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.     

流感疫苗血凝素含量测定替代方法的建立及验证

目的建立测定流感疫苗血凝素(Haemagglutinin,HA)含量的替代方法 ,并进行验证,以解决大流行流感发生初期疫苗原液中HA定量的难题。方法优化糖苷酶处理条件,将流感疫苗原液经糖苷酶处理后,以还原SDS-PAGE方法分离样品,以灰度扫描法确定HA蛋白百分比,结合样品总蛋白数值,计算样品中HA含量。以该方法和传统的单向免疫扩散(SRID)法分别测定7批流感疫苗原液中HA含量,并进行比较。结果优化的糖苷酶处理条件为:总蛋白400μg/ml,PNGaseF加入比例为1:50。样品经糖苷酶处理后,以还原SDS-PAGE法可以清晰区分各蛋白条带,经测序后确定HA两个亚基,与预期相对分子质量一致。该方法测定7批流感疫苗原液中HA含量的结果与SRID法测定结果的符合率在87.90%~122.20%之间。结论初步建立了测定流感疫苗中HA含量的替代方法 ,在WHO参考品供应不到位时,可用于大流感发生时疫苗原液中HA之定量。     

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。     

Salivary Amylase Induction by Tannin-Enriched Diets as a Possible Countermeasure Against Tannins

Tannins are characterized by protein-binding affinity. They have astringent/bitter properties that act as deterrents, affecting diet selection. Two groups of salivary proteins, proline-rich proteins and histatins, are effective precipitators of tannin, decreasing levels of available tannins. The possibility of other salivary proteins having a co-adjuvant role on host defense mechanisms against tannins is unknown. In this work, we characterized and compared the protein profile of mice whole saliva from animals fed on three experimental diets: tannin-free diet, diet with the incorporation of 5% hydrolyzable tannins (tannic acid), or diet with 5% condensed tannins (quebracho). Protein analysis was performed by one-dimensional gel electrophoresis combined with Matrix-Assisted Laser Desorption Ionization-Time of Flight mass spectrometry to allow the dynamic study of interactions between diet and saliva. Since abundant salivary proteins obscure the purification and identification of medium and low expressed salivary proteins, we used centrifugation to obtain saliva samples free from proteins that precipitate after tannin binding. Data from Peptide Mass Fingerprinting allowed us to identify ten different proteins, some of them showing more than one isoform. Tannin-enriched diets were observed to change the salivary protein profile. One isoform of α-amylase was overexpressed with both types of tannins. Aldehyde reductase was only identified in saliva of the quebracho group. Additionally, a hypertrophy of parotid salivary gland acini was observed by histology, along with a decrease in body mass in the first 4 days of the experimental period. G. da Costa and E. Lamy have contributed equally to this work.     

酸法和酶法提取鳄鱼皮胶原蛋白及性质研究

对以鳄鱼皮为原料得到的酸溶性胶原蛋白(ASC)和酶溶性胶原蛋白(psc)的性质进行比较分析.紫外扫描结果表明所提取出的胶原蛋白在232nm波长处有显著吸收峰;SDS-PAGE结果表明ASC和PSC的肽链组成具有很大的相似性,均含有两种a肽链及其交联链(β链及γ链);溶解性分析表明鳄鱼皮胶原蛋白的等电点在pH7左右;ASC和PSC的保水性经过6h (25℃)仍然高于85％; ASC的吸油性(24mL/g)和PSC的吸油性(41 mL/g)差异较大.根据上述测定结果可知,鳄鱼皮胶原蛋白ASC和PSC组成类似,符合Ⅰ型胶原蛋白的特征,但二者具体的功能性质略有差异.     

蛋白酶对大豆分离蛋白的降解模式研究

采用十二烷基酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）方法，分析了几种商品蛋白酶（包括枯草杆菌蛋白酶、胰蛋白酶、胰凝乳蛋白酶、木瓜蛋白酶和细菌碱性蛋白酶）对大豆分离蛋白（SPI）的降解模式。结果表明，大豆球蛋白酸性亚基的Ax多肽链最容易被水解，所有的酶对其均有作用，而碱性亚基和大豆伴球蛋白的B亚基最难被水解。木瓜蛋白酶对大豆分离蛋白的水解最迅速、彻底，由于大豆蛋白中含有胰蛋白酶抑制因子，胰蛋白酶和胰凝乳蛋白酶对大豆蛋白水解能力较差。