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BACKGROUND Taro (Colocasia esculenta cv. Daikichi) is believed to be one of the earliest cultivated tuber crops and it is a staple food in many parts of the world. The mother corm and side cormels (daughter and granddaughter tubers) form the major consumed parts; however, the former is rarely preferred. Taro is mainly cultivated using either unflooded or flooding cultivation, under dryland-rainfed and wetland-irrigated conditions, respectively. Although flooding cultivation has several advantages, such as lower risk of diseases, weeds, and insect pests, contributing to increased tuber yield, its effects on the quality characteristics of the tubers are largely unknown. In this study, the effects of controlled flooding cultivation on the quality of mother corm and side cormels were investigated. Their taste, color, physical properties, antioxidant activity, and starch, oxalic acid, nitrate ion, arabinogalactan (AG)/AG protein (AGP), γ-aminobutyric acid (GABA), and total polyphenol content was compared with those under unflooded cultivation. RESULTS Flooding cultivation increased polyphenol levels and antioxidant activity and decreased oxalate, nitrate ion, GABA, and AG/AGP levels. Flooding cultivation also reduced the harshness and increased the hardness and stickiness of steamed mother corm paste, generally discarded under unflooded cultivation, thus rendering it suitable for consumption. CONCLUSION Controlled flooding cultivation has economic advantages and the potential to improve the quality of cultivated taro. © 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Mulberry (Morus australis Poir.) leaves have long been consumed in the form of tea or tincture especially in Asia, owing to their high antioxidant and blood pressure-regulating properties. Although it is thought that vascular abnormal contraction may be involved in the blood pressure-suppressing effect, the effect of mulberry on vascular abnormal contraction is still unknown. Therefore, we investigated mulberry leaves as a potential source of bioactive compounds that prevent vascular abnormal contraction. Mulberry leaves were divided into fresh leaves and tea leaves and further classified according to the age of the tree: more or less than 20 years old, into roasted and unroasted. Mulberry fruits were also evaluated. We assessed the preventive effect of mulberry extracts on vascular abnormal contraction. Extracts from mulberry leaves of trees more than 20 years old showed a strong preventive effect on vascular abnormal contraction of human coronary artery smooth muscle cells. Therefore, to identify the active components in mulberry leaves, we fractionated the active fractions by gel filtration chromatography and reversed-phase high-performance liquid chromatography. The active fraction was further analyzed by mass spectrometry and nuclear magnetic resonance; an active component of the mulberry leaf extract was fisetin. In addition, our results indicated that the hydroxyl group at the C-3 position of fisetin is crucial for its activity. These results prove that fisetin is effective in preventing vascular abnormal contraction. Overall, mulberry leaves and fisetin are expected to be used in a wide range of fields such as functional foods, nutraceuticals, and drug targets.

The catechin content in green tea leaves varies according to cultivation conditions such as intensity of solar radiation, temperature, and precipitation, and thus, there is ambiguity about the best harvest time for obtaining optimal functional effects. In this study, the Yabukita (ordinary) and Benifuki varieties, which contain methylated catechin, were used to determine the difference in green tea catechins according to harvest times and tea manufacturing processes. Caffeine determination was also carried out to provide information about green tea intake for all age‐groups of children and pregnant women. Determining the quantity of each catechin was difficult because of degradation, polymerization, and isomerization that had occurred during heat‐drying in the refining process. In addition, the absorption of catechin compounds was tested using miniature swine because of their functional and physiological similarity to humans. Benifuki tea leaves contained epigallocatechin‐3‐(3”‐O‐methyl) gallate (EGCg3”Me) instead of epigallocatechin‐3‐(4”‐O‐methyl) gallate (EGCg4”Me). However, EGCg4”Me was detected during the entire intake period, but EGCg3”Me was not detected in the blood of miniature swine fed Benifuki tea. It is possible that the position of the methyl group was modified by the pig metabolism. Furthermore, caffeine from both Yabukita and Benifuki tea varieties was found to be easily accumulated in miniature swine. These results suggest that nonrefined September–October picking tea (autumn and winter tea) of the Benifuki variety is preferable over the Yabukita variety for consumption by children and pregnant women owing to its lower caffeine content and higher content of methylated catechin.

