Decrease in Rice Allergenic Proteins of Polished Rice Grains by Incubating with a Miso Solution

The effect of miso on allergenic proteins in rice seeds was investigated. When polished rice grains were incubated at 37°C for 30-120 min with a 10% miso solution, but not with heat-treated miso or 1% NaCl, the amount of soluble proteins extracted from the rice grains with 1 M NaCl markedly decreased. SDS-PAGE, immunoblotting and densitometric analyses of these soluble proteins and insoluble proteins indicate that 26 kDa globulin and 14-16 kDa allergens in the grains were decreased to 15-60% during incubation with the miso solution, especially soybean-koji miso, without any large change in the content of major insoluble proteins.     

Rice allergenic proteins, 14-16 kDa albumin and alpha-globulin, remain insoluble in rice grains recovered from rice miso (rice-containing fermented soybean paste)

The rice grains (RG) and rice seed proteins remaining in rice miso were investigated with a view point to the potential allergenicity of rice miso. RG ranging from 36 to 180 mg dry weight per g dry miso were separated from several samples of commercially available rice miso. Scanning electron microscopy of the recovered RG indicated that starch granules disappeared almost completely while protein bodies remained intact in RG. Most of the major seed proteins were extracted from RG by heating with 1% SDS/2% 2-mercaptoethanol and detected by SDS-polyacrylamide gel electrophoresis. Major rice allergenic proteins, 14-16 kDa albumin (Alb14-16) and alpha-globulin (alpha-Glb) were also detected by immunoblotting using the specific antisera, and their contents were estimated to be 1.7 to 9.0 and 1 to 7 mg protein per g dry RG respectively. However, the major rice proteins, including glutelin and prolamin, in RG were insoluble in salt, alcohol, and urea solutions, but soluble in 6 M guanidine hydrochloride (Gu-HCl). By immunoblotting and ELISA, no Alb14-16 and only a slight amount of alpha-Glb were detected even in the 6 M Gu-HCl fraction, indicating that these major allergenic proteins are denatured and are present in an insoluble form in rice miso.     

Degradation of soluble proteins including some allergens in brown rice grains by endogenous proteolytic activity during germination and heat-processing

The effect of germination and subsequent heat-processing on the degradation of soluble proteins, including some allergenic proteins, in brown rice grains was investigated. The content of soluble proteins, including 14-16-kDa and 26-kDa allergens, in the germinated and processed brown rice grains (GPR) was much lower than that of non-germinated brown rice. These proteins in brown rice grains were also much lower after subsequent heat-processing during the manufacturing process. The protease activity of germinated brown rice (GR) was detected and increased 1.5 times after germination. The optimum pH values for degradation of the 26-kDa and 14-16-kDa allergens in the GR grains were 4 and between 5 and 7, respectively. These results suggest that the decrease in the soluble proteins and allergens was induced in part by proteolytic degradation. The presence of a detergent enhanced the proteolytic degradation of the soluble proteins, especially of the 26-kDa allergen, in the brown rice grains. The degradation of the 26-kDa allergen was weakly inhibited by NEM, suggesting cysteine protease(s) may have been involved in its degradation. These results suggest that the two abundant allergens were degraded in a different manner and probably by different proteases in the grains during germination.     

Expression and purification of the recombinant SALT lectin from rice (Oryza sativa L.)

The SALT protein is a 14.5 kDa mannose-binding lectin, originally described as preferentially expressed in rice plant roots in response to NaCl stress. Recombinant SALT lectin was produced in Escherichia coli from a cDNA clone encoding protein. After isopropyl-beta-d-thiogalactopyranoside induction, the expression level achieved was 23% of the soluble protein. The recombinant agglutinin was purified by a single-step process by dialyses against a high concentrated salt solution. After purification, hemagglutination assays of rabbit erythrocytes revealed that the recombinant SALT protein is a potent agglutinin (0.078 microg ml(-1) minimal concentration). The purified recombinant lectin was also used for comparative estimation of native protein amounts in protein extracts from rice plants by Western blot assay.     

