Satoru Nirasawa

Overexpression of the wasabi defensin gene confers enhanced resistance to blast fungus (Magnaporthe grisea) in transgenic rice

Abstract.Transgenic rice (Oryza sativa cv. Sasanishiki) overexpressing the wasabi defensin gene, a plant defensin effective against the rice blast fungus, was generated by Agrobacterium tumefaciens-mediated transformation. Twenty-two T2 homozygous lines harboring the wasabi defensin gene were challenged by the blast fungus. Transformants exhibited resistance to rice blast at various levels. The inheritance of the resistance over generations was investigated. T3 plants derived from two highly blast-resistant T2 lines (WT14-5 and WT43-5) were challenged with the blast fungus using the press-injured spots method. The average size of disease lesions of the transgenic line WT43-5 was reduced to about half of that of non-transgenic plants. The 5-kDa peptide, corresponding to the processed form of the wasabi defensin, was detected in the total protein fraction extracted from the T3 progeny. Transgenic rice plants overproducing wasabi defensin are expected to possess a durable and wide-spectrum resistance (i.e. field resistance) against various rice blast races.

Konjac glucomannan-induced changes in thiol/disulphide exchange and gluten conformation upon dough mixing

Effects of konjac glucomannan (KGM) on the changes in gluten upon dough mixing were investigated in this study. Wheat flour was blended with KGM and processed into dough. Farinographic analysis showed that KGM caused a significant increase in water absorption and dough development time to reach maximum consistency. Comparison of electrophoretic protein profile from control dough and KGM-dough revealed that protein fractions were similar in molecular size distribution, but the lability of glutenin aggregates slightly differed. Addition of KGM to gluten induced negative effects on exchange between sulfhydryl groups and disulphide bonds. Fourier transform-Raman spectroscopy indicated that secondary structure of gluten proteins was differentially modified related with water absorption of flours before dough formation. This study reveals that when KGM is added to the dough, conformational behaviours of gluten proteins are changed and the hydroxyl groups of KGM might be involved in the interaction by forming strong intermolecular hydrogen bonding system.

Rapid survey of four Asp isomers in disease-related proteins by LC-MS combined with commercial enzymes.

Until relatively recently, it was considered that D-amino acids were excluded from living systems except for the cell wall of microorganisms. However, D-aspartate residues have now been detected in long-lived proteins from various tissues of elderly humans. Formation of D-aspartate in proteins induces aggregation and loss of function, leading to age-related disorders such as cataracts and Alzheimer disease. A recent study used LC-MS to analyze isomers of Asp residues in proteins precisely without complex purification of the proteins. However, to identify the four Asp isomers (Lα, Lβ, Dβ, and Dα) on the chromatogram, it was necessary to synthesize reference peptides containing the four different Asp isomers as standards. Here, we describe a method for rapidly and comprehensively identifying Asp isomers in proteins using a combination of LC-MS and commercial enzymes without synthesizing reference peptides. The protein sample is treated with trypsin, trypsin plus Asp-N, trypsin plus PIMT, trypsin plus paenidase, and the resulting peptides are applied to LC-MS. Because Asp-N hydrolyzes peptide bonds on the N-terminus of only Lα-Asp residues, it differentiates peptides containing Lα-Asp from those containing the other three isomers. Similarly, PIMT recognizes only peptides containing Lβ-Asp residues, and paenidase internally cleaves the C-terminus of Dα-Asp residues. This approach was successfully applied to the analysis of all tryptic peptides in aged lens. The comprehensive quantitative data of Asp isomer formation in age-related proteins obtained via this method might be used as biomarkers of age-related disease.

Effect of a small amount of sodium carbonate on konjac glucomannan-induced changes in wheat starch gel

Wheat starch gels were produced with konjac glucomannan (KGM) and low concentrations of Na2CO3 (0.1-0.2 wt% of starch) using a rapid viscosity analyzer (RVA). The gelling properties of wheat starch in varying ratios of KGM and Na2CO3 were characterized by differential scanning calorimetry (DSC), rheometry and confocal laser scanning microscopy (CLSM). A small amount of Na2CO3 resulted in gels with increased elasticity whereas structural ordering during retrogradation was insignificantly affected. Comparison of CLSM images of composite gels revealed that Na2CO3 at 0.2 wt% of starch allowed the formation of fiber-like extensions around scattered swollen granules by KGM and amylose interaction, making swollen granules disperse within the micro phase, which was not typical in CLSM images of gels in the absence of Na2CO3. Dynamic storage modulus and dynamic power law exponent were substantially higher than those observed for the same concentration of KGM in the presence of Na2CO3, supporting the hypothesis that Na2CO3 could promote strong interchain associations between KGM and starch components.

Effects of konjac glucomannan on heat-induced changes of wheat gluten structure

Effects of konjac glucomannan on the structure of wheat gluten were investigated at variable temperatures in this study. Dynamic oscillatory rheology study showed that konjac glucomannan conferred stiffness on gluten with a higher tan δ data at 25°C and 55°C, but this parameter was lower at 75°C and 95°C. Konjac glucomannan decreased the content of thiol equivalent groups and increased the α-helix/β-sheet content ratio, respectively. The thicker layer of gluten protein with 5% konjac glucomannan was observed by scanning electron microscopy. This study revealed that konjac glucomannan could alter the conformations of gluten proteins upon heating via non-covalent interactions and physical entanglements. It is likely that konjac glucomannan promotes protein aggregation by strengthening hydrophobic interaction at 25°C and 55°C, and alleviates heat-induced denaturation by decreasing the flexibility of polypeptide chain at higher 75°C and 95°C.

