Yoji Kato

Involvement of terminal-arabinose and -galactose pectic compounds in mealiness of apple fruit during storage

Abstract Among eight apple cultivars (Malus domestica Borkh) held at 0 or 20°C for 4 or 8 weeks, mealiness was highest in ‘Starking Delicious’ (83%) and lowest in ‘Golden Delicious’ (0%) after 20°C for 8 weeks. In ‘Starking Delicious’ and ‘Golden Delicious’ fruit, total uronic acid contents [amount of water (WSP)+hexametaphosphate (HMP)+HCl soluble (HCP) pectic fractions] of the alcohol insoluble solids (AIS) of flesh hardly changed during storage at 20°C, although the ratio of uronic acid in WSP to total uronic acid increased during storage. Total (WSP+HMP+HCP) pentose contents in AIS decreased in most cultivars during storage, and the decrease was greatest in the fruit stored at 20°C. In ‘Starking Delicious’ fruit, the ratio of pentose in WSP to total pentose increased during storage at 20°C. Among pentoses, arabinose (Ara) content in the fruit stored at 20°C decreased more than in the fruit stored at 0°C. The linkages of the arabinose residue in each fraction were mainly T (terminal)-Ara, 5-Ara and 3,5-Ara, determined by methylation analysis. The ratio of T-Ara and 3,5-Ara to total arabinose linkage decreased both in HMP and HCP. The decrease in T-Ara and 3,5-Ara was higher in ‘Starking Delicious’ fruit compared with that in ‘Golden Delicious’ fruit in storage at 20°C. The linkages of the galactose residues in each fraction identified were mainly T-Gal, 3,6-Gal, 2,4-Gal and 6-Gal. The ratio of T-Gal to total galactose linkage increased both in HMP and HCP. The increase in T-Gal was higher in ‘Starking Delicious’ fruit than in ‘Golden Delicious’ fruit in storage at 20°C. The possible effect on mealiness of the release of T-Ara and increase of T-Gal in pectic substances is discussed.

Involvement of Phenolic Esters in Cell Aggregation of Suspension-Cultured Rice Cells

Fluorescence microscopy of rice (Oryza sativa L.) callus sections showed that all of the walls fluoresced blue in water (pH 5.8) and green in ammonia (pH 10.0), both characteristics of feruloyl esters. Such fluorescence in the walls of cells cultured in Gamborgs B5 medium was much stronger than that in amino acid (AA) medium. Laser scanning microscopy showed that the level of fluorescence was higher in the intercellular layer, especially at corner junctions between cells, suggesting that ferulic acid ester derivatives are located in the middle lamella as well as in the wall. Extracellular polysaccharides appearing during cultivation in AA medium were more highly feruloylated than those in B5 medium during cultivation. Both the levels of ferulic and diferulic acid and the relative proportion of diferulic acid in the walls of cells increased on transfer of the cells cultured in AA medium to B5 medium. The walls of cells cultured in B5 medium maintained constant levels and proportions of the phenolic acids. Removal of phenolic acids from wall preparations by carboxylesterase facilitated the solubilization of noncellulosic polysaccharides. Treatment of the cell aggregates grown in AA medium with an enzyme that hydrolyzes feruloyl esters decreased the size of the aggregates to between 20 and 500 [mu]m, compared with an original size between 200 and 1000 [mu]m. These findings suggest that feruloyl and diferuloyl esters between polysaccharides are involved in the aggregation of cultured rice cells.

Identification of proteoglycan from salmon nasal cartilage.

There has been no structural information about the core protein of salmon nasal cartilage proteoglycan although its physiological activities have been investigated. Internal amino acid sequencing using nano-LC/MS/MS revealed that the salmon proteoglycan was aggrecan. Primer walk sequencing based on the amino acid information determined that the salmon aggrecan cDNA is comprised of 4207bp nucleotides predicted to encode 1324 amino acids with a molecular mass of 143,276. It exhibited significant similarities to predicted pufferfish aggrecan, zebrafish similar to aggrecan, zebrafish aggrecan, bovine aggrecan and human aggrecan isoform 2 precursor; whose amino acid identities were 56%, 55%, 49%, 31% and 30%, respectively. Salmon cartilage aggrecan had globular domains G1, G2 and G3 as in mammalian aggrecans. Neither the putative keratan sulfate attachment domain enriched with serine, glutamic acid and proline, nor the putative chondroitin sulfate attachment domain with repeating amino acid sequence containing serine-glycine, found in mammalian aggrecans were observed in salmon, however, random serine-glycine (or glycine-serine) sequences predicted to the sugar chain attachment sites were observed. Based on cDNA analysis and amino acid analysis after β-elimination, the ratio of serine attached to sugar chains was calculated to be approximately 37.7% of total serine, that is, 46 of 123 serine residues.

