Effect of aging at different temperatures on LAOS properties and secondary protein structure of hard wheat flour dough

The effects of aging from t = 0–108 h at two different temperatures (4 and 25 °C) on the non-linear viscoelastic rheological properties and secondary protein structure of hard wheat flour dough (HWD) were investigated using large amplitude oscillatory shear tests (LAOS) coupled with Fourier transform infrared spectroscopy (FTIR) and SDS-PAGE. Storage (G') and loss (G'') moduli rapidly decreased during aging at 25 °C. Subjecting HWD to progressively longer aging times at 25 °C caused dramatic changes in the non-linear viscoelastic properties demonstrated by strain softening (negative values of e₃/e₁) and shear thinning (negative values of v₃/v₁) behavior. Elastic Lissajous curves of the unaged control dough showed clockwise turn and wider elliptical trajectories as dough aging proceeds especially at higher temperatures. Other non-linear LAOS parameters (G'_M-G''_L, η'_M-η'_L, S and T) supported that aging process at higher temperature caused a progressive change in dough structure from strain stiffening to strain softening behavior while dough samples aged at 4 °C showed fairly close behavior with the control dough sample. FTIR spectra indicated that the relative content of β-sheet and β-turn structures decreased while the content of α-helix structure increased for all dough samples as a result of dough aging. SDS-PAGE results supported the breakdown of high molecular weight (HMW) and low molecular weight (LMW) glutenin subfractions. Aging at the higher temperature of 25 °C decreased the HMW/LMW ratio from 0.77 to 0.59, while the ratio was 0.73 for the dough aged at 4 °C which is fairly close to the control sample. Our results show that the degradation rate of gluten/starch network was triggered by aging at higher temperature, longer aging time, and natural fermentation which resulted in increasing acidity and increase in endogenous proteolytic and amylolytic activity, and also increasing gluten solubility and break down of intermolecular disulfide bonds at acid pH.     

湖南永州尖吻蝮蛇蛇毒的蛋白组分及毒性分析

采用Nano–LC–ESI–MS/MS蛋白鉴定技术对湖南永州尖吻蝮蛇蛇毒蛋白组分进行质谱分析,应用常规体外试管法对尖吻蝮蛇蛇毒体外溶血值进行检测,采用改良寇式法对尖吻蝮蛇蛇毒LD_(50)进行测定。结果显示:尖吻蝮蛇蛇毒含有50种蛋白,蛋白相对分子质量集中在1.0×10~4～2.0×10~4(46.9%)、4.0×10~4～5.0×10~4(22.4%)、2.0×10~4～3.0×10~4(10.6%)和7.0×10~4～8.0×10~4(7.6%),其中代表性的高丰度蛋白有蛇毒金属蛋白酶A(11.7%)、蛇毒金属蛋白酶H1(9.8%)、蕲蛇类凝血酶–2(7.3%)、抗凝血酶A–A亚基(6.8%),低丰度蛋白(0.1%)有Ecto–5'–核苷酸酶、碱性磷脂酶A2 DAV–N6、生长分化因子11;蛇毒引起红细胞溶血的最低质量浓度为60.00μg/mL,尖吻蝮蛇蛇毒对KM小鼠的LD_(50)为7.1796mg/kg,攻毒小鼠出现呼吸急促、躁动不安、注射部位出现奇特瘙痒和大面积溃烂,至死亡。     

利用SDS-PAGE鉴定转基因小麦HMW-GS的表达类型和后代遗传

采用SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)在蛋白质水平上对T2～T5代小麦转基因株系高分子量谷蛋白亚基(HMW—GS)的遗传表达及其分离进行了鉴定和分析。结果发现，外源1Dx5和1Dy10基因在T2代部分株系中表达；在有的株系中出现了原亚基(8亚基)的缺失或沉默和新亚基(暂定8^*)的产生，因而检测出2 5 10 12，2 5 7 8^* 10 12，5 7 10，2 7 10 12的多种类型。对2 5 10 12型株系进行了多代跟踪分析，发现其在T2～T5代陆续发生分离，存在于不同单株、不同单穗的子粒和同一单粒后代之间。经筛选，获得了天然不存在的亚基类型为2 5 10 12，2 10 12，2 5 12等稳定表达的种质创新株系。     

Allelic variation of the HMW glutenin subunits in Aegilops tauschii accessions detected by sodium dodecyl sulphate (SDS-PAGE), acid polyacrylamide gel (A-PAGE) and capillary electrophoresis

