Chemical Studies on Components of Dried Bonito, “Katsuobushi”

Volatile components obtained by the extraction of “Katsuobushi” with 80% ethanol and by the subsequent steam distillation of the extract were fractionated by the usual methods, and the resulting hydrocarbon fraction was investigated. Gas chromatographic study on this fraction originated from “Katsuobushi” of bonito (Katsuwonus pelamis) revealed 9 hydrocarbons, including n-tetradecane, n-pentadecane, n-hexadecane, n-heptadecane, n-octadecane, n-nonadecane, n-eicosane, n-heneicosane and n-docosane, which were tentatively identified by the retention times with the aid of authentic hydrocarbons. n-Pentadecane and n-heptadecane that were main components among these hydrocarbons were identified further by NMR and IR spectrometry. “Katsuobushi” of frigate mackerel (Auxis thazard), mackerel (Scomber Japonicus Houttuyn) or muroaji (Decapterus muroadsi) also contained n-penta-decane and n-heptadecane in large amounts, but did other hydrocarbons in negligible amounts.

Possible mechanisms of the hydrocarbon formation during the processing of “Katsuobushi” were discussed.     

Chemical Studies on Components of Dried Bonito, “Katsuobushi”

The flavor concentrate obtained by the extraction of “Katsuobushi” of bonito (Katsuwonus pelamis) with 80% ethanol and by the subsequent steam distillation of the extract was fractionated by the usual method and the resulting neutral, non-carbonyl oxygenated fraction was investigated by gas chromatography. The following components were tentatively identified: 2-pentanol and 2-methyl-1-heptanol as free alcohols, and 4 alcohols of n- and isobutanol, n-pentanol and n-dodecanol and 9 carboxylic acids of propanoic, n-butanoic, n-pentanoic, n-octanoic, n-nonanoic n-decanoic, n-dodecanoic, n-tetradecanoic and n-hexadecanoic acid as the constituents of esters. A constituent alcohol existing in the largest amount was isolated by gas chromatography and identified as 2-methyl-1-heptanol by elemental analysis, NMR, IR, and MS. A constituent acid existing in large amount was also isolated and investigated similarly, and the structure was partially estimated. 2-Methyl-1-heptanol holds a fresh woody aroma and seems to have a major effect on “Katsuobushi” flavor.     

Studies on the Fluorescence of Saké

By spectrofluorophotometric investigation on various kinds of Saké it was found that they have at least two kinds of fluorescent colors, the one is blue, the other yellowish green. The former is always more dominant than the latter, but is unstable although the intensities of both color decrease remarkably by treatment of active charcoal. Ferulic acid and harman as the blue fluorescent components are isolated, the former from Saké in young, the latter from Saké kept for a long time under direct sun light.     

Studies on the Flavors in Saké

A very small amount of vanillin was found in Saké, but the mechanism of its formation during Saké brewing has not yet been elucidated. Therefore, shaking culture of a Saké yeast (Kyokai No. 7 strain) was carried out in the Hayduck’s solution containing ferulic acid which was considered to be a precursor of vanillin. By the analysis of the fermentation products, formation of p-hydroxybenzoic acid and vanillic acid was elucidated. On the other hand, in the similar experiment using vanillin in place of ferulic acid, p-hydroxybenzoic acid, p-hydroxybenzaldehyde and vanillic acid were identified.

On these results, it was suggested that vanillin might be formed as an intermediate of the degradation reaction of ferulic acid, and also, the demethoxylation of vanillin might be occurred in the fermentation of yeast.     

Chemical Studies on Amino—Carbonyl Reaction

When N-n-butyl-D-xylosylamine was heated with acetic acid in methanol at 55~70°G, it decomposed to N-n-butyIpyrrole-2-aldehyde,** through 3-deoxy-d-pentosulose as an intermediate. d-Xylose and methylamine in neutralized aqueous solution at 65~100°C also formed N-methylpyrrole-2-aldehyde. N-n-Butyl-l-rhamnosylamine, in a mixture of methanol and acetic acid, formed the corresponding pyrrolealdehyde, l-n-butyl-5-methylpyrrole-2- aldehyde, at the almost same rate as did N-xyloside. On the contrary, N-n-butyl-d-glucosylamine, under the same condition, did not form any detectable amount of the corresponding pyrrolealdehyde, but formed complicated products. A formation mechanism of the pyrrolealdehydes from 3-deoxyosulose and amine was proposed.     

