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Yuki Y.,Tokyo Medical University | Yuki Y.,Tokyo International University | Mejima M.,Tokyo Medical University | Kurokawa S.,Tokyo Medical University | And 23 more authors.
Plant Biotechnology Journal | Year: 2013

Plants have been used as expression systems for a number of vaccines. However, the expression of vaccines in plants sometimes results in unexpected modification of the vaccines by N-terminal blocking and sugar-chain attachment. Although MucoRice-CTB was thought to be the first cold-chain-free and unpurified oral vaccine, the molecular heterogeneity of MucoRice-CTB, together with plant-based sugar modifications of the CTB protein, has made it difficult to assess immunological activity of vaccine and yield from rice seed. Using a T-DNA vector driven by a prolamin promoter and a signal peptide added to an overexpression vaccine cassette, we established MucoRice-CTB/Q as a new generation oral cholera vaccine for humans use. We confirmed that MucoRice-CTB/Q produces a single CTB monomer with an Asn to Gln substitution at the 4th glycosylation position. The complete amino acid sequence of MucoRice-CTB/Q was determined by MS/MS analysis and the exact amount of expressed CTB was determined by SDS-PAGE densitometric analysis to be an average of 2.35 mg of CTB/g of seed. To compare the immunogenicity of MucoRice-CTB/Q, which has no plant-based glycosylation modifications, with that of the original MucoRice-CTB/N, which is modified with a plant N-glycan, we orally immunized mice and macaques with the two preparations. Similar levels of CTB-specific systemic IgG and mucosal IgA antibodies with toxin-neutralizing activity were induced in mice and macaques orally immunized with MucoRice-CTB/Q or MucoRice-CTB/N. These results show that the molecular uniformed MucoRice-CTB/Q vaccine without plant N-glycan has potential as a safe and efficacious oral vaccine candidate for human use. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Mimura Y.,Forestry and Fisheries Technology Research Center | Minamiyama Y.,Forestry and Fisheries Technology Research Center | Sano H.,Kyoto Prefectural University | Hirai M.,Forestry and Fisheries Technology Research Center | Hirai M.,Kyoto Prefectural University
Journal of the Japanese Society for Horticultural Science | Year: 2010

A quantitative trait locus (QTL) analysis of pepper growth traits was performed using a doubled haploid (DH) population derived from a cross between two Capsicum annuum genotypes, a bell-type cultivar 'California Wonder' and a Malaysian small-fruited cultivar 'LS2341'. Simple sequence repeats and amplified fragment length polymorphism markers were used to construct a genetic map for this population. The map spans 1,213 cM, and consists of 15 linkage groups (LGs). The axillary shooting, flowering date, primary axis length, number of leaves on primary axis and mean internode length were evaluated in 94 F 1DH families. Twelve QTLs were identified by interval analysis, and each QTL accounts for 14 to 34% of the phenotypic variation. Markers on chromosomes 2, 3, 12, and linkage group 8 (LG8) were associated with QTL for these traits. The present analysis revealed 2 loci for the growth traits in LG 8. One affected mainly axis length and had a minor effect on flowering, while the other had a large effect on flowering date and a smaller effect on axis length. © 2010 JSHS.

Kurokawa S.,Tokyo Medical University | Kurokawa S.,Kyoto Prefectural University | Kuroda M.,Japan National Agriculture and Food Research Organization | Mejima M.,Tokyo Medical University | And 13 more authors.
Plant Cell Reports | Year: 2014