Variability of high molecular weight glutenin subunits (HMW-GS) was studied in198 accessions of Ae. Tauschii (2n=2x=14, DD) by sodium dodecyl sulphate(SDS-PAGE) and acid polyacrylamide gel electrophoresis (A-PAGE) and capillary electrophoresis (CE). A high allelic variation of HMW-GS, including some novel x- and y-type subunits and variable subunit combinations were observed. One accession(TD159) showed a x-type null form. The results by A-PAGE analysis revealed that the subunits Dx5 ^t and Dy10 ^t encoded by Glu-D ^t 1 locus in Ae. tauschii were different in relative mobilities in comparison with the subunits Dx5 and Dy10 found in bread wheats, whereas they had the same mobilities, respectively, when separated by SDS-PAGE. The higher resolution of Ae. tauschii HMW-GS separated by CE method showed two clear peaks in accordance with x- and y-type subunits, respectively,except the accession TD151 which possessed only subunit Dy12.1^*t. The electro elution time of the x-type and y-type subunits were about 13–14 and 7–8minutes, respectively. Characterization of wheat HMW-GS was facilitated by using CE which provides high resolution and increases the speed of analysis in conjunction with the traditional gel electrophoretic methods. A total of 42HMW-GS alleles were identified, among which were several alleles not presently detected in bread wheats. Hence Ae. tauschii is potentially a valuable genetic resource for quality improvement of bread wheat. This revised version was published online in August 2006 with corrections to the Cover Date.     

Glu-B1位点亚基色谱鉴定及7OE对面团强度的影响

准准确鉴定高分子量谷蛋白亚基（HMW-GS）及探讨其对面团特性的影响是小麦品质研究的重要内容。用聚丙烯酰胺凝胶电泳（SDS-PAGE）和反相高效液相色谱（RP-HPLC）对62份品种（系）的HMW-GS组成进行鉴定。结果表明，结合SDS-PAGE和RP-HPLC两种方法可以对Glu-B1位点，尤其是Glu-B1（7+8）进行有效鉴定。选用13份姊妹系为材料，使用RP-HPLC和凝胶色谱（SE-HPLC）方法，研究了Glu-B1al（7^OE+8*）以及贮藏蛋白组分含量对面团强度的影响。结果表明，Glu-B1al（7^OE+8*）可显著提高HWM-GS总量和面团强度，7OE可作为优质亚基用于强筋小麦育种。相关性分析表明，谷蛋白总量、HWM-GS、LMW-GS以及x-HMW含量与最大抗阻力呈1%显著正相关，相关系数分别为0.76、0.76、0.77和0.72。SDS-不溶性谷蛋白大聚体（UPP）占谷蛋白聚合体总量的百分比与揉面仪峰值时间、粉质仪形成时间和稳定时间以及最大抗阻呈1%或0.1%显著正相关，相关系数分别为0.77、0.90、0.89和0.87。醇溶蛋白与谷蛋白含量的比值与揉面仪峰值时间呈1%显著负相关，相关系数为－0.69；与粉质仪稳定时间和最大抗阻呈5%显著负相关，相关系数分别为－0.58和－0.64。HPLC可有效分离和量化HWM-GS，并作为小麦品质育种有效的辅助手段。     

The effect of introgressions of wheat D-genome chromosomes into 'Presto' triticale

Hexaploid triticale (X Triticosecale Wittmack) (2n= 6x= 42, AABBRR) and wheat (Triticum aestivum L.) (2n= 6x= 42, AABBDD) differ in their R and D-genomes. This produces differences in both agronomic and end-use quality characteristics. Our objective was to determine how introgressions of individual chromosomes from the D-genome of wheat affect these characteristics of a winter triticale 'Presto'. We studied the effects of 18 D-genome chromosome substitution lines, 15 sib-lines as controls, and five check cultivars at Lincoln, NE in 1996, using a randomized complete block design with two replications. The experiment was repeated at Lincoln and Mead, NE in 1997 and 1998 with 15 substitution lines that survived the first winter in Lincoln, along with their 12 control sibs and five check cultivars. Few D-genome chromosomes had positive effects. Chromosomes 2D, 4D, and 6D significantly reduced plant height when substituted for 2R, 4B, and 6R, respectively. No grain yield increases were associated with any of the D-genome chromosomes tested, but three substitutions decreased the grain yield. Depending on the allele of the hardness gene present, chromosome 5D increased or decreased kernel hardness when substituted for 5R or 5A, respectively. Introgressions of chromosomes 1D and 6D improved end-use quality characteristics of Presto. These results suggest that apart from beneficial effects of individual loci located on the D-genome chromosomes, no major benefit can be expected from D-genome chromosome substitutions.     

