Seventeen alkyl-substituted quaternary pyridinium compounds were synthesized by the condensation reaction of amino acids and primary amines with alkanals. The growth-inhibitory capabilities of these pyridiniums were investigated against gram-negative and gram-positive bacteria (Escherichia coli and Streptococcus thermophilus), as well as against a mold (Neurospora crassa) and yeast (Candida utilis). Most of the pyridiniums appreciably inhibited the growth of the microorganisms tested. l-Carboxymethyl-2-hexyl-3, 5-dipentylpyridinium betaine and l-(2hydroxyethyl)-2-hexyl-3, 5-dipentylpyridiriium chloride showed the greatest inhibitory activity. Least growth inhibition by pyridinium ions was observed for E. coli.

View full abstract

Protoplasts of Pyricularia oryzae P₂, a rice blast mold, were prepared in high yield from the young mycelium of the fungus using lytic enzymes from Bacillus circulans WL12. The majority of the protoplasts had one nucleus per cell. The protoplasts formed a cell wall and eventually reverted to normal mycelial form in liquid medium. The process of regeneration was studied under phasecontrast and electron microscopes. The protoplast built a very thick wall prior to the protrusion of a germ-tube like hypha. Golgi apparatus-like structures appeared in the early stage of regeneration and disappeared later. Electron-transparent amorphous structures accumulated during regeneration. Lomasomes were observed in the regenerated cell walls.

View full abstract

All positional isomers of methyl methylcyclohexaneacetates were prepared. Their diastereomeric pairs were separated from one another by preparative GLC. Either the cis or trans configuration was assigned to each of these GLC fractions on the basis of the chemical shift values of the ring methyl carbons in their ¹³C NMR spectra, which were found closely approximate to those of the corresponding dimethylcyclohexanes. The conformational compositions of the thermodynamically less stable diastereomers were determined using the chemical shift values of the axial and equatorial methyl carbons as parameters. The isomers of propyl methylcyclohexaneacetates for the biological test were separated similarly, and their configurations were determined by comparing the shift values of their ring methyl carbons with those of the methyl esters. The relative activity between the diastereomeric pairs was evaluated by a comparative trap test. The result was : cis-4-methyl isomer > trans-4-methyl isomer; trans-3-methyl isomer > cis-3-methyl isomer; and, trans-2-methyl isomer > cis-2-methyl isomer. The cis-4-methyl isomer exhibited activity comparable to that of the parent compound.

View full abstract

A search was undertaken for microorganisms that produce an enzyme capable of deacylating 7β-(4-carboxybutanamido)cephalosporanic acid (glutaryl 7-ACA, GL-7-ACA) to 7-aminocephalosporanic acid. Pseudomonas putida ATCC 950 was selected from our stock cultures, and Pseudomonas SY-77-1 was isolated from soil. Adding glutaric acid to the growth media enhanced the production of GL-7-ACA acylase by Pseudomonas SY-77-1. A β-lactamase-deficient mutant, Pseudomonas NS-2, was derived from Pseudomonas SY-77-1 treated with a mutagen. The cell suspension of Pseudomonas NS-2 specifically hydrolyzed cephalosporin compounds having aliphatic dicarboxylic acid in the acyl side chain.

View full abstract

The components and structure of the cell wall of Rhizopus delemar were investigated using purified lytic enzymes, protease and chitosanase from Bacillus R-4 and chitinase II from Streptomyces orientalis. When these enzymes were used individually they only partially lysed the cell wall, but when allowed to react on the cell wall together, a complete lysis was achieved by cooperative action. These modes of action on the cell wall and the chemical and morphological data suggested that the cell wall structure was different in Rhizopus delemar of Zygomycetes from filamentous fungi of Euascomycetes and that its wall structure might be composed mainly of chitin fibers cemented by chitosan and protein or peptides scattered in a mosaic manner.