Key message: RNAi-mediated suppression of the endogenous storage proteins in MucoRice-CTB-RNAi seeds affects not only the levels of overexpressed CTB and RAG2 allergen, but also the localization of CTB and RAG2. A purification-free rice-based oral cholera vaccine (MucoRice-CTB) was previously developed by our laboratories using a cholera toxin B-subunit (CTB) overexpression system. Recently, an advanced version of MucoRice-CTB was developed (MucoRice-CTB-RNAi) through the use of RNAi to suppress the production of the endogenous storage proteins 13-kDa prolamin and glutelin, so as to increase CTB expression. The level of the α-amylase/trypsin inhibitor-like protein RAG2 (a major rice allergen) was reduced in MucoRice-CTB-RNAi seeds in comparison with wild-type (WT) rice. To investigate whether RNAi-mediated suppression of storage proteins affects the localization of overexpressed CTB and major rice allergens, we generated an RNAi line without CTB (MucoRice-RNAi) and investigated gene expression, and protein production and localization of two storage proteins, CTB, and five major allergens in MucoRice-CTB, MucoRice-CTB-RNAi, MucoRice-RNAi, and WT rice. In all lines, glyoxalase I was detected in the cytoplasm, and 52- and 63-kDa globulin-like proteins were found in the aleurone particles. In WT, RAG2 and 19-kDa globulin were localized mainly in protein bodies II (PB-II) of the endosperm cells. Knockdown of glutelin A led to a partial destruction of PB-II and was accompanied by RAG2 relocation to the plasma membrane/cell wall and cytoplasm. In MucoRice-CTB, CTB was localized in the cytoplasm and PB-II. In MucoRice-CTB-RNAi, CTB was produced at a level six times that in MucoRice-CTB and was localized, similar to RAG2, in the plasma membrane/cell wall and cytoplasm. Our findings indicate that the relocation of CTB in MucoRice-CTB-RNAi may contribute to down-regulation of RAG2. © 2013 Springer-Verlag Berlin Heidelberg.

Sasou A.,Kyoto Prefectural University | Shigemitsu T.,Kyoto Prefectural University | Saito Y.,Kyoto Prefectural University | Tanaka M.,Kyoto Prefectural University | And 4 more authors.
Plant Cell Reports | Year: 2016

Key message: Prolamin–GFP fusion proteins, expressed under the control of native prolamin promoters, were localized in specific layers of PB-Is. Prolamin–GFP fusion proteins were gradually digested from outside by pepsin digestion.Abstract: In rice seed endosperm, protein body type I (PB-I) has a layered structure consisting of prolamin species and is the resistant to digestive juices in the intestinal tract. We propose the utilization of PB-Is as an oral vaccine carrier to induce mucosal immune response effectively. If vaccine antigens are localized in a specific layer within PB-Is, they could be protected from gastric juice and be delivered intact to the small intestine. We observed the localization of GFP fluorescence in transgenic rice endosperm expressing prolamin–GFP fusion proteins with native prolamin promoters, and we confirmed that the foreign proteins were located in specific layers of PB-Is artificially. Each prolamin–GFP fusion protein was localized in specific layers of PB-Is, such as the outer-most layer, middle layer, and core region. Furthermore, to investigate the resistance of prolamin–GFP fusion proteins against pepsin digestion, we performed in vitro pepsin treatment. Prolamin–GFP fusion proteins were gradually digested from the peripheral region and the contours of PB-Is were made rough by in vitro pepsin treatment. These findings suggested that prolamin–GFP fusion proteins accumulating specific layers of PB-Is were gradually digested and exposed from the outside by pepsin digestion. © 2016 The Author(s)

Morita T.,Fisheries Research Agency | Niwa K.,Fisheries Research Agency | Fujimoto K.,Fisheries Research Agency | Kasai H.,Fisheries Research Agency | And 15 more authors.
Science of the Total Environment | Year: 2010

Iodine-131 (physical half-life: 8.04days) was detected in brown algae collected off the Japanese coast. Brown algae have been extensively used as bioindicators for radioiodine because of their ability to accumulate radionuclides in high concentration factors. The maximum measured specific activity of 131I in brown algae was 0.37±0.010Bq/kg-wet. Cesium-137 was also detected in all brown algal samples used in this study. There was no correlation between specific activities of 131I and 137Cs in these seaweeds. The specific activity of 137Cs ranged from 0.0034±0.00075 to 0.090±0.014Bq/kg-wet. Low specific activity and minimal variability of 137Cs in brown algae indicated that past nuclear weapon tests were the source of 137Cs. Although nuclear power stations and nuclear fuel reprocessing plants are known to be pollution sources of 131I, there was no relationship between the sites where 131I was detected and the locations of nuclear power facilities. Most of the sites where 131I was detected were near big cities with large populations. Iodine-131 is frequently used in diagnostic and therapeutic nuclear medicine. On the basis of the results, we suggest that the likely pollution source of 131I, detected in brown seaweeds, is not nuclear power facilities, but nuclear medicine procedures. © 2010 Elsevier B.V.

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