Anti-aging effects of high molecular weight proteoglycan from salmon nasal cartilage in hairless mice

Proteoglycans comprise a family of complex macromolecules consisting of a core protein with covalently attached glycosaminoglycan (GAG) chains. The skin anti-aging effects of oral administration of proteoglycan fractions with different molecular weights from salmon nasal cartilage were investigated in a hairless mouse model of skin aging; aging was caused by repeated ultraviolet B (UVB) irradiation. Three proteoglycan fractions of different molecular weights were prepared from salmon nasal cartilage water extract by ion-exchange column chromatography and gel filtration column chromatography. Physiological and histological analysis of the skin indicated that oral administration of high molecular weight proteoglycan inhibited UVB-induced skin aging, defined as increased erythema, increased transepidermal water loss (TEWL), decreased hydration, and epidermal and dermal hypertrophies. The serum and dorsal skin inflammatory cytokine levels indicated that high molecular weight proteoglycan acts on gut immunity and improves skin by inhibiting surplus inflammatory cytokines produced by UVB irradiation. These results suggest that high molecular weight proteoglycan from salmon nasal cartilage is effective in preventing skin aging.

Anti-aging effects of extracts prepared from salmon nasal cartilage in hairless mice

The skin anti-aging effects of orally administered salmon nasal cartilage extract (SNCE), which includes abundant proteoglycan, were investigated using a hairless mouse skin-aging model, in which aging was caused by repetitive ultraviolet B (UV-B) irradiation. Physiological analysis of the skin surface following repetitive UV-B irradiation of 8xa0weeks revealed inhibition of erythema levels and reduction of transepidermal water loss (TEWL) due to oral administration of SNCE. Similarly, inhibitory actions of epidermal and dermal hypertrophy were revealed by hematoxylin and eosin staining. Furthermore, effects on the hydration level of the skin surface by SNCE administration were indicated at 4xa0weeks of UV-B irradiation, but greater effects were not apparent. These results indicate that SNCE may serve as an anti-aging agent for healthy skin.

Filamentous fungus Aspergillus oryzae has two types of α-1,2-mannosidases, one of which is a microsomal enzyme that removes a single mannose residue from Man9GlcNAc2

Abstractα-Mannosidase activities towards high-mannose oligosaccharides were examined with a detergent-solubilized microsomal preparation from a filamentous fungus, Aspergillus oryzae. In the enzymatic reaction, the pyridylaminated substrate Man9GlcNAc2-PA was trimmed to Man8GlcNAc2-PA which lacked one α-1,2-mannose residue at the nonreducing terminus of the middle branch (Man8B isomer), and this mannooligosaccharide remained predominant through the overall reaction. Trimming was optimal at pH 7.0 in PIPES buffer in the presence of calcium ion and kifunensine was inhibitory with IC50 below 0.1[emsp4 ]μM. These results suggest that the activity is the same type as was previously observed with human and yeast endoplasmic reticulum (ER) α-mannosidases. Considering these results together with previous data on a fungal α-1,2-mannosidase that trimmed Man9GlcNAc2 to Man5GlcNAc2 (Ichishima, E., et al. (1999) bit>Biochem J, 339: 589–597), the filamentous fungi appear to have two types of α-1,2-mannosidases, each of which acts differently on N-linked mannooligosaccharides.

Structural analysis of the oligosaccharide units of xyloglucan and their effects on growth of COLO 201 human tumor cells

Abstract The oligosaccharide units of xyloglucans from some fruits were analyzed by enzymatic digestion followed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The oligosaccharide units of the polysaccharides were XXXG, XXLG, XLXG, XXFG, XLLG, and XLFG [where each (1→4)-β-linked d -glucosyl residue in the backbone is given a one-letter code according to its substituents: G, β- d -Glc; X, α- d -Xyl-(1→6)-β- d -Glc; L, β- d -Gal-(1→2)-α- d -Xyl-(1→6)-β- d -Glc; F, α- l -Fuc-(1→2)-β- d -Gal-(1→2)-α- d -Xyl-(1→6)-β- d -Glc] in an approximate molar ratio of 10:51:4:13:11:11 for kiwi, of 23:3:40:27:2:5 for peach, of 31:1:9:19:1:39 for avocado, of 35:3:12:19:5:26 for apple, of 29:5:4:33:4:25 for fig, of 29:17:1:29:13:11 for pineapple, and of 27:4:8:21:4:36 for mandarin. In addition, different types of xyloglucan oligo- and polysaccharides were tested for effects on the growth of COLO 201 human tumor cells. The cell growth was reduced by fucose-containing oligo- and polysaccharides.