View full abstract

Partially purified soybean β-conglycinin (7S), glycinin (11S) and acid precipitated proteins (APP) were acetylated to various degrees. A hybrid program was established; (i) a cyclic temperature test (20→90→20°C) under a constant shear rate and (ii) a cyclic shearing test (48.7→243.7→48.7 sec^-1) under isothermal conditions. The apparent viscosity of the soy protein suspending system (12%, wt/vol) gradually increased with an increase in acetyl content. A: considerable increase in viscosity was found in the cooling period with an increase in acetyl content. Although native 1IS globulin showed nearly no increase in viscosity, the highest acetylated US globulin showed a high viscosity, as much as that of7S globulin and APP, in the cooling period and in the cyclic shearing test after heating. There was little variation among highly acetylated heated gels.

View full abstract

A study was made of rats fed a high fructose diet to determine whether males and females responded differently to lipoprotein lipase activity and how this response was affected by sex. The rats were meal-fed with a high fructose or a high glucose diet for two weeks. After two days of starvation, each diet-group was divided into three subgroups: (a) fasted, (b) injected with physiological saline solution, and (c) injected with mannoheptulose solution. Subgroups (b) and (c) were then immediately refed their respective meals of the high fructose or the high glucose diet for three hours and then sacrificed. The weights of the liver and adipose tissues, amounts of liver lipids, lipoprotein lipase activities in the adipose tissues and serum immunoreactive insulin concentrations were determined. Males fed the high fructose diet had a significantly higher hepatic lipid content than those fed the high glucose diet. The weight of adipose tissues of females fed the high fructose diet was less than that of those fed the high glucose diet (p<0.01). In males fed the high fructose diet, lipoprotein lipase activity, expressed in units per lOOg body weight per hour, was less than that of males fed the high glucose diet; the females showed a similar trend. Moreover, females on the fructose diet had lower lipoprotein lipase activity than males in same subgroups. The serum immunoreactive insulin level increased in rats fed the high glucose diet after the three hours refeeding, but the serum immunoreactive insulin level of the rats fed the high fructose diet showed a lower elevation.

View full abstract

Resting Escherichia coli B and K-12 cells suspended in a phosphate buffer solution were exposed to alternating current (AC) of 50 Hz. The surviving fractions of cells exposed to AC were decreased with an increase in current density from 0 to 300mA/cm² at 29±3°C for a definite exposure time. At a definite current density the fractions were decreased proportionally with exposure time. Even for conditions of definite exposure time and current density, the fractions varied depending on the concentration of cells initially suspended in buffer solution in AC-exposure chamber. E. coli cells were more resistant to AC-exposure under anaerobic conditions than aerobic conditions. Addition of catalase to the cell suspension during AC-exposure effectively protected both strains. Methionine and albumin had mild protective effects, but chemicals, such as cysteine, ehanol or a-tocopherol had little protective effect.

View full abstract

The light-emitting species of chemiluminescence produced in rat liver homogenate on adding autoxidized linseed oil (AOLO) were investigated. The chemiluminescent intensity of liver homogenate was strongly enhanced by the addition of AOLO and showed a proportional relationship to the amount of AOLO. The chemiluminescence was reduced with singlet oxygen (¹0₂) quenchers and free radical scavengers. Among them, β-carotene showed the most effective quenching. The emission spectrum had broad bands in the visible region with eminent chemiluminescent lines at 520, 575 and 640nm due to the simultaneous transition, 2[¹Δ_g]→2[³Σ_g^-]. An additional weak line was found at 480 nm corresponding to [¹Δ_g] +[¹Σ_g⁺]→2[³Σ_g^-]. In the presence of β-carotene, lines corresponding to the simultaneous transition of ¹0₂ disappeared. These results indicate that the liver homogenate with AOLO generated singlet molecular oxygen as one of the major light-emitters of the chemiluminescence. A possible mechanism for the generation of XO2 is by decomposition of peroxy radicals derived from AOLO in the liver homogenate.

View full abstract

The rates of hydrolysis of aryl-β-D-xylosides (e.g. p-NO₂-phenyl-, p-Cl-phenyl-, phenyl-, p-CH₃-phenyl- and p-CH₃O-phenyl-β-D-xylosides) by Malbranchea β-xylosidase were investigated. The Michaelis constants, Km, were almost independent of aglycone, whereas maximum velocity, V_max, showed a marked dependence. In all hydrolysis reactions studied, the molecular activities, k0 (V_max/e₀, where e₀ is the molar concentration of enzyme), were markedly increased by introducing both electron-withdrawing and electron-donating substituents in the para position of the phenyl ring. Between the values of log k₀ and inductive sigma constants, σ, V-shaped Hammett plots were obtained. When electron-withdrawing substituents were introduced, the Hammett reaction constant, ρ, was + 1.27, when electron-donating substituents were introduced, its value was -2.07. On enzymic hydrolysis of p-nitrophenyl- and phenyl-β-D-xyloside, the reaction products, in both cases, were found to be α-D-xylose with inversion of configuration.

