Terpenoid Induction in Sweet Potato Roots by Cyclic-3′, 5′-Adenosine Monophosphate

The bar-headed goose, a specialized high-altitude species, has a capacity for high oxygen uptake from a hypoxic environment. It thus has a higher oxygen affinity than other bird species of lower-altitude environments. Oxygen affinity is determined by molecular structures and genetic mutations of hemoglobin (Hb), which can also influence the coordinating structures and dynamics of oxygen-Hb. To explore the structural differences in Hbs as between high and low altitude species, photolysis dynamic parameters, including quantum yield, enthalpy, and conformational volume changes in carboxy-Hbs (HbCO) for the bar-headed goose and low altitude counterparts (the Chinese goose and chicken) were investigated by the laser pumping-probing technique and photoacoustic calorimetry. Comparing the photolysis results for HbCO of the three species, the enthalpy and conformational volume changes of the bar-headed goose were much smaller than those of the others, although the quantum yields of all three species are similar. To explain the possible mechanisms of these differences, modifications of salt bridges and key residue mutations at the α β subunit interfaces of the proteins are described and discussed briefly.     

Comparison of Soybean Tissue Extracted with Different Solvents

The enantioselective hydrolysis of (R,S)-3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4H-[1,4]benzoxazine (I) with enzymes was investigated. Optically active I and its hydrolyzate, 7,8-difluoro-2,3-dihydro-3-hydroxymethyl-4H-[1,4]benzoxazine (II), are the intermediates for preparing optically active ofloxacins, whose racemate is known to be an excellent antibacterial agent. Lipoprotein lipase from Pseudomonas fluorescens (LPL Amano 3) was found to predominantly hydrolyze (S)-I, giving (R)-I in 54% e.e. and (R)-II in 44% e.e. On the other hand, lipase from Candida cylindracea was found to predominantly hydrolyze (R)-I, giving (S)-I in 24% e.e. and (S)-II in 20% e.e. Since, the optical purities of I and II thus obtained were not particularly high, these optically active I and II were converted into 3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4-(3,5-dinitrobenzoyl)-4H-[1,4]benzoxazine (IV). After recrystallizing IV from ethyl acetate-hexane, (S)- and (R)-II were obtained with high enantiomeric excess by removing the crystallized racemic IV and subsequently hydrolyzing the resulting optically active IV with alkali. The reduction of II afforded 7,8-difluoro-2,3-dihydro-3-methyl-4H-[1,4]benzoxazine (III), for which the optical purity was estimated to be >96%e.e. by HPLC analysis. (R)- and (S)-ofloxacin were prepared from (R)- and (S)-III with retention of their configuration.     

Purification and Properties of Dimethylglycine Oxidase from Cylindrocarpon didymum M–1

Dimethylglycine oxidase was purified to homogeneity from the cell extract of Cylindrocarpon didymum M–1, aerobically grown in medium containing betaine as the carbon source. The molecular weight of the enzyme was estimated to be 170,000 by the gel filtration method and 180,000 by the sedimentation velocity method. The enzyme exhibited an absorption spectrum characteristic of a flavoprotein with absorption maxima at 277, 345 and 450 nm. The enzyme consisted of two identical subunits with a molecular weight of 82,000, and contained two mol of FAD per mol of enzyme. The flavin was shown to be covalently bound to the protein. The enzyme was inactivated by Ag⁺, Hg²⁺, Zn²⁺ and iodoacetate. The enzyme oxidized dimethylglycine but was inert toward choline, betaine, sarcosine and alkylamines. Km and Vmax values for dimethylglycine were 9.1 mm and 1.22 μmol/min/mg, respectively. The enzyme catalyzed the following reaction: Dimethylglycine+O₂+H₂O → sarcosine+formaldehyde+H₂O₂.     

Distribution and Formation of Acyl-CoA Synthetase in Microorganisms

The distribution of acyl-CoA synthetase was investigated among microorganisms. High enzyme activity was found in some strains in genera of Pseudomonas, Fusarium, Gibberella and Cylindrocarpon, and in many strains of basidiomycetes. There were two groups in respect to enzyme formation. The enzyme activities of Escherichia, Klebsiella, Enterobacter, Citrobacter and Serratia were detected only when they were grown with fatty acids as the carbon source. On the other hand, the activities of many fungal strains and pseudomonads were easily detected regardless of the carbon source for growth.

Gel filtration on Sephadex G-200 showed that the enzymes of Escherichia coli and Gibberella fujikuroi were mostly present around the void volume of the column and retarded by the gel after treatment with Triton X-100. Pseudomonas aeruginosa produced two kinds of enzymes, one was eluted around the void volume of the column and the other retarded by the gel. This elution pattern did not change upon treatment with Triton X-100. Some catalytic properties of acyl-CoA synthetases from P. aeruginosa and G. fujikuroi were also described.     

