Chemical screening of an inhibitor for gibberellin receptors based on a yeast two-hybrid system

We applied a yeast two-hybrid (Y2H) system to the high-throughput monitoring of two proteins’ interaction, a receptor for phytohormone gibberellin (GA) and its direct signal transducer DELLA. With this system, we screened inhibitors to the interaction. As a result, we discovered a chemical, 3-(2-thienylsulfonyl)pyrazine-2-carbonitrile (TSPC), and we confirmed that TSPC is an inhibitor for GA perception by in vitro and in planta evaluations.     

Design and synthesis of a series of α-benzyl phenylpropanoic acid-type peroxisome proliferator-activated receptor (PPAR) gamma partial agonists with improved aqueous solubility

In the continuing study directed toward the development of peroxisome proliferator-activated receptor gamma (hPPARγ) agonist, we attempted to improve the water solubility of our previously developed hPPARγ-selective agonist 3, which is insufficiently soluble for practical use, by employing two strategies: introducing substituents to reduce its molecular planarity and decreasing its hydrophobicity via replacement of the adamantyl group with a heteroaromatic ring. The first approach proved ineffective, but the second was productive. Here, we report the design and synthesis of a series of α-benzyl phenylpropanoic acid-type hPPARγ partial agonists with improved aqueous solubility. Among them, we selected (R)-7j, which activates hPPARγ to the extent of about 65% of the maximum observed with a full agonist, for further evaluation. The ligand-binding mode and the reason for the partial-agonistic activity are discussed based on X-ray-determined structure of the complex of hPPARγ ligand-binding domain (LBD) and (R)-7j with previously reported ligand-LDB structures. Preliminal apoptotic effect of (R)-7j against human scirrhous gastric cancer cell line OCUM-2MD3 is also described.     

Separation and Purification of Molecular Species of Galactolipids by High Performance Liquid Chromatography

The NaCl concentration of the growth medium affected hydrogen production by Lyngbya sp. (No. 108) strain. Cells grown in medium containing 3% NaCl produced the most hydrogen. The carbohydrate content of this strain also increased with increasing NaCl concentration of the growth medium up to 720 fig/mg cells at 5 % NaCl. In the presence of 20 finlol/ml MFA (monofluoroacetic acid), inhibition of hydrogen production was observed. We extracted the glycogen from this nonheterocystous filamentous cyanobacterium, Lyngbya sp. (No. 108), and observed that glycogen and carbohydrate consumption of this strain is coincident with hydrogen production.

These results led us to the conclusion that the reserve glycogen or other carbohydrate were used as sources of electron donors for hydrogen production, and that the NaCl concentration of the medium affected the hydrogen production by this strain.     

Glycolipids Isolated from Spirulina maxima: Structure and Fatty Acid Composition

Several glycolipids were isolated from Spirulina maxima, an edible blue-green algae, by systematic fractionation with different solvents. Structural investigation by using methylation, GC-MS, and enzymic techniques indicated that the major glycolipids are O-β-d-galactosyl-(1→l′)-2′, 3′-di-O-acyl-d-glycerol, O-α-d-galactosyl-(l-→6)-O-β-d-galactosyl-(1→l′)-2′,3′-di-O-acyl-d-glycerol and 6-sulfo-O-α-quinovosyl-(l→l′)-2′, 3′-di-O-acyl-d-glycerol. Main fatty acid components of these glycolipids were identified as palmitic acid and linoleic or linolenic acid. Based on-these fatty acid compositions, Spirulina glycolipids were compared with those in higher plants.     

Conformation of Some Hexuronic Acids and d-Galactopyranosiduronic Acid Moiety in the Peracetylated and Permethylated Derivatives of Orange Pectic Acid in Solutions

1. The 1C conformation was estimated for α-d-galactopyranosiduronic acid moiety of pectic acid in the permethylated derivative dissolved in 1 n NaOD-D₂O and in the peracetylated derivative dissolved in dimethyl sulfoxide-d₆, and the C1 conformation was estimated for some derivatives of d-galactopyranuronic acid in chloroform-d by NMR spectroscopy.