Fractionation of globulins of milled rice

Globulins were prepared by repeated precipitation with 1.3 M (NH₄)₂SO₄ from a 0.7 M NaCl extract of milled rice. Isoelectric precipitation at pH 4.5 did not effectively remove the α-globulin from the others. A major fraction that remained in solution during dialysis of the globulin precipitate against water was similar in properties to the globulin soluble at pH 4.5 during the isoelectric precipitation process. Some properties of this water-soluble globulin fraction are reported. Proteins extracted from milled rice at 50° with 0.5 M NaCl and precipitated as 1- to 3-μm particles on cooling were verified to be globulins.     

Generation of transgenic rice lines with reduced contents of multiple potential allergens using a null mutant in combination with an RNA silencing method

Rice seed proteins are known to be a causative antigen in some patients with food allergy, especially cereal allergy, with clinical symptoms such as eczema and dermatitis. The α-amylase/trypsin inhibitors (14-16 kDa), α-globulin (26 kDa) and β-glyoxalase I (33 kDa) are regarded as major potential allergens of rice (Oryza sativa L.) seed based on specific recognition by serum IgE from allergy patients. In order to suppress the production of these major allergens in rice grains, a mutant in the 'Koshihikari' background lacking the 26 kDa allergen (GbN-1) was used as a host for RNA silencing. A binary vector harboring two RNA interference (RNAi) gene cassettes for suppression of 14-16 kDa and 33 kDa allergens driven by the 13 kDa and 10 kDa prolamin endosperm-specific promoters, respectively, was introduced into the GbN-1 genome by Agrobacterium-mediated transformation. In the most promising transgenic line, the content of the three potential allergens was remarkably reduced to a very faint level without a change in seed phenotype. IgE binding of 15 patients' sera to the transgenic rice seed mostly deficient in the three major allergens was on average only about 10% that of the control wild-type rice, suggesting that these three accounted for the great majority of rice seed causative allergens recognized by patients' IgE and that the sequential allergen deletion/reduction strategy works in the development of hypo-allergenic rice lines.     

Ca2+-protease--an enzyme of the metabolism of cytoskeletal proteins in the olfactory membrane of vertebrates

Two forms of Ca(++)-activated protease (calpain I and calpain II) associated with an endogenous inhibitor (calpastatin) were detected in a cytosolic fraction of the olfactory tissue of vertebrates (pig, rat). Using ion exchange chromatography on DEAE-cellulose column, calpain I is divided into 2 peaks (eluting by 0.07-0.15 and 0.22-0.25 M NaCl), and calpain II is eluted by 0.35-0.40 M NaCl. The calpain activity was detected in fractions eluted by 0.1-0.17 M NaCl. The Ca(++)-activated protease was demonstrated also in a fraction of cytoskeleton of olfactory tissue insoluble in a 1% solution of Triton X-100. The activity can be detected by Ca(++)-dependent destruction of exogenous substrate (casein), and by Ca(++)-dependent degradation of cytoskeletal endogenous proteins (16, 18 and 20 kDa), of which one may be calmodulin.     

Effects of NaCl Salinity on Nitrogenous Compounds and Proteases during Germination of Vigna sinensis Seeds

Serido bean (Vigna sinensis (L.) Savi) seeds were sown in water and in NaCl solution of —4.3 bars water potential. Total, insoluble and soluble proteins as well as soluble amino nitrogen and proteolytic activity of cotyledons were studied after 0, 1, 3, 5, 7, and 9 days of germination. Protein breakdown and turnover was delayed by the NaCl treatment as compared to the control. This was not due to the total amount of proteolytic activity, which was unchanged by salinity; even though the specific activity decreased due to the delayed breakdown of proteins under salt stress. The inhibitory effects of salinity on seed protein reserve mobilization may be due more to inhibition of translocation of hydrolysis products than to inhibition of protease activity.     