Vascular disease poses a major public health problem worldwide. Trigonelline isolated from Raphanus sativus cv. Sakurajima Daikon (Sakurajima radish) induces nitric oxide production from vascular endothelial cells and enhances vascular function. Here, we investigated the characteristics of trigonelline and its effects on endothelial function after consumption of Sakurajima radish by humans. Our results show that Sakurajima radish contains approximately 60 times more trigonelline than other radishes and squashes. Additionally, no significant differences were observed between varieties of Sakurajima radish, suggesting that any type of Sakurajima radish can be ingested for trigonelline supplementation. The effects of cooking and processing Sakurajima radish were also evaluated, as were the effects of freezing, and changes in osmotic pressure and pH. A first-in-human trial using Sakurajima radish showed that ingestion of 170 g/day of Sakurajima radish for ten days increased blood trigonelline concentrations and significantly improved flow-mediated dilation, which is a measure of vascular endothelial function. Overall, our findings suggest that the trigonelline contained in Sakurajima radish may contribute to improved human vascular endothelial function. Hence, Sakurajima radish may enhance vascular endothelial function as a functional food.

Purpose Mushroom waste medium can be used as a source of enzymes, but few studies on animal feed improvement using it have been found. In this study, the potential as additives for improving feed efficiency was examined. Extracts from waste media containing shochu lees used for growing three mushrooms cultivation: monkey head mushroom (MHM) Hericium erinaceus, oyster mushroom Pleurotus ostreatus, and shiitake mushroom Lentinula edodes were investigated. Methods Several polysaccharide hydrolase activities in the extracts were measured and the following feedstuffs were partially digested by the extracts: Italian ryegrass, rice straw, and sweet potato runner. Digestibility of neutral detergent fiber (NDF) in the three feedstuffs was compared by the analysis of sugar composition of residual NDF after hydrolysis with sulfuric acid and trifluoroacetic acid. Antioxidant activity of the extract from waste media was also determined by DPPH radical method. Results The polysaccharide hydrolase activity differed depending on the kind of mushroom and waste medium composition. The waste media containing sweet potato shochu lees showed higher enzyme activities than the standard media did. The additives reduced NDF content in the feedstuffs, especially in treating sweet potato runner with MHM extract. MHM remarkably decreased xylose in NDF of rice straw, and glucose and xylose in NDF of sweet potato runner. Conclusions The results suggested that the additives might be effective in improving the efficiency of the feedstuffs for heat-stressed or weakened livestock.

Vascular diseases, such as myocardial and cerebral infarctions, are the leading causes of death. Some vascular diseases occur as the result of decreases in vascular endothelial function. The innermost layer of the vasculature is formed by vascular endothelial cells (VECs), which are critical for nitric oxide (NO) synthesis. In our search for active constituents in farm products with the potential for improving the vascular system, we examined the effect of Raphanus sativus cv. Sakurajima Daikon on NO production in VECs. In this study, we found that the underlying mechanism for stimulating NO production by Sakurajima Daikon extract involves endothelial-NO-synthase (eNOS) activation by the phosphorylation of Ser1177 and the dephosphorylation of Thr495, which are triggered by elevated concentrations of cytoplasmic Ca2+ resulting from the activation of Ca2+ channels in VECs. We observed that trigonelline, an active constituent of Sakurajima Daikon, improves NO production in VEC cultures.

Growing crops in sabulous soils is often challenging due to their limited oligotrophy and weak water retention. However, some plants adapt to these adverse growth conditions, and in some cases, favorable properties are imparted to the fruit. This study investigated the influence of the cultivation environment on Capsicum by assessing the levels and functions of both pungent components and antioxidants when cultivated in sandy soils in Southern Japan; these parameters were then compared to those in traditional tropical-origin Capsicum. In seven varieties of Capsicum, the distribution of capsaicin and dihydrocapsaicin differed between the pericarp and seeds within the placenta. The leaves and fruits of Habanero orange and Tabasco, which are the most suitable for cultivation in sandy soil, were collected during the cultivation period and analyzed in terms of their size, color, and pungent component composition. Pungent components were detected in fruits only, and not in leaf or flower samples. In particular, we found that pungent components were generally present within the seeds and placenta. Antioxidant activity and nitric oxide production within human vascular endothelial cells were also evaluated to compare the differences in their functionality. Satsuma-Habanero orange cultivated under adverse conditions possessed the highest antioxidant activity. Furthermore, Satsuma-Capsicum cultivated under adverse conditions exhibited higher levels of antioxidants than traditional tropical-origin peppers, and induced similar levels of nitric oxide production in the vascular endothelial cells. We concluded that Capsicum cultivated in harsh environments produced beneficial effects such as higher levels of antioxidants and capsaicinoids in seeds and placenta. Moreover, the fruits from these plants could be harvested for a significantly longer period and took longer to spoil than traditional Capsicum; thus, they show merit as a viable commercial crop in Japan.