Isoenzyme and cotyledon protein variation for identification of black beans (Phaseolus vulgaris L.) with similar seed morphology

Summary Methods developed to identify genetically diverse varieties of beans (Phaseolus vulgaris L.) were applied to closely related lines that were difficult to distinguish on the basis of seed morphology. Seedling tissues and seeds of black beans, were examined electrophoretically for isoenzyme and cotyledon proteinn protein patterns. Seven enzymes, extracted from seeds or from seedling stem, root or leaf tissues, were compared for polymorphism. Peptidase, polyphenol oxidase, phosphoglucoisomerase and glutamate oxaloacetate transaminase patterns were the same for all lines. Some differences were observed for acid phosphatase, peroxidase and esterase patterns, but complete discrimination of the six selected lines was not possible on the basis of isoenzyme patterns alone. Using polyacrylamide gel electrophoresis (PAGE) of dissociated 0.1 M acetic acid soluble proteins at pH 3.1 (acid-PAGE), or sodium dodecyl sulphate PAGE (SDS-PAGE) of residual protein extracts, all but one pair of samples in each case could be distinguished from the other samples. Using both techniques all of the lines could be identified unequivocally.     

患出血病鲫鱼主要组织可溶性蛋白质变化的研究

采用SDS-聚丙烯酰胺凝胶电泳法(SDS-PAGE)对正常和患出血病的鲫鱼心、肝、肾、鳃、脑、肌肉、肠、眼8种组织中可溶性蛋白质进行比较分析。结果表明：病鱼的心、肝、肾、鳃、脑、肌肉6种组织的蛋白质电泳谱带与正常鱼相比有较大变化，心和肾各缺失5条谱带；肝新诱导出1条谱带，另有1条谱带缺失；鳃中新诱导出2条谱带，有2条谱带缺失；脑中增加了4条新谱带，1条谱带缺失；肌肉新增5条谱带。说明鱼在遭受病原菌侵袭后，蛋白质的合成代谢受到较大影响。这些变化可作为辅助疾病诊断的生化指标。     

Variation among maize (Zea mays L.) accessions of Bendel State,Nigeria

Summary Variability in maize zein protein band mobilities in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was characterized to classify 27 maize accessions (OTUs) collected from Bendel State, Nigeria. The classification of the OTUs was achieved using two numerical procedures: average linkage cluster analysis and principal component analysis (PCA). Five clusters were delineated by the cluster analysis whereas the PCA complemented the cluster analysis by separating the OTUs with yellow kernels into one group and OTUs with early maturity into another. OTUs from the same geographical contiguity commonly grouped together. However some regional overlappings of the OTUs occurred. Results of the PCA revealed that zein bands that stained less intensely more strongly separated the OTUs into various clusters than did those that stained more intensely.     

聚乙二醇化重组人白细胞介素6的放射性标记

分别用常规氯氨T和改进的双相氯氨T法对PEG-rhIL-6进行放射性碘标记,用柱层析法和离心超滤法对标记物分离纯化,同时用三氯乙酸沉淀法和SDS-PAGE电泳法鉴定纯化后的标记化合物放射化学纯度,采用rhIL-6依赖细胞7TD1,用MTT比色法测定标记物的生物学活性。结果表明:1.改进的双相氯氨T法标记率为74.5%,高于常规氯氨T法的62.3%,放射性比活度分别为5.513×105和4.610×105Bq/μg。2.经柱层析法和离心超滤法对标记物分离纯化后,用三氯乙酸沉淀法测得放射化学纯度均能达到99%以上。3.用SDS-PAGE电泳法检测两种标记方法所得的标记物与非标记物的结果表明,常规氯氨T法标记物比非标记物多1条高分子量蛋白带,认为是标记过程中蛋白质受到部分损伤;改进的双相氯氨T的标记物与非标记物蛋白质条带一致,认为蛋白质标记后基本没有受到损伤。4.生物活性检测表明改进的双相氯氨T标记物与非标记物活性之间无显著差异,常规氯氨T法标记物活性略低于双相氯氨T的标记物活性。