Spermidine and abscisic acid-mediated phosphorylation of a cytoplasmic protein from rice root in response to salinity stress

Importance of higher polyamines, spermidine, and spermine, in relation to the mechanism and adaptation to combat abiotic stress has been well established in cereals. Owing to their polycationic nature at physiological pH, polyamines bind strongly to negative charges in cellular components such as nucleic acids, various proteins, and phospholipids. To study the physiological role of polyamine during salinity stress, phosphorylation study was carried out in cytosolic soluble protein fraction isolated from the roots of salt tolerant (Nonabokra) and salt sensitive (M-1-48) rice cultivars treated with none (control), NaCl (150 mM, 16 h), spermidine (1 mM, 16 h) or with abscisic acid (100 μM, 16 h). A calcium independent auto regulatory 42 kDa protein kinase was found to phosphorylate myelin basic protein and casein but not histone. Interestingly, this was the only protein to be phosphorylated in root cytosolic fraction during NaCl/abscisic acid/spermidine treatment indicating its importance in salinity mediated signal transduction. This is the first report of polyamine as well as abscisic acid induced protein kinase activity in rice root in response to salinity stress.     

The influence of environmental factors on the adhesion of combinations of probiotics to rice fibre fractions

Nine co-cultures of the Bifidobacterium and Lactobacillus species were tested for their ability to adhere to insoluble dietary fibre (IDF1, IDF2), soluble dietary fibre (SDF1, SDF2), and total dietary fibre (TDF1, TDF2) from two rice varieties (RR1 and RR2). Combinations of the same genus (BB + BL and LA + LR) showed 30-40 % (poor) adhesion, and combinations of different genera showed 40-50 % (moderate) adhesion, which is significantly higher (p < 0.05) than the combinations of same genus. The increase in adhesion with species from different genera suggests some synergistic activity. The microbial combinations had the ability to adhere to dietary fibre fractions as early as 30 min. Colonization of rice fibre by bacterial cells was affected by the temperature, with adhesion being higher at 37 °C than at room temperature. The optimal pH value for adhesion was 4.2-4.5. This study observed that the combinations tested had a moderate percentage of adhesion in the presence of bile, low pH (4.3-4.5) and pancreatin, irrespective of the type of co-culture. In addition, adhesion was not affected by an increase in NaCl and Tween 80. Adhesion was affected by disaccharides and polysaccharides. The amount of adhesion of co-cultures was not significantly affected by the substrate (p > 0.05). Results indicated that rice fibre fractions are suitable hosts for the probiotics tested.     

Alleviation of the adverse effects of NaCl on germination of maize grains by calcium

The lengths of roots and shoots, fresh and dry matter yield, and the contents of insoluble saccharides and free amino acids were reduced with the rise in NaCl concentration. However, under combination of NaCl with Ca²⁺ ions, these parameters generally raised. Contents of soluble saccharides, proline and quaternary ammonium compounds increased with increasing NaCl concentration, but under addition of CaCl₂ or CaSO₄, contents of these compounds were decreased. Low concentrations of NaCl stimulated soluble proteins, production, but higher concentrations decreased the content of soluble proteins. Addition of Ca²⁺ in the media did not improve the soluble protein production. Insoluble proteins content was increased with the rise of salinity level, but these effects were more pronounced with NaCl and CaCl₂ or CaSO₄ than with NaCl only.     

Growth of nisin-producing lactococci in cooked rice supplemented with soybean extract and its application to inhibition of Bacillus subtilis in rice miso

Lactic acid fermentation of cooked rice and rice koji by supplementation with soybean extract (SBE) and its application to rice miso fermentation were investigated. By supplementing the cooked rice with SBE, lactic acid bacteria (LAB) grew well without any unfavorable effects on the rice such as off-flavor or coloration. Lactococcus lactis subsp. lactis IFO12007 (Lc. lactis, a producer of the bacteriocin nisin) proliferated at 10(8 to approximately 9) cells/g after 24 h of incubation and produced high activity of nisin. The fermented rice with Lc. lactis strongly inhibited not only Bacillus subtilis ATCC19659 but also the other Bacillus strains. While some strains of LAB markedly inhibited the growth of Asp. oryzae, resulting in failure of koji fermentation, Lc. lactis did not affect the growth of these molds. When Lc. lactis was used for rice miso fermentation as a lactic acid starter culture, Lc. lactis rapidly proliferated and produced high nisin activity of 6,400 IU/g, in the steamed rice, resulting in complete growth inhibition of B. subtilis, which had been inoculated at the beginning of the koji fermentation. The rice miso after 12 weeks of aging had a suitable pH, and favorable taste and color. Furthermore, hyposalting of rice miso could be done without difficulty by lactic acid fermentation of both rice and soybeans.     

