitrogen source was diminished except when their homologues were used.
7) The production was inhibited remarkably by the addition of various substances contain-ing sulfur, while 8-Azaguanine, base analogue, did not affect the production.

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It was ascertained by the tracer method that calcium is a constituent of the proteinase of Ps. myxogenes sp. However, the activity is not altered by the treatment of EDTA until the temperature reaches to 50°C. This might suggest that calcium is firmly combined to the enzyme protein. From these results, the indispensable role of calcium in proteinase production has been discussed. The author wishes to express his sincere thanks to Prof. H. Katagiri of Kyoto Univ. and also to Mr. E. Masuo of this Laboratory, for their constant guidance and encouragement in the course of this work.

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