View full abstract

Temperature-induced structural changes in chicken egg white ovomucoid were investigated by circular dichroism (CD) and ultraviolet absorption (UV). A major unfolding transition was brought about between 60 and 90°C with a rise in temperature, as monitored by variations in CD at 222nm and UV at 287nm. Thermal transition was reversible when there was no prolonged exposure to the denaturing condition. The Van't Ho ff plot for the major unfolding process of the ovomucoid yielded the following thermodynamic parameters at pH 7.0 between 60 and 90°C: ΔH_VH=73 kcal•mol^-1 at 74°C and ΔS_VH=209 cal•deg^-1 •mol^-1 at 74°C, on the assumption of a two-state transition. The equilibrium CD spectra at various temperatures suggested that the secondary structure change caused by heating from 20 to 60°C was qualitatively different from that from 60 to 90°C. By prolonged exposure to high temperature of 90°C, the reversible denatured state was slowly transformed into an irreversible denatured state.

View full abstract

Ferredoxin-nitrite reductase (EC 1.7.7. 1), an enzyme which catalyzes the 6-electron reduction of nitrite to ammonia, has been isolated from Spinacia oleracea. The isolated enzyme was homogeneous by disc electrophoresis with polyacrylamide gel. The molecular weight of the enzyme was estimated to be 86, 000 by Ultrogel AcA 34 gel filtration. In the oxidized form, the enzyme had absorption maxima at 278, 388 (Soret band), 573 (α band) and 690nm, indicating that siroheme is directly involved in the catalysis of nitrite reduction. This absorption spectrum was modified by sulfite, hydroxylamine and cyanide. The enzyme exhibited electron paramagnetic resonance signals with g values of 6.9 and 5.2, which are characteristic ofa high spin Fe³⁺-siroheme in the molecule. These signals disappeared upon the addition of dithionite or nitrite. This isolated enzyme also contained four moles of labile sulfide and 7g-atoms of iron per 86, 000g of protein.

View full abstract

This work is a new attempt to modify the functionality of food proteins by the use of a potential activity of papain. Fish protein concentrate, soy protein isolate, casein, ovalbumin and gelatin were succinylated prior to using them as substrates. Each of the L-leucine n-alkyl esters with different alkyl-chain lengths was covalently incorporated into each substrate by applying the unusual papain-catalyzed reaction [Agric. Biol. Chem., 43, 1065 (1979)]. The resulting products posessed high whippability and/or emulsifying activity. The highest whippability was observed when leucine butyl ester or hexyl ester was incorporated, while the incorporation of higher alkyl esters generally caused an increase in emulsifying activity.

View full abstract

Strains producing high levels of ubiquinone 10 were isolated from tobacco cell suspension cultures by a cell cloning technique, and the UQ content of these cell strains was analyzed by high performance liquid chromatography. Three strains with high UQ yields in suspension culture were selected and used for investigations on the necessary cultural conditions for UQ production. Sucrose concentration, 2, 4-d concentration and culture temperature did not have marked effects on UQ formation. These results differed from those of the original cell lines. Furthermore, the addition of yeast extract to the medium promoted cell growth in these strains remarkably but did not enhance UQ production in the cell strains. According to these investigations, UQ production was increased to 15mg/liter and the UQ level to 1890μg/g dry wt. of cells after 11 days of incubation.

