检测脑膜炎球菌菌苗中内毒素方法的研究

作者应用聚丙烯酰胺凝胶电泳结合银染(SDS—PAGE银染)和2—酮基—3—脱氧辛糖(KDO)方法来测定脑膜炎球菌外膜蛋白和多糖菌苗中的脂多糖(LPS)。这二种方法能比较真实地测定出存在于蛋白或多糖制品中的LPS实际含量。其结果和家兔热原质试验一致。KDO法并能定量测定蛋白样品中的LPS含量。     

Quality Monitoring of Porous Zein Scaffolds: A Novel Biomaterial

Our previous studies have shown that zein has good biocompatibility and good mechanical properties. The first product from a porous scaffold of zein, a resorbable bone substitute, has passed the biological evaluation of medical devices (ISO 10993) by the China Food and Drug Administration. However, Class III medical devices need quality monitoring before being placed on the market, and such monitoring includes quality control of raw materials, choice of sterilization method, and evaluation of biocompatibility. In this paper, we investigated four sources of zein through amino acid analysis (AAA) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in order to monitor the composition and purity, and control the quality of raw materials. We studied the effect of three kinds of sterilization method on a porous zein scaffold by SDS-PAGE. We also compared the changes in SDS-PAGE patterns when irradiated with different doses of gamma radiation. We found that polymerization or breakage did not occur on peptide chains of zein during gamma-ray (γ-ray) sterilization in the range of 20–30 kGy, which suggested that γ-ray sterilization is suitable for porous zein scaffolds. Regarding cell compatibility, we found a difference between using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and a cell-counting kit-8 (CCK-8) assay to assess cell proliferation on zein film, and concluded that the CCK-8 assay is more suitable, due to its low background optical density.     

Effect of heating and acidic pH on characteristics of wheat gluten suspension

The effects of different pH treatments with and without heating on the characteristics of wheat gluten suspension was investigated. At pH 1, maximum changes in colour were observed with a concurrent 65% decrease in protein free-thiol content compared to the control gluten. The SDS-Extractability of protein (SDS-EP) chromatogram eluted at lower retention time and the presence of bands at the top lane even during reducing conditions in SDS-PAGE gel suggested complex formation involving bonds other than disulphides. An increase in the free-amino group content and the presence of an additional peak at a higher retention time in the SDS-EP chromatogram was suggestive of hydrolysis. At pH 2 and 3, similar decreases in SDS-EPs and free-thiol content indicated formation of complexes. When heated, the free-thiol content of the dispersions increased compared to the non-heated dispersions indicating disruption of disulphide bonds with changes in gluten structure and size distribution.     

Proteome Analysis of Subsarcolemmal Cardiomyocyte Mitochondria: A Comparison of Different Analytical Platforms

Mitochondria are complex organelles that play critical roles in diverse aspects of cellular function. Heart disease and a number of other pathologies are associated with perturbations in the molecular machinery of the mitochondria. Therefore, comprehensive, unbiased examination of the mitochondrial proteome represents a powerful approach toward system-level insights into disease mechanisms. A crucial aspect in proteomics studies is design of bioanalytical strategies that maximize coverage of the complex repertoire of mitochondrial proteins. In this study, we evaluated the performance of gel-based and gel-free multidimensional platforms for profiling of the proteome in subsarcolemmal mitochondria harvested from rat heart. We compared three different multidimensional proteome fractionation platforms: polymeric reversed-phase liquid chromatography at high pH (PLRP), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and isoelectric focusing (IEF) separations combined with liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), and bioinformatics for protein identification. Across all three platforms, a total of 1043 proteins were identified. Among the three bioanalytical strategies, SDS-PAGE followed by LC-MS/MS provided the best coverage of the mitochondrial proteome. With this platform, 890 proteins with diverse physicochemical characteristics were identified; the mitochondrial protein panel encompassed proteins with various functional roles including bioenergetics, protein import, and mitochondrial fusion. Taken together, results of this study provide a large-scale view of the proteome in subsarcolemmal mitochondria from the rat heart, and aid in the selection of optimal bioanalytical platforms for differential protein expression profiling of mitochondria in health and disease.     

Thermal markers arising from changes in the protein component of milk

Classification of heat load applied to milk requires the detection of parameters appropriately related to the intensity of the heat treatment. Current analytical methods based on heat-induced changes in the protein component of milk have been directed either to determine the amount of protein-derived products arised from heat treatments or to evaluate the extent of thermal denaturation of milk proteins. Lately, a new analytical strategy has been developed according to the occurrence of three major whey proteins, namely bovine serum albumin (BSA), beta-lactoglobulin (βlg) and alfa-lactalbumin (αla), normally soluble at pH 4.6 in raw milk, in the pH 4.6 insoluble protein fraction recovered from heat-treated milk. The results have shown that pH 4.6 insoluble BSA, βlg and αla, as detected by ELISA in milk, can be regarded as thermal markers suited for either dairy process control or regulation purposes.     