不同类型镉积累水稻细胞镉化学形态及亚细胞和分子分布

利用水培试验结合亚细胞组分分级分离和凝胶过滤等技术,研究了水稻根和叶中镉的化学结合形态及其亚细胞和分子分布,比较了低镉积累品种“广源占No.3”和高镉积累品种 “珍桂矮”的差异.结果表明：随着营养液中镉浓度的升高,根和叶亚细胞镉含量显著上升,大部分镉积累在细胞壁（F_Ⅰ）和细胞可溶部分（F_Ⅲ）.高镉积累品种“珍桂矮”根和叶中可溶部分镉含量显著高于低镉积累品种“广源占No.3”.根和叶各种形态镉中,以氯化钠提取态占优势,其次是醋酸提取态,盐酸提取态镉含量最低.与“广源占No.3”相比,“珍桂矮”中迁移性较强的去离子水和乙醇提取态镉比例较高.凝胶过滤结果表明,两种类型的水稻可溶部分镉的出峰位置与样品流份中可溶性蛋白的出峰位置大致相同.可溶部分中的镉大多与分子量为3kD的物质结合,属于植物鳌合肽（PCs）或低分子量物质.“广源占No.3” 根系中镉与PCs配合的组分（Cd-PCs）含量远小于“珍桂矮”.“广源占No.3”细胞可溶部分较低的镉含量以及根系中较少的Cd-PCs形成量,降低了镉的移动及其向地上部转运的可能性.     

生物炭调控盐胁迫下水稻幼苗耐盐性能

土壤盐渍化降低土壤生产力.探索生物炭对盐胁迫下水稻幼苗耐盐性能的影响,对调控盐渍区水稻生产潜力具有重要意义.本研究通过生物炭介入盐胁迫稻田土壤的盆栽试验,调查了生物炭对盐胁迫下土壤环境和水稻幼苗耐盐性能的影响.盐胁迫设置4个水平,分别为0 g NaCl·kg-1土(S0),1 g NaCl·kg-1土(S1),2 g ...     

幼苗期不同水稻材料应答盐胁迫的生理差异分析

为了解耐盐水稻HD96-1幼苗期耐盐生理调控特性，该研究以籼型水稻HD96-1(耐盐性强)和93-11(耐盐性弱)为材料，采用营养液水培法，设置3种NaCl盐浓度(0、60、120 mmol·L^-1),对3叶期幼苗进行了7 d盐处理，测定和分析了两个材料的生长参数和生理生化指标。结果表明：(1)在盐胁迫下，水稻幼苗均表现为株高和假茎宽减小，根冠比增加；与93-11比，HD96-1株高和茎宽减小幅度低，根冠比增加幅度高；地上部和根系干重，HD96-1增加，而93-11减少。(2)盐胁迫后，水稻幼苗的丙二醛(MDA)、超氧阴离子(O₂^-)和过氧化氢(H₂O₂)含量均上升，但HD96-1增幅较93-11低。(3)在盐胁迫下，水稻幼苗体内的超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)的活性，以及抗坏血酸(AsA)、谷胱甘肽(GSH)、脯氨酸、可溶性糖和可溶性蛋白的含量均升高，HD96-1增幅高于93-11。综上表明，两个水稻材料幼苗...     

Distribution patterns of 104 kDa stress-associated protein in rice

A 104 kDa protein (SAP 104) accumulates in rice seedlings in response to several abiotic stress conditions and immunological homologues of rice SAP 104 have been detected in several monocot and dicot species, as also Neurospora crassa, a fungus. In this report, we show that the amino acid sequence of a tryptic peptide generated from purified SAP 104 bears significant homology with an ATP-binding domain of the HSP 100 family proteins of Arabidopsis thaliana and Glycine max. It is further shown that differential uninduced and induced (by high-temperature stress) levels of this protein are accumulated in various organs of the mature rice plant grown under field conditions. Significant uninduced levels of this protein were in particular found in developing and mature rice grains. Seeds/grains of several other plant genera (i.e. Triticum aestivum, Zea mays, Brassica juncea) were also found to contain high uninduced levels of SAP 104. Importantly, the levels of uninduced SAP 104 in rice grains were found to decline during the seed germination phase: after two days of germination, this protein was undetectable in tissues representing pooled sample of seeds and just-emerged seedlings. Tissue print-immunoblotting analysis has indicated that in seeds high levels of this protein are specifically present in the embryo portion.     