The pericarp of passion fruit is typically discarded before consumption. We aimed to demonstrate that pigments extracted from the pericarp could be used as natural colourants. We focused on the stability of the dyes in response to heat, light, and storage, which is the most pressing problem associated with natural colourants. The stability of the pigment in solutions was affected by certain factors. However, colour measurements confirmed that for test samples in powder form or those covered by a coloured commercial plastic bag or light shield, the pigments remain stable with no noticeable colour change. The pigment extracted from the pericarp included anthocyanins such as cyanidin-3-glucoside, peonidin-3-glucoside, callistephin, and cyanidin-3-rutinoside, which have 3-O-glycoside bonding of the C-ring. They were not found in the passion fruit pulp or seeds. The colour value of the pigment was lower than that of the same amount of red cabbage dye or purple sweet potato dye, which are typically used in food manufacturing. The pigment extracted from passion fruit pericarp was used to dye processed food such as jelly, which was coloured pink to red depending on the amount of pigment used. The passion fruit pericarp could be used as a low-cost natural red colourant.

Growing crops in sabulous soil is challenging owing to limited oligotrophy and low water retention. Nonetheless, some plants adapt well, imparting favorable properties to the fruit. This study investigated the influence of sandy soil (southern Japan) on red pepper by assessing the levels of pungent components, antioxidant activity, and vascular endothelial function. Leaves and fruits of Habanero orange and Tabasco pepper, the two varieties most suitable for cultivation in sandy soil, were analyzed for size, color, and pungent component composition. Pungent components were detected in the seeds and placenta of fruits but not in leaves or flowers. Antioxidant activity and nitric oxide production in human vascular endothelial cells were evaluated to detect differences in functionality. Capsicum peppers cultivated in sandy soil exhibited higher levels of antioxidants than peppers cultivated under nutrient-rich conditions (control) and induced nitric oxide levels in vascular endothelial cells similar to control peppers. Especially Satsuma-Habanero orange peppers cultivated in sandy soil exhibited the highest antioxidant activity. The fruits from pepper plants cultivated in sabulous soil could be harvested for a significantly longer period and were slower to spoil than control peppers; therefore, Satsuma-Capsicum plants may be commercially viable in oligotrophic areas.

The root bark of Melia toosendan afforded four new limonoids with a C-19/C-29 bridged acetal structure, together with two known limonoids, 12α-hydroxyamoorastatone and its 12-acetate. The new compounds were elucidated as 1-O-acetyltrichilin H and 29-O-substituted amoorastatone derivatives, named neoazedarachins A, B and D, by spectroscopic and chemical means. Their antifeedant activity was also studied.

A new mannose-recognizing lectin (MOL) was purified on an asialofetuin-column from fruiting bodies of Marasmius oreades grown in Japan. The lectin (MOA) from the fruiting bodies of the same fungi is well known to be a ribosome-inactivating type lectin that recognizes blood-group B sugar. However, in our preliminary investigation, MOA was not found in Japanese fruiting bodies of M. oreades, and instead, MOL was isolated. Gel filtration showed MOL is a homodimer noncovalently associated with two subunits of 13 kDa. The N-terminal sequence of MOL was blocked. The sequence of MOL was determined by cloning from cDNA and by protein sequencing of enzyme-digested peptides. The sequence shows mannose-binding motifs of bulb-type mannose-binding lectins from plants, and similarity to the sequences. Analyses of sugar-binding specificity by hemagglutination inhibition revealed the preference of MOL toward mannose and thyroglobulin, but asialofetuin was the strongest inhibitor of glycoproteins tested. Furthermore, glycan-array analysis showed that the specificity pattern of MOL was different from those of typical mannose-specific lectins. MOL preferred complex-type N-glycans rather than high-mannose N-glycans.