Chemical Studies on Amino––Carbonyl Reaction

The 3-deoxy-n-pentosone (I) was isolated from the browning degradation mixture of N-n-xy1osy1-n-butylamine by the action of acetic acid at 55°C. The 3-deoxy-d-erythrohexosone (IIa) and the 3-deoxy-n-threohexosone (IIb) were also prepared by degradation of the corresponding N-glycosyl-n-butylamine. The 3-deoxy-d-pentosone was characterized as the 2,4-dinitrophenylosazone and its diacetate, and the p-nitrophenylosazone. The two 3-deoxy-d-hexosones were also characterized as the analogous derivatives. The three 3-deoxyosones gave positive color reactions with 2-thiobarbituric acid.

As one of the intermediates in 3-deoxyosone formation from N-glycoside, 1,2-eno1 form of 1-deoxy-1-n-butylamino-2-ketose (IV) was proposed.     

Chemical Studies on “Clover Sickness”

Two new isoflavonoids were isolated from red clover as germination inhibitors for the same plant and their structures were determined as a glucoside of biochanin A (7-d-β-glucosyl-5,7-dihydroxy-4′-methoxyisoflavone) (II) and its 5-malonate (I), respectively. Besides these compounds the following substances were also isolated as inhibitors: trifolirhizin (III), ononin (IV), daidzein (V) and its 7-glucoside (VI), formononetin (VII), genistein (VIII) and biochanin A (IX).     

Studies on the thermostability of the α-amylase of Bacillus caldovelox

Summary Molecular mechanisms of thermoinactivation of the thermostable -amylase of Bacillus caldovelox were examined. Monomolecular conformational processes were found to be the major causes of thermoinactivation at both pH 4.5 and 8.0. The enzyme possessed considerable additional thermostability at pH 8.0, with half-lives of 0.75 and 7.0 min at 90° C and pH 4.5 and 8.0, respectively. The amino acid composition was examined with respect to the underlying thermostability exhibited by this enzyme. The inherent thermostability exhibited may be due to the high proline content (4.47 mol%), but more likely due to the high content of residues forming hydrophobic bonds (60.89 mol%) allied to a low content of residues responsible for ionic interactions (28.34 mol%). Offprint requests to: C. T. Kelly     

Studies on the oxidation–reduction systems of the erythrocyte

1. Starvation for 3 days produces a decrease in methaemoglobin-reductase and glutathione-reductase activities, but it does not alter the glucose 6-phosphate-dehydrogenase activity of the rat erythrocyte. 2. The feeding of a protein-free diet for 11 days causes greater changes in the first two enzymes and also a diminution of the third. Under this experimental condition slight decreases in protein and haemoglobin contents were noted. 3. The experimental animals did not show methaemoglobinaemia, probably because the activity of methaemoglobin diaphorase is preserved. 4. The GSH content was not affected but the stability of the tripeptide in the presence of an oxidizing agent was diminished.     

Studies on Growth Inhibition of Hiochi-bacteria,Specific Saprophytes of Saké

The authors have succeeded in isolating an antibiotic, from the cultured filtrate of Asp. oryzae, responsible for, at least, the majority of the active substances against hiochi-bacteria, as pale yellow crystals of m.p. 152~153°C. It has been proved that this antibiotic is identical with hydroxyaspergillic acid. The minimum concentration for complete inhibition in diluted Saké-peptone medium against true hiochi-bacilli, Lactobacillus homohiochii H-42 and Lactobacillus heterohiochii H-l, was 10 γ/ml, and that against hiochi-lactobacilli H-7 and H-34 20 γ/ml.     