View full abstract

Furano-terpene production in sweet potato root tissue treated with chemicals, such as HgCl₂, L-alanine and _CAMP was inhibited by antibiotics, such as cycloheximide, blasticidin S, puromycin and chloramphenicol when the antibiotics were administered to the tissue immediately after cutting. However furano-terpene production was not inhibited by antibiotics when they were administered to the 18-hr incubated cut tissue together with chemical elicitors of furano-terpene production. Furano-terpenes were accumulated in the tissue in 5 hr after HgCl₂ treatment to the 18hr incubated cut tissue and the content reached a maximum in lO hr and then decreased gradually. The rate of protein synthesis in the tissue was enhanced in 6 hr after cutting and decreased by 15 hr, then reached a constant rate. HgCl₂ treatment to the 24-hr incubated cut tissue did not affect the rate of protein synthesis but cycloheximide administered to the tissue with HgCl₂ strongly suppressed the rate of protein synthesis. These data suggest that protein synthesis is induced by cutting, and furano-terpene production is induced by HgCl₂ treatment to the cut and incubated tissue without synthesis of the bulk protein.

View full abstract

A strain of yeast was isolated from the exudate ofa tree at Ishigaki Island near Taiwan. It was found to produce 3-hydroxy-3-methylglutaric acid extracellularly as the primary metabolic product of glucose and ethanol metabolism under aerobic conditions. The yeast was identified as Candida sorbosa Hedrick et Burke ex van Uden et Buckley. The acid was produced at a concentration of approximately 10mg per ml of culture nitrate.

View full abstract

Lysinoalanine was determined by gas chromatography. Lysinoalanine formation in lysozyme depended on alkali concentration, pH, temperature and exposure time. The upper limits of lysinoalanine formation in lysozyme and a-lactalbumin were related to the number of lysine residues with a cystine disulfide bond in the adjacent position rather than the individual contents of these residues. In cases of α_s1- and β-caseins, the upper limits were related to the number of phosphoserine residues, regardless of their sequential relationship to lysine residues. Gel electrophoresis suggested that intermolecular cross-linking took place in the a-lactalbumin and caseins.

View full abstract

Titration curves of ovalbumin and s-ovalbumin were compared in studies of changes in properties of ovalbumin on conversion to s-ovalbumin. Although there are 49 carboxyl groups in ovalbumin, two were not titrated in 0.25 M KCl but on conversion to s-ovalbumin. A similar change was also noted in the two carboxyl groups of ovalbumin in denaturation with guanidine hydrochloride. Liberation ofcarboxyl groups was noted in ovalbumin samples which were assumed to contain various amounts of intermediate and the isoelectric focusing patterns of these samples also changed. No difference was noted in the both amounts of ionizable amino groups and phenolic hydroxyl groups between ovalbumin and s-ovalbumin. This seems to show that these two groups are not concerned with the liberation of carboxyl groups during ovalbumin-s-ovalbumin transformation.

View full abstract

Eleven-S globulin heated in the presence of N-ethylmaleimide (NEM) retained its buffersoluble state. Its gel filtration gave two peaks. Two kinds of electrophoresis indicated that the first peak contained soluble aggregates consisting of intermediary subunits as the major component, and the second peak contained a monomer of acidic subunit and a monomer of intermediary subunit. With an increase in heating time, soluble aggregates polymerized through disulfide bond occurred a little. The temperature ofthe endothermic peak of 1 IS globulin heated in the presence of NEM was slightly higher than that in the absence of NEM.

View full abstract

Fourteen new and three known ο-arylethenylbenzoic acids (10-18), required for testing as potential plant growth regulators, have been synthesized by the Wittig reaction. The cis- and trans- isomers were separated by fractional crystallization and identified by NMR spectroscopy. The preliminary assay results on cress seedlings are reported and show that both geometric isomers appear to have nearly identical anit-geotropic activity in this test system.

View full abstract

Three phytotoxins named pyrenochaetic acids A, B and C were isolated from culture filtrates of Pyrenochaeta terrestris, and the structures were determined to be 4-crotonoyl-3-methoxy-5-methylbenzoic acid (I), 4-(3-hydroxybutyroyl)-3-methoxy-5-methylbenzoic acid (II) and 4-butyroyl-3-methoxy-5-methylbenzoic acid (III), respectively. Their phytotoxicities were demonstrated in bioassays.