Antimicrobial peptides are important components of the host innate immune responses by exerting broad-spectrum microbicidal activity against pathogenic microbes. Cy-AMP1 found in the cycad (Cycas revoluta) seeds has chitin-binding ability, and the chitin-binding domain was conserved in knottin-type and hevein-type antimicrobial peptides. The recombinant Cy-AMP1 was expressed in Escherichia coli and purified to study the role of chitin-binding domain. The mutants of Cy-AMP1 lost chitin-binding ability completely, and its antifungal activity was markedly decreased in comparison with native Cy-AMP1. However, the antimicrobial activities of the mutant peptides are nearly identical to that of native one. It was suggested that the chitin-binding domain plays an essential role in antifungal, but not antimicrobial, activity of Cy-AMP1.

Novel antimicrobial peptides (AMP), designated Cy-AMP1, Cy-AMP2, and Cy-AMP3, were purified from seeds of the cycad (Cycas revoluta) by a CM cellulofine column, ion-exchange HPLC on SP COSMOGEL, and reverse-phase HPLC. They had molecular masses of 4583.2 Da, 4568.9 Da and 9275.8 Da, respectively, by MALDI-TOF MS analysis. Half of the amino acid residues of Cy-AMP1 and Cy-AMP2 were cysteine, glycine and proline, and their sequences were similar. The sequence of Cy-AMP3 showed high homology to various lipid transfer proteins. For Cy-AMP1 and Cy-AMP2, the concentrations of peptides required for 50% inhibition (IC(50)) of the growth of plant pathogenic fungi, Gram-positive and Gram-negative bacteria were 7.0-8.9 microg/ml. The Cy-AMP3 had weak antimicrobial activity. The structural and antimicrobial characteristics of Cy-AMP1 and Cy-AMP2 indicated that they are a novel type of antimicrobial peptide belonging to a plant defensin family.

Ovalbumin is a serpin without inhibitory activity against proteases. During embryonic development, ovalbumin in the native (N) form undergoes changes and takes a heat-stable form, which was previously named HS-ovalbumin. It has been known that N-ovalbumin is artificially converted to another thermostable form called S-ovalbumin by heating at an alkaline pH. Here, we characterized further the three ovalbumin forms, N, HS, and S. The epitope of the monoclonal antibody 2B3/2H11, which recognizes N- and HS-ovalbumin but not S-ovalbumin, was found to reside in the region Glu-Val-Val-Gly-Ala-Ser-Glu-Ala-Gly-Val-Asp-Ala-Ala-Ser-Val-Ser-Glu-Glu-Phe-Arg, which corresponds to 340-359 of amino acid residues and is contained in the reactive center loop (RCL). Removal of RCL by elastase or subtilisin mitigated binding of the antibody. Dephosphorylation experiments indicated that the phosphorylated Ser-344 residue located on RCL is crucial for the epitope recognition. We suggest that the shift to the heat-stable form of ovalbumin accompanies a movement of RCL.

Cycas revoluta leaf lectin (CRLL) of mannose-recognizing jacalin-related lectin (mJRL) has two tandem repeated carbohydrate recognition domains, and shows the characteristic sugar-binding specificity toward high mannose-glycans, compared with other mJRLs. We expressed the N-terminal domain and C-terminal domain (CRLL-N and CRLL-C) separately, to determine the fine sugar-binding specificity of each domain, using frontal affinity chromatography, glycan array and equilibrium dialysis. The specificity of CRLL toward high mannose was basically derived from CRLL-N, whereas CRLL-C had affinity for α1-6 extended mono-antennary complex-type glycans. Notably, the affinity of CRLL-N was most potent to one of three Man 8 glycans and Man 9 glycan, whereas the affinity of CRLL-C decreased with the increase in the number of extended α1-2 linked mannose residue. The recognition of the Man 8 glycans by CRLL-N has not been found for other mannose recognizing lectins. Glycan array reflected these specificities of the two domains. Furthermore, it was revealed by equilibrium dialysis method that the each domain had two sugar-binding sites, similar with Banlec, banana mannose-binding Jacalin-related lectin.