Studies on the Clausena dunniana Lévl Rutaceae,A New Resource of Isoanethole

The plant Clausena dunniana Levl. With leaves of anethole fragrance is growing in limestone area of north Guangdong, The essential oils in leaves of anethole fragrance were extracted by steam distillation with a yield of 0.7%. The oils contained isoanethole (methyl chavcol) 93.10% and may be used in the synthesis of anethole and for medicinal purpose. Isoanethole and other 15 chemical constituents were identified by GC,GC/MS/DS, IR and NMR.     

Studies on the cyanophytes of Cuba 4–6

The article comprises three subsequently published studies of the taxonomy of Cuban cyanophyte/cyanobacterial flora: (4)Lyngbyopsis willei: This oscillatorialean genus and species, described byGardner (1927) from mountain creeks in Puerto Rico, was found more than 50 years later in similar localities in Cuba. The morphological variability of the Cuban populations is described and similarity with the genusSchizothrix (sect.Inactis) discussed. —(5)Cylindrospermum-species: The morphological variability of twenty-oneCylindrospermum-populations collected in Cuba was studied, documented by graphical methods and compared with the published data. Four new taxa were recognized (C. minutissimum v.rinoi, C. zonatum, C. bourrellyi, andC. desikacharyi). The other populations belong to the variation ranges ofC. breve, C. minutissimum, C. michailovskoense, andC. muscicola v.kashmiriense.— (6)Gomphosphaerioideae-species: Fifty eight populations (9 species) of the subfamilyGomphosphaerioideae (Microcystaceae, Chroococcales) from freshwater biotopes of Cuba were evaluated: Four planktic species with a probable cosmopolitan distribution were found (Coelosphaerium kuetzinginianum, C. minutissimum, Snowella lacustris andCoelomoron pusillus), and from the genusCoelomoron Buell two new species,C. microcystoides andC. vestitus were described. The tropical planktic speciesWoronichinia fremyi forms occasionally water blooms in larger reservoirs. Two tropical species from the genusGomphosphaeria Kütz, were recognized,G. multiplex (Nyg.) c. n. andG. semen-vitis sp. n.     

Studies on the Cell Wall of Piricularia oryzae†

The chemical constituents of the cell wall of Piricularia oryzae, the pathogenic fungus of rice blast disease, were studied with the aids of chemical analysis, X-ray diffraction, infra-red absorption and enzymatic degradation. The sugar constituents were identified by chromatography as glucose (62%), mannose (4%), galactose (0.5%), and hexosamine (13%). The acidic amino acid rich protein was comprised 4.6% in the cell wall. The cell wall consists of at least three different polysaccharide complexes: a) α-Heteropolysaccharide protein complex containing mannose, glucose and galactose, b) β-1,3-Glucan containing β-1, 6-linked branch, c) Chitin like substance.     

Studies on the Decomposition of Raffinose by α-Galactosidase of Actinomycetes

α-Galactosidase was isolated from the culture broth of Streptomyces olivaceus and was partially purified by chromatography on a DEAE-sephadex column. The optimum pH of the preparation was found to be 5.2 for raffinose and the preparation was inactivated completely by maintaining it at 60°C for 15 minutes. p-Chloromercuribenzoate, HgCl₂ and AgNO₃ caused complete inhibition of the enzyme activity at 2 × 10^?5 M concentration. The preparation showed transglycosylase activity. A sugar spot, chromatographically identical with that of stachyose, appeared in the digest of raffinose. However, the preparation hydrolyzed raffinose completely into galactose and sucrose after a prolonged incubation.

A simple raffinose estimation method was developed using the enzyme preparation, and it was found that the method allowed to estimate 125~500 μg of raffinose with an accuracy of ±5%. The method was applied to the estimation of raffinose in beet molasses.     

Chemical Studies on Smelling Compounds in Hen’s Egg

Volatile smelling compounds of freezed egg white, freshness of which was kept by freezed storage, were collected by steam-distillation. After DNP-hydrazones of volatile carbonyl compounds were separated into four classes by column chromatography, DNP-hydrazones contained in each class were separated by thin layer chromatography. Rf and melting point of recrystalized compounds were compared with those of authentic compounds. Volatile basic compounds were collected as hydrochlorides and detected by paper and thin layer chromatography.