硅素分期施用对镉污染水稻光合特性及物质积累的影响

硅(Si)可有效提高水稻对镉(Cd)的抗性,但关于优化硅肥管理对水稻耐Cd性、光合及物质积累等响应机制尚不明晰。采用受污染的农田土壤,通过盆栽试验研究移栽期施Si(T)、拔节期施Si(J)、移栽和拔节期等量分期施Si(TJ)对Cd污染水稻光合作用及物质积累的影响,以不施Si(CK)为对照。结果表明：施Si可明显提高Cd污染下水稻净光合速率,延长叶片光合功能,促进叶片Si沉积,增加Cd在叶中的固定,减少其向籽粒转移。与CK相比,TJ处理在全生育期具有较高的光合速率,产量显著高于其他处理。T、TJ、J处理叶片细胞壁Cd的固持量分别增加11.45%、24.16%和30.15%,且叶片Cd更多以惰性形态(包括果胶和蛋白质结合态Cd、不溶解性磷酸Cd和残渣态Cd)存在,导致叶片Cd转移系数降低,T、TJ、J较CK分别降低33.91%、56.67%和52.16%。此外,主成分分析结果表明,3种Si处理对水稻耐Cd性和光合特性的综合影响大小为TJ>J>T。综合考虑Si调控光合作用、产量、叶片和籽粒Cd浓度的效应,推荐Si素于移栽期与拔节期分期施用。     

Expression of natural antimicrobial human lysozyme in rice grains

In the present study, we explored the expression of human lysozyme in maturing rice grains. Particle bombardment-mediated transformation was utilized to deliver the codon-optimized structural gene for human lysozyme to the callus of rice cultivar Taipei 309. Lysozyme expression is controlled by the promoter and signal peptide sequence for rice storage protein Glutelin 1. A total of 33 fertile plants were regenerated from independent transformation events and 12 of them with significant expression levels of lysozyme were advanced to further generations. The transgenes were characterized by PCR and Southern blot analysis. Segregation analysis indicated a typical Mendelian 3: 1 inheritance, suggesting a single locus or closely linked loci of gene insertion. The expression levels of lysozyme reached 0.6% of the brown rice weight or 45% of soluble proteins. Seven transgenic breeding lines have been selected and followed over six generations. Lysozyme expression levels were maintained in all generations. Biochemical, biophysical and functional comparisons of native and recombinant human lysozyme revealed identical N-terminal sequence, molecular weight, pI and specific activity. Similar thermal and pH stability was observed for lysozyme from two sources. Furthermore, similar bactericidal activity was displayed towards a laboratory strain of E. coli. The possibility of improving medical and nutritional quality of infant formulas and baby foods with rice flour or rice extract containing recombinant human lysozyme is discussed.     

Unique proteins from the statoliths of Lolliguncula brevis (Cephalopoda: Loliginidae)

Information concerning the role of the organic matrix (OM) in statolith mineralization may contribute to resolving problems currently facing the use of increments in squid statoliths to estimate the age of individuals. A preliminary study aimed at purifying and characterizing the OM proteins from statoliths of the loliginid squid Lolliguncula brevis is described. Proteins extracted from the statoliths were separated into two fractions, insoluble and soluble in aqueous solutions. Gel electrophoresis indicated that the insoluble fraction is composed of at least eight major proteins, ranging in size from 25 to over 200 kDa. The five largest insoluble proteins were glycosylated, as shown by immunoassay. In contrast, the soluble fraction is composed of a single dominant protein of about 100 kDa, and two other major proteins of higher molecular weight. All three soluble proteins were glycosylated. Molecular weight and partial sequence data of peptides from five of the insoluble and one of the soluble proteins were used to search databases for possible homologs. No matches were found, suggesting that these proteins may belong to a class of hitherto undescribed OM compounds. Different proteins are involved in biomineralization processes in different organisms. We propose that the insoluble matrix proteins are responsible for regulating the appearance of increments in squid statoliths.