The inhibitory activity of six groups of flavonoids against yeast and rat small intestinal alpha-glucosidases and porcine pancreatic alpha-amylase was compared, and chemical structures of flavonoids responsible for the inhibitory activity were evaluated. Yeast alpha-glucosidase was potently inhibited by the anthocyanidin, isoflavone and flavonol groups with the IC50 values less than 15 microM. The following structures enhanced the inhibitory activity: the unsaturated C ring, 3-OH, 4-CO, the linkage of the B ring at the 3 position, and the hydroxyl substitution on the B ring. Rat small intestinal alpha-glucosidase was weakly inhibited by many flavonoids, and slightly by the anthocyanidin and isoflavone groups. 3-OH and the hydroxyl substitution on the B ring increased the inhibitory activity. In porcine pancreatic alpha-amylase, luteolin, myricetin and quercetin were potent inhibitors with the IC50 values less than 500 microM. The 2,3-double bond, 5-OH, the linkage of the B ring at the 3 position, and the hydroxyl substitution on the B ring enhanced the inhibitory activity, while 3-OH reduced it.

We succeeded in the in situ observation of liquid-liquid phase separation of glass under microgravity at the Japan Microgravity Center (JAMIC), which provides a 10 s microgravity duration. The microgravity level is 10 - 5 g (g ≡ gravitational field at Earth's surface). We constructed an apparatus for the observation of phase separation suitable for the experimental facility. The melt of 3BaO.97B 2 O 3 (mol%) was held as a film on a Pt wire loop. The sample was initially heated above the immiscibility temperature and held at that temperature. After the homogenization stage, the melt was cooled. Liquid-liquid phase separation of the melt was observed directly with a video camera. It was found that the nucleation and growth of phase-separated particle under microgravity are slower than in the case of 1-g, and the delay time of phase separation between the case of microgravity and 1-g depends on the flow rate of the melt under microgravity. These observations could be interpreted in terms of Marangoni convection and a change of diffusion.

Novel antimicrobial peptides (AMP), designated Fa-AMP1 and Fa-AMP2, were purified from the seeds of buckwheat (Fagopyrum esculentum Moench.) by gel filtration on Sephadex G75, ion-exchange HPLC on SP COSMOGEL, and reverse-phase HPLC. They were basic peptides having isoelectric points of over 10. Fa-AMP1 and Fa-AMP2 had molecular masses of 3,879 Da and 3,906 Da on MALDI-TOF MS analysis, and their extinction coefficients in 1% aqueous solutions at 280 nm were 42.8 and 38.9, respectively. Half of all amino acid residues of Fa-AMP1 and Fa-AMP2 were cysteine and glycine, and they had continuous sequences of cysteine and glycine. The concentrations of peptides required for 50% inhibition (IC50) of the growth of plant pathogenic fungi, and Gram-positive and -negative bacteria were 11 to 36 microg/ml. The structural and antimicrobial characteristics of Fa-AMPs indicated that they are a novel type of antimicrobial peptides belonging to a plant defensin family.

A new method is proposed to improve the adsorption properties of basic, macroreticular (MR) anion exchange resins by forming a material composite with chitosan. In this study, one strong basic and one weak basic MR resins were used as matrixes for the new composites. Chitosan was accumulated in the macropores of the resins, and breakthrough curves for adsorption of CO2 on the new adsorbents were measured. The resulting equilibrium and kinetic data were compared with those of the original resins. The composite of strong basic MR resin and chitosan showed a more than three times higher adsorption capacity for CO2 than the pure resin. On the other hand, the mass transport in the adsorbent slowed down due to a narrowing of the pores by deposited chitosan.