Acetone, acetaldehyde, formaldehyde, 2-pentanone, 2-butanone diacetyl except two unknown compounds as volatile carbonyl compounds, and ammonia, methylamine, dime-thylamine and putrescine as volatile basic compounds were tentatively identified.

Correlations between these compounds and smell of freezed egg white were discussed.     

Studies on the Chemical Synthesis of Potential Antimetabolites. 32.1 Synthesis of β-D-Pentofuranosyldeazaadenines as Candidate Inhibitors for S-Adenosylhomocysteinases and Methyltransferases

Abstract

9-β-D-Arabinofuranosyldeazaadenines [1-deaza-araA (4a) and 3-deaza-araA (4b)] were prepared from 6-chloro-β-D-ribofuranosyl-1- (6a) and -3-deazapurine (6b), respectively. Synthesis of 2′-deoxy-1-deaza-adenosine (5a) from 1-deazaadenosine (6c) is also described.     

Studies of the effects of aminopterin on the crypts of Lieberkühn in rats

Summary Studies have been made on the effects of an intramuscular injection of aminopterin on the crypts of Lieberkühn in rats. A decrease in the mitotic counts was accompanied by a rapid increase in the number of abnormal cells present in the epithelium of the crypt. Three hours after administration of the aminopterin, an almost complete absence of true metaphase chromosoms was found. By 24 hours, a partial return towards normal mitotic activity was observed but the number of abnormal cells present was still very high. It is suggested that the mitotic changes are in keeping with the conclusion of Grampa and Dustin (1952) of an arrest at interphase but that a secondary arrest at metaphase cannot be excluded.A morphological feature of some of the abnormal cells was the presence of a Feulgen positive granule in the cytoplasm, which by electron microscopy was also shown to contain many cytoplasmic constituents. It is suggested that material is lost from the nucleus and incorporated into a granule in the cytoplasm. A possible explanation of the purpose and function of the granule, as a means of disposing of unwanted or aberrant material, is put forward.Acknowledgements. I am grateful to Professor R. J. Brocklehurst for his continued interest and support of this work, and to the Stage II, B. Sc. students (1964) who counted the cells in many of the specimens as a laboratory exercise. My thanks are also due to Mr. J. Clements for technical assistance.     

Effect of “Chemical” Hypoxia on the Potassium Conductance of the Membrane of Pheochromocytoma Cells

We studied the effect of “chemical” (induced by the action of sodium thiosulfate, STS) hypoxia on the potassium conductance of the membrane of pheochromocytoma cells. Application of 1 to 10 mM STS decreased in a dose-dependent manner the amplitude of integral potassium current without changes in the voltage dependence of its activation. The concentration dependence of the action of STS on the amplitude of potassium current was estimated using the Boltzmann equation. The value of concentration for 50% inhibition was 2.7 ± 0.2 mM, while the slope coefficient was 0.9 ± 0.2 mM⁻¹. In the presence of 10 mM STS, the decrease in the amplitude of potassium current reached, on average, 55%. Therefore, “chemical” hypoxia influences rather significantly the potassium conductance of the membrane of pheochromocytoma PC12 cells.     

Studies of the effect of organic solvents on the stability of β-glucosidase fromFusarium oxysporum

Summary The effect of organic solvents on the stability of -glucosidase in a powder form, isolated fromFusarium oxysporum, has been studied using several organic solvents of different degree of hydrophobicity. It was found that -glucosidase remains quite stable after a prolonged incubation in the presence of most of the organic solvents used, even at temperatures as high as 70°C. Only dimethyformamide (DMF) and tetrahydrofuran (THF) reduce considerably the enzyme activity in a short preincubation period. Studies on the effect of the pH of the buffer used prior to lyophilization, as well as of exogenous added water to the incubation mixture, on enzyme stability show that it is more stable in pH 5.0 and in the lowest water content. In addition it was found that the presence of glucose in the lyophilization procedure gives a significant protection to the enzyme when it is incubated for 30 h in pentanol and n-hexane.