Koh-Ichi Yoza

High-performance liquid chromatographic determination of naturally occurring flavonoids in Citrus with a photodiode-array detector

Abstract High-performance liquid chromatography (HPLC) coupled with ultraviolet-visible spectrophotometry using a photodiode-array detector was used as a routine method for the simultaneous separation and determination of 25 naturally occurring Citrus flavonoids. The separation system consisted of a C18 reversed-phase column, a gradient system of 0.01 M phosphoric acid (A) and methanol (B), and a photodiode-array detector. Each of the 25 flavonoids was eluted from the column with a gradient system composed of three periods: (1) 0–55 min, 70-55% (v/v) A in B, (2) 55–95 min, 55-0% A in B, and (3) 95–100 min, isocratic, 100% B, and quantified by spectrophotometric detection at 285 nm. Identifications of specific flavonoids were made by comparing their retention times (tR) and UV spectra with those of standards. The relative standard deviations of tR, values were 0.029–0.321%. The recoveries of pure eriocitrin, naringin, hesperidin and tangeretin added to tissues prepared from Unshiu (Citrus unshiu Marc.) and Hirado-buntan (Citrus grandis Osbeck f. Hirado) and subsequent extraction were 97.47–103.13% from the mesocarp and 96.87–104.93% from the juice with standard deviations of 2.32–5.72% and 2.18–5.96%, respectively.

Avidin Expressed in Transgenic Rice Confers Resistance to the Stored-Product Insect Pests Tribolium confusum and Sitotroga cerealella

Rice (Oryza sativa var. Nipponbare) was transformed with an artificial avidin gene. The features of this construct are as follows: (1) a signal peptide sequence derived from barley alpha amylase was added at the N-terminal region, (2) codon usage of the gene was optimized for rice, and (3) the gene was driven by rice glutelin GluB-1, an endosperm-specific promoter. Avidin was produced in the grain of the transgenic rice but not in the leaves. The concentration of avidin in the kernels was about 1,800 ppm. All larvae of the confused flour beetle (Tribolium confusum) and Angoumois grain moth (Sitotroga cerealella) died when fed transgenic avidin rice powder or kernels, respectively, whereas most of the test insects developed into adults when they were fed a nontransgenic rice control diet. Avidin extracted from the transgenic rice kernel lost most biotin-binding activity after 5 min heating at 95 °C.

High-performance liquid chromatographic determination of polyamines in selected vegetables with postcolumn fluorimetric derivatization

Abstract A rapid high-performance liquid chromatographic analysis for the simultaneous separation of five naturally occurring polyamines (agmantine, putrescine, cadaverine, spermidine and spermine) is described. Postcolumn derivatization with o-phthalaldehyde is used. The separation systems consisted of a strong cation-exchange column, an elution buffer consisting of 1 M sodium citrate (pH 5.4), a mixing coil for the chemical reaction and a spectrofluorimetric detector. The derivatized fluorescent compounds were detected at 340 nm (excitation) and 455 nm (emission). The recoveries of the polyamines were 94.5–107.0% with a standard deviation of 0.83–7.65%.

Molecular Cloning and Functional Expression of cDNA Encoding a Cysteine Proteinase Inhibitor, Cystatin, from Job's Tears (Coix lacryma-jobi L. var. Ma-yuen Stapf)

A λZAP II cDNA library was constructed from mRNA in immature seeds of the grass Jobs tears. A cDNA clone for a cysteine proteinase inhibitor, cystatin, was isolated from the library. The cDNA clone spanned 757 base pairs and encoded 135 amino acid residues. The deduced amino acid sequence was similar to that of cystatins from the gramineous plants rice, sorghum, and corn. The central Gln-Val-Val-Ala-Gly sequence thought to be one of the binding sites of cystatins was found. A remarkable characteristic of the peptide sequence of Jobs-tears cystatin was the putative signal peptide that has been found in sorghum and corn but not in rice. The cystatin cDNA was expressed in Escherichia coli as a His-tagged recombinant protein. The purified recombinant protein inhibited papain.

Changes in molecular weight and carbohydrate composition of cell wall polyuronide and hemicellulose during ripening in strawberry fruit

Abstract Polyuronides and hemicelluloses from the cell wall of developing strawberry fruit ( Fragaria ananasa , Duch. cv. Toyonoka) were examined for molecular weight distribution, and carbohydrate composition prior to and following gel filtration chromatography. In non-fractionated polyuronides, the galacturonic acid and arabinose concentrations decreased during development. The concentration of rhamnose markedly increased, whereas that of arabinose declined in low-molecular-weight polyuronides ( M r kDa ). A significant reduction of hemicelluloses was detected in the high-molecular-weight region ( M r >170 kDa ), whereas little change was observed in the sugar composition of these polymers. Polygalacturonase activity was consistently reduced during ripening.