Two jacalin-related lectins (JRLs) were purified by mannose-agarose and melibiose-agarose from seeds of Treculia africana. One is galactose-recognizing JRL (gJRL), named T. africana agglutinin-G (TAA-G), and another one is mannose-recognizing JRL (mJRL), TAA-M. The yields of the two lectins from the seed flour were approximately 7.0 mg/g for gJRL and 7.2 mg/g for mJRL. The primary structure of TAA-G was determined by protein sequencing of lysyl endopeptic peptides and chymotryptic peptides. The sequence identity of TAA-G to other gJRLs was around 70%. Two-residue insertion was found around the sugar-binding sites, compared with the sequences of other gJRLs. Crystallographic studies on other gJRLs have shown that the primary sugar-binding site of gJRLs can accommodate Gal, GalNAc, and GalNAc residue of T-antigen (Galβ1-3GalNAcα-). However, hemagglutination inhibition and glycan array showed that TAA-G did not recognize GalNAc itself and T-antigen. TAA-G preferred melibiose and core 3 O-glycan.

Effects of chemical modifications on the protein-synthesis inhibitory (PSI) activities of momordin-a and luffin-a were investigated. Treatment with a 50-fold excess of diethylpyrocarbonate at pH 6.5 modified one histidine residue in momordin-a and luffin-a and reduced their PSI activities to 10% and 8.3%, respectively. Modifications with a 20-fold excess of KI3 at pH 7.0 at 0 degree C greatly reduced their PSI activities to 10% by iodination of nearly one tyrosine residue. The PSI activity of momordin-a was rapidly reduced to 6.4% by the modification of one lysine residue with trinitrobenzensulfonic acid as in the case of luffin-a reported previously. By analyses of the tryptic peptides from the modified momordin-a and luffin-a, the modified residues were identified as His140, Tyr165, and Lys231. Furthermore, the amounts of three modified momordin-a binding to rat liver ribosomes were reduced to about half or less than half of that of native momordin-a. From these results, it was suggested that His140, Tyr165, and Lys231 are highly exposed on the surface of momordin-a and luffin-a molecules and are involved in their PSI activities, probably by binding to ribosomes.

A protein-synthesis inhibitor, designated RPSI, was isolated from the seeds of rye (Secale cereale) using gel filtration and S-Sepharose column chromatography. RPSI is a basic protein with an isoelectric point of over 10, and the concentration of protein required for 50% inhibition of protein synthesis (IC50) of purified RPSI was about ten-fold the concentration of ricin A-chain. The complete amino acid sequence of RPSI was discovered by analyzing the peptides and fragments obtained from the proteolytic digests and by the cyanogen bromide- and hydroxylamine-cleavages of RPSI. RPSI consists of 280 amino acid residues and has a molecular weight of 30,171. RPSI has only 21% sequence identity with that of ricin A-chain, but all five amino acid residues involved in the active site of ricin A-chain are conserved in RPSI.

Three different types of ring C-seco limonoids, 3-O-acetylohchinolal, ohchinolide C and nimbolidin F, were isolated as insect antifeedants from Melia toosendan along with three known limonoids, salannin, azadirone and acetyltrichilenone, and a known protolimonoid, 20, 21, 22, 23-tetrahydro-23-oxotrichilenone. Their structures were elucidated by spectroscopic studies and their antifeedant properties were examined with the larvae of Spodoptera eridania.

A lectin was isolated from fruiting bodies of Agrocybe cylindracea by two ion-exchange chromatographies and gel filtration on Toyopearl HW55F. The lectin was homogeneous on polyacrylamide gel electrophoresis and its molecular mass was determined to be 30 000 by gel filtration, and 15 000 by sodium dodecylsulfate polyacrylamide gel electrophoresis, signifying a dimeric protein.

Since November 1994 comprehensive field study has been performed in the Ara-River estuary three times to simultaneously measure the hydraulic and water-quality indices for 24 consecutive hours. The data are here analyzed to ultimately establish a method to protect the water quality in the area. For this purpose special emphasis is laid on the flux of water-quality indices, and the water-quality management is proposed with respect to such indices as COD, DO and TN that can be measured instantaneously.