Phytoestrogenic activity of blackcurrant (Ribes nigrum) anthocyanins is mediated through estrogen receptor alpha

SCOPEnBlackcurrants (Ribes nigrum L., Grossulariaceae) contain high amounts of anthocyanin polyphenols, which have antioxidant and anti-carcinogenic health benefits. This study analyzed the potential phytoestrogenic effects of blackcurrant extract (BCE) in breast cancer (MCF-7) and human endometrial cancer (Ishikawa) cell lines that over-express estrogen receptor alpha (ERα), as well as in immature female rats.nnnMETHODS AND RESULTSnMicroarray analysis and Ingenuity® Pathway Analysis showed that BCE activated the ERα pathway, whereas quantitative-PCR confirmed that BCE and four types of anthocyanins up-regulated genes downstream of ERα. BCE (0.1-1.0 μg/mL) and anthocyanins (0.1-10 μM) induced MCF-7 cell proliferation; however, this effect was blocked by ER antagonist fulvestrant. Flow cytometry showed that anthocyanins reduced and increased the number of MCF-7 cells in the G0/G1 and G2/M phases, respectively. Anthocyanins stimulated ERα transcriptional activity in human ERα reporter assays and induced alkaline phosphatase activity in Ishikawa cells. Competition assays and in silico analysis indicated that anthocyanins bind to ERα. Finally, BCE focally induced stratification of columnar epithelial cells in the rat uterus and increased cytoplasmic mucin levels in these cells.nnnCONCLUSIONnThese results suggest that blackcurrant anthocyanins act as phytoestrogens in vitro and in vivo.

Biochemical and atomic force microscopic characterization of salmon nasal cartilage proteoglycan

Biological activities of salmon nasal cartilage proteoglycan fractions are known, however, structural information is lacking. Recently, the major proteoglycan of this cartilage was identified as aggrecan. In this study, global molecular images and glycosaminoglycan structure of salmon nasal cartilage aggrecan purified from 4M guanidine hydrochloride extract were analyzed using HPLCs and atomic force microscopy with bovine tracheal cartilage aggrecan as a control. The estimated numbers of sulfates per disaccharide unit of chondroitin sulfate chains of salmon and bovine aggrecans were similar (approximately 0.85). However, the disaccharide composition showed a higher proportion of chondroitin 6-sulfate units in salmon aggrecan, 60%, compared to 40% in bovine. Gel filtration HPLC and monosaccharide analysis showed the salmon aggrecan had a lower number (approximately one-third), but 1.5-3.3 times longer chondroitin sulfate chains than the bovine aggrecan. Atomic force microscopic molecular images of aggrecan supported the images predicted by biochemical analyses.

Protective effects of raw and cooked blackcurrant extract on DNA damage induced by hydrogen peroxide in human lymphoblastoid cells

Abstract Context: Blackcurrant (Ribes nigrum L.) is a classical fruit that has long been used to make juice, liqueur and sometimes medicines in Europe. The beneficial effects of blackcurrant, which are inhibition of lipopolysaccharide-stimulated inflammatory, anticarcinogenesis and other health effects, have been reported. Objective: Previously, we reported the antimutagenic activities of blackcurrant using a yeast gene mutation assay. In this study, we investigated whether this antimutagenicity of blackcurrant was confirmed in human cells. Materials and methods: We prepared four types of aqueous blackcurrant extracts (BCE) from mature and premature with or without heat treatment by microwave. Antioxidant activities of BCE were measured by the DPPH radical scavenger assay. In the DPPH radical scavenger assay, the maximum concentration of BCE was 1.6u2009mg/reaction. We investigated the antigenotoxic activities of BCE by the comet assay and micronucleus test using the human lymphoblastoid cell line TK6. In the comet assay, TK6 was treated with 300u2009μMu2009H2O2 without or with BCE at concentrations of 0.5,u20091.0,u20092.0 and 3.0u2009mg/mL. In the micronucleus test, TK6 was treated with 1u2009mg/mL BCE without or with H2O2. Results: All BCEs exhibited more than 90% of inhibition rates of DPPH radicals at the maximum concentration of BCE. DNA damage and micronuclei induced by H2O2 significantly decreased in the each BCE-treated condition. Conclusion: The results suggest that BCE treatment can reduce the genomic instability induced by H2O2 in human cells. We consider that these antigenotoxic effects are related to polyphenols, l-ascorbic acid and other antioxidant compounds.