The effect of enzyme systems and processing on the hydrolysis of peanut (Arachis hypogaea L.) protein

In vitro protein digestion studies were carried out on raw and roasted peanut flour as the starting material in the production of peanut protein hydrolysate. Peanut flour was hydrolyzed with alcalase and alternately in a sequential digestion with pepsin-pancreatin, both for up to 24 h. The degree of hydrolysis (DH) at different times of hydrolysis was determined using the trinitrobenzenesulfonic acid (TNBS) method. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to indicate destruction of native protein units in the enzymatic digests.Hydrolysis with alcalase was very rapid for the first 6 h after which a plateau was reached, whereas that with pepsin–pancreatin was more gradual reaching a plateau after 12 h of hydrolysis. Raw peanut hydrolyzed with alcalase and pepsin–pancreatin had 23% and 21% DH after 24 h respectively, whilst roasted peanut hydrolyzed with alcalase had 21% DH, with the pepsin–pancreatin hydrolysate recording the highest value of 25% after 24 h of hydrolysis.SDS-PAGE results showed that raw peanut samples behaved differently from the roasted samples; increasing hydrolysis time reduced larger peanut protein subunits, with only peptides of <20 kDa visible after hydrolysis for raw peanut, and virtually no distinct visible bands for the roasted peanut after 3 h of hydrolysis.     

Characterisation of the S-Carboxymethyl Extract of Wool Using Small Format Alkaline Urea-PAGE/SDS-PAGE Followed by Silver Staining

Abstract

Alkaline urea-PAGE/SDS-PAGE, when used in a novel format which was significantly smaller than that employed by earlier workers, and when followed by silver staining, resulted in the detection of excellently resolved protein components from S-carboxymethyl reductive extracts of very small wool samples, even samples as small as an individual wool fibre. The silver stained two-dimensional gel patterns exhibited significant improvements compared to the fluorograph gel patterns of earlier workers based on the presence of radiolabel incorporated within the S-carboxymethyl moiety. The silver staining resulted in the visualisation of numerous protein components, and in the region of the gel pattern expected to contain the high-sulfur protein fraction, there appeared more major components than the number of high-sulfur protein components usually displayed in fluorograph patterns. The relative amount of the high-sulfur protein fraction in the final silver stained gel pattern could be boosted, if desired, if the gel was loaded not with the whole wool extract but with the filtrate resulting from the passage of the whole extract through a centrifugal ultrafilter.     

唐菖蒲两变异株的同功酶及SDS-聚丙烯酰胺凝胶电泳的比较分析

为探讨电子束对唐菖蒲诱变的可行性及不同剂量电子束对其花性状的影响，用不同剂量电子束辐照唐菖蒲“江山美人”球茎，在40Gy和160Gy处理组分别得到了1株花色和花序变异株(M1’和M2’)。对这两变异株和对照以及其相应辐照剂量（40Gy和160Gy）处理组进行了研究。M1代植株叶片的过氧化物酶、过氧化氢酶、淀粉酶和酯酶4种同功酶电泳结果表明，40Gy和160Gy处理组的酶带与对照组的相同，变异株与对照组相比，酶带有所增减。基于同功酶谱带带型，使用SPSS11.5进行了聚类分析并得到聚类树状图。图中显示，供试材料被分成了3个组：对照组（包括40Gy和160Gy处理组与对照），M1’组和M2’组。SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析表明，蛋白表达明显被电子束辐照所抑制，但在这两个变异株中观测到3条特异表达的蛋白条带，分子量分别为96kDa、115.4kDa和137.2kDa，这些特异表达的蛋白可能与花色与花序的调控有关。由此表明，电子束辐照诱导花色与花形突变体是1种有效的途径。     

部分小麦品种的高相对分子质量谷蛋白亚基组成分析

利用电泳技术分析了94个国产小麦样品的高相对分子质量谷蛋白亚基组成,结果表明:这些小麦品种大部分具有1A上的优质亚基1,1B上的优质亚基7 8/17 18,1D上的5 10,个别品种还同时具有1A、1B、1D上的优质亚基;我国的小麦品种高相对分子质量谷蛋白亚基组成类型较丰富,共检测到16种亚基和28种亚基组合类型,品质评分4~10分,平均为6.9分;在小麦品种中,含有优质亚基5 10、17 18或2*,分别有44、5和13个品种,可供优质小麦育种应用.