A galactose specific lectin (CpL) was purified from the Clavaria purpurea mushroom by affinity chromatography. CpL agglutinated only trypsin-treated rabbit erythrocytes. On enzyme linked lectin sorbent assay (ELLSA), the lectin bound with thyroglobulin and asialo bovine submaxillary mucin (BSM). The fine sugar binding specificity of CpL was elucidated using inhibition of hemagglutination and sugar immobilized gold nano-particles (SGNP). The results indicated a preference of CpL towards α-galactosyl sugar chains. Among several monosaccharides and disaccharides assayed for dissociation effect on the SGNP-CpL complex, Galα1-3Gal and raffinose were the best inhibitors. The partial amino acid sequence showed two QXW motifs in CpL and similarity towards members of the ricin B superfamily.

A new galectin was characterized in the Amethyst deceiver mushroom Laccaria amethystina. The complete amino acid (AA) sequence of the lectin, which exhibited β-galactoside specificity, was deduced from its peptide sequences. The AA sequence of L. amethystina galectin (LAG) showed high homology with those of the same genus, at 75.6% identity to Laccaria bicolor, and 35.5-65.0% to galectins of Agrocybe spp. and Coprinopsis cinerea. The AA sequence of LAG contained all but one conserved residue known to be involved in β-galactoside binding, with His at the position 57 residue replaced by Thr in LAG. Analysis of binding specificity by hemagglutination inhibition assay and enzyme-linked lectin-sorbent assay revealed high specificity of LAG towards O-glycoproteins.

A terahertz chemical microscope (TCM) has been carried out to visualize the chemical potential distribution in μ-TAS. Since the TCM can detect the chemical potential at exactly where the femtosecond laser was irradiated, the spatial resolution less than 1μm, which is limited by the wavelength of the femtosecond laser, can be achieved. The μ-TAS was fabricated on the sensing chip and the distribution of chemical potential was successfully visualized using TCM.

Terahertz chemical microscope was carried out to visualize the distribution of the redox reaction of the immobilized-enzyme. The urease films with the spot size of 3, 2, 1 mm in diameter were immobilized on a SiO 2 /Si/Sapphire layered device and the laser-excited terahertz radiation from the Si layer was observed, which can be related to the chemical potential shift of the SiO 2 surface. The redox reaction between the urease and the urea solution was successfully measured.

Antimicrobial peptides are important components of the host innate immune responses by exerting broad-spectrum microbicidal activity against pathogenic microbes. Cy-AMP1 found in the cycad (Cycas revoluta) seeds has chitin-binding ability, and the chitin-binding domain was conserved in knottin-type and hevein-type antimicrobial peptides. The recombinant Cy-AMP1 was expressed in Escherichia coli and purified to study the role of chitin-binding domain. The mutants of Cy-AMP1 lost chitin-binding ability completely, and its antifungal activity was markedly decreased in comparison with native Cy-AMP1. However, the antimicrobial activities of the mutant peptides are nearly identical to that of native one. It was suggested that the chitin-binding domain plays an essential role in antifungal, but not antimicrobial, activity of Cy-AMP1.

Three new proteins which inhibit protein synthesis in rabbit reticulocyte lysates were isolated from an extract of sponge gourd (Luffa cylindrica) seeds by chromatography on a AF-Blue Toyopearl column followed by FPLC with a Mono S column. These three protein-synthesis inhibitory proteins (PSIs) have molecular masses of 19 kDa, 15 kDa, and 9 kDa, and were designated 19K-PSI, 15K-PSI, and 9K-PSI, respectively. Although the 19K-PSI had no effect on protein synthesis in HeLa cells, its inhibitory activity on the cell-free protein synthesis was 340- and 83-fold stronger than those of ricin A-chain and luffin-a, respectively, probably due to hydrolyzing mRNA. The inhibitory activities of 15K- and 9K-PSIs on the cell-free protein synthesis were weaker than those of ricin A-chain and luffin-a. The 19K-PSI was a glycoprotein having an ordinary amino acid composition, three intramolecular disulfide bonds and a blocked N-terminal residue, while the 15K-PSI was extraordinarily rich in glycine and the 9K-PSI in arginine and glutamic acid (and/or glutamine). The amino acid composition of 19K-PSI was: Ser27Glx3Gly164Tyr7Lys9His6, and that of 9K-PSI was: Asx3Glx25Pro2Gly5Lys2His2Arg25Trp3.