View full abstract

A major proportion of selenium in bovine milk was found in fluorornetric analysis to be associated with the casein fraction, largely in alkali-labile form, and the rest with the whey fraction mostly in free selenite form. This uneven distribution of milk selenium seems to provide an explanation for selenium deficiency in purified caseins. The activity of glutathione peroxidase, a selenoprotein, in the liver of growing male rats fed ad libitum low-selenium diets containing either vitamin-free casein or Torula yeast 0.065 ±0.012 or 0.015 ±0.004μg Se/g diet, respectively) for 3 weeks decreased to 4 to 6% of that of the control rats fed a commercial stock diet (0.185±0.092fig Se/g diet). Selenium status was evaluated by three different parameters for the rats assigned under pair-feeding regimen to those vitamin-free casein-based diets which were supplemented with graded levels of selenium as sodium selenite. The hepatic levels of the thiobarbituric acid-reactive substance, an indication of lipid peroxidation, decreased to control level with selenium supplementation per g diet of 0. 1 μg and over. The hepatic glutathione peroxidase activity reached a plateau above a 0. 1 μg/g diet of selenium supplementation, whereas the erythrocyte enzyme activity increased with increasing levels of supplementary selenium. These results support the notion that semi-purified diets containing vitamin-free casein as a prime protein source would not satisfy the selenium requirement of growing animals unless deliberately supplemented with additional selenium.

View full abstract

The structure of fusariocin C, C₂₇H₂₈O₆ has been established by X-ray diffraction, PMR and IR studies. It is a pseudo dimer structure containing the tropolone skeleton.

View full abstract

A new aryl-peptidyl amidase has been isolated from a Lactobacillus casei homogenate. Its ribosomal localization was shown by fractionation and its general properties studied after purification on Sepharose 6B and DEAE-Sephacel. The enzyme requires 1 mM Mg²⁺ for stability, while Zn²⁺, Mn²⁺, Co²⁺ and Ca²⁺ result in only partial stability. No inhibitory effects were noted after treatment with phenylmethylsulfonylfluoride or EDTA. Enzymatic activity was totally inhibited by 5 mM p-hydroxymercuribenzoate; activity was restored by dithiothreitol. The only substrates hydrolyzed by this enzyme were the succinyl-L-phenylalanine-p-nitroanilide type, with a pH optimum between 6 and 7 and a Michaelis constant of 0.76 mM. No hydrolysis could be detected using proteins, peptides, amides or esterase substrates. This enzyme would thus not be an endopeptidase (E.C. 3.4.21), but would to rather be considered as belonging to the group of amidases (E.C. 3.5.1).

View full abstract

The effect of protein depletion and repletion on the contents of rat body collagen and muscle protein has been investigated. Adult male rats were placed on a protein-free diet for 64 days, and thereafter fed with a 20% casein diet for 83 days. Total nitrogen, hydroxyproline and N^t-methylhistidine were determined in several tissues. Skin collagen was fractionated into neutral saltsoluble, acid-soluble and insoluble collagen.
In prolonged protein depletion, the total amounts of liver protein, skin collagen and muscle protein were markedly decreased, while carcass collagen (excluding skin) showed little effect. The concentrations of skin and carcass collagen were significantly increased in protein depleted rats. Some differences in metabolism between skin collagen and other carcass collagen were discussed.
After 64 days of depletion, refeeding on an adequate protein diet brought about a rapid increase in body weight, liver protein and muscle protein. Skin collagen did not increase in the early period of refeeding, but increased rapidly at a later stage. A remarkable change in the solubility pattern of skin collagen was observed in protein depletion and repletion.
These results indicate that feeding depleted rats with an adequate protein diet brings about an almost complete recovery of muscle protein and collagen which are the main sources of the amino acid pool for protein synthesis during prolonged protein deprivation.

View full abstract

(±)-Methyl jasmonate (JA-Me) was resolved by preparative high performance liquid chromatography of its diastereomeric ketal with (-)-2, 3-butanediol to give optically pure (+)-JA-Me, while the optical purity of the (-)-form obtained by the resolution was 81.2-81.9%. The inhibitory activity of (+)-JA-Me on the growth of rice seedlings was found to be lower than that of the naturally occurring (-)-form, the (+)-form being still fairly potent.

View full abstract

Using a method consisting of two repetitions of "prophage transformation, " the thermostable a-amylase gene in Bacillus subtilis has been cloned in temperate phage p11.

View full abstract