Comparative Study between Gas Chromatography and Ion-exchange Chromatography on Amino Acid Analysis of Foods

Lactobacillus brevis and Saccharomyces cerevisiae were completely sterilized by the supercritical (SC) CO2 micro-bubble method. Gaseous (G) and liquid (LQ) CO2 were used in a similar manner to compare the sterilizing effect. Among the three treatments, the microorganisms were only effectively sterilized by the SC CO2 treatment at 25 MPa and 35°C.     

Identification of catalytic residues of a very large NAD-glutamate dehydrogenase from Janthinobacterium lividum by site-directed mutagenesis

We previously found a very large NAD-dependent glutamate dehydrogenase with approximately 170?kDa subunit from Janthinobacterium lividum (Jl-GDH) and predicted that GDH reaction occurred in the central domain of the subunit. To gain further insights into the role of the central domain, several single point mutations were introduced. The enzyme activity was completely lost in all single mutants of R784A, K810A, K820A, D885A, and S1142A. Because, in sequence alignment analysis, these residues corresponded to the residues responsible for glutamate binding in well-known small GDH with approximately 50?kDa subunit, very large GDH and well-known small GDH may share the same catalytic mechanism. In addition, we demonstrated that C1141, one of the three cysteine residues in the central domain, was responsible for the inhibition of enzyme activity by HgCl₂, and HgCl₂ functioned as an activating compound for a C1141T mutant. At low concentrations, moreover, HgCl₂ was found to function as an activating compound for a wild-type Jl-GDH. This suggests that the mechanism for the activation is entirely different from that for the inhibition.     

Isolation of Escherichia coli B Mutant Deficient in Glutathione Biosynthesis

Certain edible large jellyfishes belonging to the order Rhizostomeae are consumed in large quantities in China and Japan. The exumbrella part of the edible jellyfish Stomolophus nomurai was cut and soaked in dilute hydrochloric acid solution (pH 3.0) for 12 h, and heated at 121 °C for 20 min. The immunostimulation effects of the jellyfish extract were examined. The jellyfish extract enhanced IgM production of human hybridoma HB4C5 cells 34-fold. IgM and IgG production of human peripheral blood lymphocytes (PBL) were also accelerated, 2.8- and 1.4-fold respectively. Moreover, production of interferon (IFN)-γ and tumor necrosis factor (TNF)-α by human PBL was stimulated 100- and 17-fold respectively. Collagenase treatment inactivated the immunostimulation activity of the jellyfish extract. In addition, purified collagen from bovine Achilles’ tendon accelerated IgM production of hybridoma cells. These facts mean that collagen has an immunostimulation effect, and that the active substance in jellyfish extract is collagen.     

Yamogenin in fenugreek inhibits lipid accumulation through the suppression of gene expression in fatty acid synthesis in hepatocytes

Yamogenin is a diastereomer of diosgenin, which we have identified as the compound responsible for the anti-hyperlipidemic effect of fenugreek. Here, we examined the effects of yamogenin on the accumulation of triacylglyceride (TG) in hepatocytes, because yamogenin is also contained in fenugreek. It was demonstrated that yamogenin also inhibited TG accumulation in HepG2 hepatocytes and suppressed the mRNA expression of fatty acid synthesis-related genes such as fatty acid synthase and sterol response element-binding protein-1c. Indeed, yamogenin also antagonized the activation of the liver X receptor (LXR) in luciferase ligand assay similar to diosgenin. However, yamogenin could not exert such effects in the presence of T0901713, a potent agonist of LXR. These findings indicate that the effects of yamogenin on TG accumulation would be weaker than those of diosgenin, suggesting that the structural difference between yamogenin and diosgenin would be important for the inhibition of LXR activation.     

Growth Profile of Crown Gall Cells of Tobacco in Suspension Culture

In order to obtain a basic information of plant cell suspension culture as a step toward the development of large scale culture, culture conditions of crown gall cells (auxin non-requiring cells) were investigated. Addition of yeast extract to culture medium was significantly effective for the growth and cell dispersion.

In experiments on the ability of the cultured cells to utilize sugars as the carbon source, it was observed that galactose, added to the culture medium, markedly inhibited the cell growth.

Pasteurization of the medium containing fructose as carbon source made it brownish by Maillard reaction and the medium apparently restrained the cell growth. However, the fructose medium sterilized by filtration was excellent for the cell growth as well as sucrose or glucose medium. In a jar fermentor, even the glucose medium became brownish by heat sterilization and the brown colored medium restrained the cell growth. Under optimum conditions, the doubling time was 1.1 day in exponential phase and 2.0 g of cell (dry weight) per 100 ml culture was obtained as the maximum yield.