An antimicrobial peptide, designated Pa-AMP, was purified by gel filtration on Sephadex G-75 followed by S-Sepharose, Cosmosil-SP, and reverse-phase HPLC from the seeds of pokeweed (Phytolacca americana). Pa-AMP is a basic peptide having an isoelectric point of over 10 and its extinction coefficient at 280 nm of 1% aqueous solution was 7.7. Pa-AMP has a molecular mass of 4 kDa and 3.4 kDa on tricine SDS-PAGE under nonreducing and reducing conditions, respectively. The N-terminal amino acid of Pa-AMP was blocked. The concentrations of peptide required for 50% inhibition (IC50) of the growth of plant pathogenic fungi, Gram-positive, and Gram-negative bacteria were 3 to 41 μg/ml. Differing from other peptides, Pa-AMP inhibited the growth of some Gram-negative bacteria.

Novel antimicrobial peptides (AMP), designated Tu-AMP 1 and Tu-AMP 2, were purified from the bulbs of tulip (Tulipa gesneriana L.) by chitin affinity chromatography and reverse-phase high-performance liquid chromatography (HPLC). They bind to chitin in a reversible way. They were basic peptides having isoelectric points of over 12. Tu-AMP 1 and Tu-AMP 2 had molecular masses of 4,988 Da and 5,006 Da on MALDI-TOF MS analysis, and their extinction coefficients of 1% aqueous solutions at 280 nm were 3.3 and 3.4, respectively. Half of all amino acid residues of Tu-AMP 1 and Tu-AMP 2 were occupied by cysteine, arginine, lysine, and proline. The concentrations of peptides required for 50% inhibition (IC(50)) of the growth of plant pathogenic bacteria and fungi were 2 to 20 microg/ml. The structural characteristics of Tu-AMP 1 and Tu-AMP 2 indicated that they were novel thionin-like antimicrobial peptides, though Tu-AMP 2 was a heterodimer composes of two short peptides joined with disulfide bonds.

An attempt to synthesize 6-hydroxypyridoxine (OPN), hydroxylation on C-6 of pyridoxine (PN) by hydroxyl radical (OH*). was conducted. Application of two well-known OH* generating reactions, i.e. the Fenton reaction and the Fe2+-EDTA/ascorbate reaction, were unsuccessful, as large amounts of by-products were formed. Although generation of OH* by autoxidation of ascorbic acid in the absence of metal ions was slow, by-products were formed in small quantities, and OPN was easily obtained in colorless crystals. Its structure was confirmed by spectral analyses. OPN was comparable to polyphenols such as (+)-catechin, rutin and gallic acid in the antioxidative activity against linoleic acid peroxidation, and was an effective DPPH radical scavenger, though the DPPH radical-scavenging activity of OPN was somewhat lower than that of the polyphenols. PN was relatively inactive under the conditions used here, indicating that the introduction of a hydroxyl group on C-6 of PN greatly enhanced both activities.

Calluses were produced from plant tissues of Japanese cycad ( Cycas revoluta Thunb.). Its toxic component named cycasin, β-glucoside of methylazoxymethanol, was formed by the calluses only in the presence of exogenous methylazoxymethanol. A glucosyltransferase catalysing the formation of cycasin from UDP-glucose and methylazoxymethanol occurred in the calluses. The properties of this enzyme in embryo-derived callus and in leaf tissue were similar.

There was an ionic interaction between acidic polysaccharides (APS) and proteins at the pH range in which APS were negatively charged and proteins were positively charged, and in enzymes the interaction was detected as a change in the enzyme activity. At pH 4.7, acid phosphatase (pI, 5.4), alpha-glucosidase (pI, 5.7), and beta-glucosidase (pI, 7.3) were inhibited by APS to various extents. On the other hand, alpha-glucosidase and alkaline phosphatase (pI, 4.5) were not inhibited by APS at pH 6.8 and 9.8, respectively, most of these two enzymes being negatively charged at the respective pHs. Sulfated polysaccharides combined with hemoglobin (pI, 6.8 to approximately 7.0) by an ionic bond at pH 2 to make hemoglobin unsusceptible to proteolysis by pepsin, but polyuronides which were not charged at this pH did not affect hydrolysis of hemoglobin.