The study of heat stress in tomato fruits by NMR microimaging

The ripening of the tomato fruit was delayed for several days (average 5 days) by a 1-day heat treatment at 42 degrees C. Ethylene production increased during the first 3 h, but, after 6 h inhibition was almost total in tomato fruit incubated at 42 degrees C. However, recovery of ethylene production was rapid if fruits were returned to a temperature of 25 degrees C after heating. In NMR microimaging, three imaging pulse sequences with different repetition and echo times at 42 degrees C were used to obtain the proton density (TR = 6000 ms, TE = 15 ms), the T1 weighted image (TR = 1000 ms, TE = 15 ms) and the T2-weighted image (TR = 6000 ms, TE = 120 ms). After 12 h heating, the water in locular tissues began to show shorter T1 and T2 values. Though the tomatos were returned to 25 degrees C and preserved one more day, the water having a shorter T2 value in locular tissues, did not change. These results show that tomato fruit do not fully recover from heating even after one day, although ethylene production is recovered almost immediately. For this reason, we suggest that some denaturation event inside the tomato, which goes on after the end of heating, is the cause of the delay in tomato ripening.

Putrescine accumulation in banana fruit with ripening during storage.

Abstract Considerable accumulation of the free polyamine, putrescine (Put), was found in both pulp and peel tissue of unchilled and rewarmed bananas (Musa AAA group) during normal fruit ripening with climacteric ethylene production, while free Put remained at the original level in continuously chilled fruit. The bound Put content in both tissues of the unchilled and rewarmed fruits increased ca 3–10 fold. No competitive relationship was observed between Put and ethylene production in the banana fruit.

A Simple Determination of Thiamine in Rice (Oryza Sativa L) by High-Performance Liquid Chromatography with Post-Column Derivatization

Abstract We report a rapid high-performance liquid chromatographic method for the determination of thiamine in rice with fluorimetric post-column derivatization. The analysis system consisted of a combination of both thiamine extraction with a mixture of 0.1 M hydrochloric acid-40% methanol (0.1 M HCI-40% MeOH) solution from rice flour, and chromatographic separation and determination. To extract thiamine, the rice flour was refluxed for 30 min at 60 °C with 0.1 M HCl-40% HCI. The separation systems constituted of a Sorbax TMS column, column oven (55°C), elution system containing a mixture of 0.01 M sodium dihydrogen phosphate and 0.5 M sodium perchlorate solution, a mixing coil for chemical reaction and a spectrofluorimetric detector. The thiamine derivative compound was detected at 375 nm for excitation and 435 nm for emission. The recovery of thiamine was 94 -101%. The contents of thiamine in rice of six cultivars produced in Japan were determined by the above-mentioned HPLC method and AOAC method. The...

Putrescine accumulation in wounded green banana fruit

The concentrations of putrescine (Put) and ethylene, and the effects of biosynthesis inhibitors on the production of polyamines and ethylene were examined in wounded pulp tissue of green banana fruit. The Put concentration in the sliced banana fruit, and the arginine decarboxylase (ADC, EC 4.1.1.19) activity increased with the increase of ethylene evolution. Difluoromethylarginine (DFMA) suppressed Put accumulation by 72% while difluoromethylornithine (DFMO) had no effect on the Put content. Methylglyoxal bis(guanylhydrazone) (MGBG) decreased ethylene production and suppressed Put accumulation. Aminooxyacetic acid (AOA) and norbornadiene (NBD) also depressed Put accumulation. These findings suggest that ethylene produced in the wounded pulp tissue affects the induction of ADC and the accumulation of Put.

Molecular Cloning and Structural Characterization of the Hagfish Proteinase Inhibitor of the Alpha-2-Macroglobulin Family

The “most primitive” living vertebrate the hagfish has a dimeric proteinase inhibitor, a protein homologous to human α2-macroglobulin, in its plasma at high concentration. Although the hagfish proteinase inhibitor has been isolated and its function and quaternary structure studied, its primary structure, subunit composition and fragmentation process remain unclear. In this study, hagfish proteinase inhibitor cDNA was cloned, sequenced and cDNA-deduced amino acid sequence was analyzed. A large fraction of homosubunits in the dimeric structure of the protein has undergone a cleavage at a specific arginyl residue (Arg833) while the rest retained their chain integrity without being processed. Thus random combinations of processed and nonprocessed subunits in the dimeric structure of this protein result in different molecular conformers and generate a complicated multiband pattern in SDS-PAGE. It was further demonstrated by proteolytic analysis that the hagfish inhibitor has no susceptible arginyl residues within its bait region and thus incapable of trapping arginine specific proteinases. This implies that the specific subunit cleavage at Arg833 was caused by an unknown arginine specific proteinase which escaped from the entrapment by the hagfish inhibitor.