Prorenin Processing Enzyme (PPE) Produced by Baculovirus-Infected Sf-9 Insect Cells: PPE Is the Cysteine Protease Encoded in the AcMNPV Gene

In infection cultures of Spodoptera frugiperda (Sf-9) insect cells with a recombinant baculovirus, vhpR, carrying human preprorenin cDNA in the polyhedrin locus of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), the expressed inactive recombinant human (rh)-prorenin is reported to be proteolytically processed to yield active rh-renin in the very late phase of culture (Takahashi et al., Biosci. Biotechnol. Biochem., 71, 2610–2613 (2007)). To identify the enzyme that catalyzes the processing of rh-prorenin, referred to as prorenin processing enzyme (PPE), we purified potential PPE from virus-infected Sf-9 culture supernatant by the use of an internally quenched fluorescent (IQF) substrate for PPE. The 32-kDa protein band agreed well with PPE activity on the final Mono Q FPLC. By N-terminal amino acid sequence analysis, the protein was revealed to be a cysteine protease encoded by the AcMNPV gene. Enzyme activity was inhibited by cysteine protease inhibitors but not by other protease inhibitors. When the purified rh-prorenin was incubated with the 32-kDa protein, renin activity appeared concomitant with the disappearance of rh-prorenin. The N-terminal amino acid sequence of the activated product was identical to that of the rh-renin that had accumulated in the infection cultures. These results indicate that the 32-kDa cysteine protease derived from the AcMNPV gene is the enzyme PPE of virus-infected Sf-9 cells.

Purification and Characterization of Aspartic Protease Derived from Sf9 Insect Cells

An aspartic protease that is significantly produced by baculovirus-infected Spodoptera frugiperda Sf9 insect cells was purified to homogeneity from a growth medium. To monitor aspartic protease activity, an internally quenched fluoresce (IQF) substrate specific to cathepsin D was used. The purified aspartic protease showed a single protein band on SDS–PAGE with an apparent molecular mass of 40 kDa. The N-terminal amino acid sequence of the enzyme had a high homology to a Bombyx mori aspartic protease. The enzyme showed greatest affinity for the IQF substrate at pH 3.0 with a K m of 0.85 μM. The k cat and k cat⁄K m values were 13 s−1 and 15 s−1 μM−1 respectively. Pepstatin A proved to be a potent competitive inhibitor with inhibitor constant, K i, of 25 pM.

In vivo anti-fatigue activity of sufu with fortification of isoflavones

Background: Sufu is a traditional Chinese fermented soybean food. Isoflavones are abundant in soybean and products incorporated with isoflavones exert many health benefits. The aim of this study was to investigate anti-fatigue effect of sufu fortified with isoflavones. Materials and Methods: In vivo anti-fatigue activity of sufu with fortification of isoflavones (IF) was investigated in this study via exhaustive swimming test using ICR mice and determination of biochemical parameters. Factors relating to fatigue, including hepatic glycogen, blood lactic acid (BLA), blood urea nitrogen (BUN) were determined. The isoflavone composition in the IF sufu was also determined to explore the anti-fatigue activity of isoflavones. Results: During fermentation, isoflavone glucosides were converted into aglycones and both sufu with and without fortification of IF prolonged the exhaustive swimming time of ICR mice. Intake of sufu also increased the hepatic glycogen content, while it decreased the levels of both the blood lactic acid (BLA) and blood urea nitrogen (BUN) content. A dose-response relationship was observed in both exhaustive swimming and BLA clearance test, with medium dose (1%) fortification of IF revealing the highest activity. Conclusion: IF sufu could possess high anti-fatigue activity.

Physicochemical changes in myofibrillar proteins extracted from pork tenderloin thawed by a high-voltage electrostatic field

The application of a high-voltage electrostatic field (HVEF) is a novel method for thawing. To determine the effects of HVEF thawing (voltage range: -25kV to 0kV) on myofibrillar protein oxidation and/or denaturation and to provide a theoretical understanding of this process, pork tenderloin was thawed by traditional and HVEF methods. Based on the total sulfhydryl and carbonyl contents, further protein oxidation did not occur during HVEF thawing. It was proposed that the free radical-mediated oxidation of myofibrillar proteins was hindered by HVEF. The results of dynamic rheological analysis, protein aggregation and gel texture studies showed that HVEF thawing, especially -10kV HVEF thawing, led to better indicators than those achieved under air thawing. A higher abundance of proteins extracted from -10kV HVEF-thawed samples compared with air-thawed samples was found. Finally, this study showed that thawing under -10kV conditions did not affect the structure of myofibrillar proteins.

New insights into the antioxidant activity and components in crude oat oil and soybean oil

Developing new antioxidants and using natural examples is of current interest. This study evaluated the antioxidant activities and the ability to inhibit soybean oil oxidation of oat oil obtained with different solvents. Oat oil extract obtained by ethanol extraction gave the highest antioxidant activity with a DPPH radical (1,1-diphenyl-2-picrylhydrazyl) scavenging activity of 88.2xa0% and a reducing power (A700) of 0.83. Oat oil extracted by ethanol contained the highest polyphenol and α-tocopherol content. Significant correlation was observed between the total polyphenol contents, individual phenolic acid, α-tocopherol, and DPPH radical scavenging activity. Soybean oil with 2xa0% added oat oil showed low malondialdehyde content (8.35xa0mmolxa0mL−1), suggesting that the added oat oil inhibited oxidation. Oat oil showed good antioxidant activity, especially when extracted with ethanol which could also retard the oxidation of soybean oil . DPPH radical scavenging activity was the best method to evaluate the antioxidant activity and components of oat oil.