2. Random conformation of the whole macromolecule was estimated for pectic acid in water on the basis of no appearance of any induced Cotton effects in the 200 ~ 700 mμ region in the ORD spectra of pectic acid-anionic dye complexes.

3. The conformation was supported by the fact that the rate of periodate oxidation of pectic acid at 5° was slightly decreased in comparison with that of amylase in 7 m urea solution.

     

Determination of 2-Aminoethylphosphonic Acid and Its Y-iM ethyl Derivative in Animal Tissues by Gas Chromatography with Flame Photometric Detection

A sensitive and selective method was developed for the determination of 2-aminoethylphosphonic acid (AEP) and W-methyl AEP in animal tissues by gas chromatography (GC). These compounds were converted into their A-isobutoxycarbonyl methyl ester derivatives and measured by GC with flame photometric detection (FPd-GC), using 0.5 % FFAP on Uniport HP as the GC column packing. The calibration curves for AEP and A-methyl AEP in the range of 0.02 ~ 2 μg were linear, and the detection limit was about 20 pg as an injection amount. AEP and A^-methyl AEP in animal tissues were found in the free form and bound form with lipid and other biological macromolecules, and they could be measured without any influence from coexistent substances by FPd-GC. The recoveries of AEP and A'-methyl AEP added to the tissue samples were 92 —105 %, and their reproducibility was found to be satisfactory. The distribution of these compounds in various animals was also studied by using this new method.     

Applying Proteolytic Enzymes on Soybean

Soy proteins were incubated with a microbial acid protease (Molsin) under the following condition: substrate concentration, 1%; enzyme-substrate ratio (by weight), 1/100; pH, 2.8; and temperature, 40°C—flavor components and related impurities are removable from crude soy-protein concentrates by their incubation for 2 hr under the above condition. The acid-precipitated fraction of soy protein incubated for 2 hr with Molsin (i.e. 2 hr-proteolyzate) showed the following composition: 10% trichloroacetic acid (TCA) insoluble fraction, 47.52%; 10% TCA soluble peptide fraction, 52.02%; and free amino acid fraction, 0.46%. Gel filtration of the 2 hr-proteolyzate gave an elution pattern showing its molecular weight distribution.

In the process of the incubation of the acid-precipitated protein, the 10% TCA insoluble fraction showed increase in amino nitrogen content, its solubility in a phosphate buffer increased to change at 6 hr, and a hydrophobic amino acid share in this fraction increased gradually.

In vitro digestibility of the acid-precipitated fraction were improved and the lipoxygenase activity in this fraction decreased through the Molsin treatment.

Ultracentrifugal analysis showed a decreasing tendency of the cold-insoluble fraction of soy protein during its incubation with Molsin. Optical rotatory dispersion and circular dichroism study elucidated conformational changes in this fraction during its incubation either with or without Molsin.     

Stimulation of Methionine Sulfoxide Reduction and Ethylene Production by Isoprothiolane in Relation to Its Activity against Non-parasitic Damping-off (MURENAE Disease) of Rice Seedlings

A rice blast controlling agent, isoprothiolane (diisopropyl 1,3-dithiolan-2-ylidenemalonate), stimulated the reduction of methionine sulfoxide to methionine by the rice plant. In the presence of isoprothiolane, the methionine/(methionine + its sulfoxide) ratio was increased to 129~208% of the control. The ethylene production by the plant was also enhanced by isoprothiolane, probably because methionine is an important precursor of ethylene. The non-parasitic damping-off caused by chilling stress on rice seedlings was effectively prevented with the application of isoprothiolane as well as ethephon, which easily decomposes to ethylene and acids. Therefore, the ethylene level modified by isoprothiolane and ethephon can contribute to their protective activity against the non-parasitic damping-off of rice seedlings. Indeed, a close relationship between the ethylene level and the protective activity against damping-off was obtained with isoprothiolane, but not with ethephon. Endogenous ethylene seems to be more effective in controlling the damping-off than exogenous ethylene from ethephon.