沙蚕活性蛋白酶诱导人肺癌SPC-A-1细胞凋亡的机制研究

本文探讨了沙蚕活性蛋白酶(Nereis active protease,NAP)诱导SPC-A-1细胞凋亡机制。采用MTT法检测NAP对SPC-A-1细胞的抑制作用,倒置显微镜及AO/EB染色观察SPC-A-1细胞的形态学的变化,采用流式细胞术检测细胞早期凋亡率和细胞膜电位的变化;并通过Western Blotting检测细胞中凋亡相关蛋白的表达变化。NAP对SPC-A-1细胞活性具有明显的抑制作用且呈现剂量和时间的依赖性;NAP作用后细胞出现凋亡的形态学特征;经流式细胞术检测结果显示,随着NAP作用浓度的增加,SPC-A-1细胞的早期凋亡率从13.50%提高到22.98%,且线粒体膜电位下降所占的百分率从12.95%提高到25.28%。Western Blotting结果显示,50μg/mL NAP作用24h后,Bax/Bcl-2的比率相对对照组明显增加了6.05倍;Cyt-C、Cleaved-Caspase 9、Cleaved-Caspase 3、Cleaved-PARP等含量显著上调,相对表达量分别达到对照组的2.32、3.07、3.68、1.36倍。NAP诱导SPC-A-1细胞凋亡的作用机理有可能是通过下调Bcl-2蛋白的表达、上调Bax蛋白的表达,进而诱导线粒体膜电位的下降,促使Cyt-C的转移以及激发Caspase家族发生级联反应最终导致SPC-A-1细胞的凋亡。     

海水养殖凡纳滨对虾肠道产蛋白酶菌株的筛选、鉴定

为开发海洋来源的微生物蛋白酶,本文以海水养殖凡纳滨对虾肠道为原料,利用蛋白酶水解圈法初筛得到27株产蛋白酶菌株,测定菌株的透明圈直径与菌落直径比值(即D/d)初步估计其产酶能力,通过发酵液酶活测定复筛得到一株高产蛋白酶菌株,编号为xc10,通过形态学观察、生理生化鉴定、16S r DNA基因序列分析及系统发育树分析进行菌种鉴定,并对其发酵液粗蛋白酶的酶学性质进行了初步探讨。结果表明:菌株xc10为蜡状芽孢杆菌(Bacillus cereus),蛋白酶的最适温度为50℃;最适p H为7.5;Fe3+、Cu2+、EDTA对该酶有抑制作用,但Zn2+对该酶有激活作用。因此,从海水养殖的凡纳滨对虾肠道中分离得到的产蛋白酶的菌株xc10为一株中性、中温蛋白酶菌,为开发利用海洋微生物蛋白酶在水产品加工的应用提供了理论基础。     

糖萜素及不同温度对异育银鲫消化酶活力的影响

选取相同生长阶段、体重均匀的异育银鲫160尾,随机分为5个组,每个组16尾,每个组设2个重复。糖萜素的添加水平为0、0.5、0.9、1.2和1.5 g/kg。结果表明:在适宜的温度范围内,试验2组异育银鲫的蛋白酶、淀粉酶和脂肪酶活力最高,其次是试验1组和试验3组,对照组最低。蛋白酶适宜温度为40~50℃,淀粉酶适宜温度为30~40℃,脂肪酶适宜温度为30~40℃。糖萜素对异育银鲫消化酶活性具有正向促进作用,但在不同添加水平上,消化酶活性增强的程度不同,其适宜的添加量为0.5~0.9 g/kg。     

复合蛋白酶处理对小麦胚芽粉品质特性的影响

利用复合蛋白酶对小麦胚芽粉进行酶解处理,分析料液比、酶量以及酶解时间对小麦胚芽粉品质特性的影响。同时选取小麦胚芽酶解粉中水溶性指数最高的三组酶解粉与奶粉进行复配并作感官评定,以确定最佳复配比。结果表明,与小麦胚芽超微粉相比,蛋白酶酶解得到的小麦胚芽粉具有更好的品质。在料液比为1:5 g/mL、酶量为4400 U/g、反应时间为2.5 h时,小麦胚芽粉的水溶性指数达到最大值,为60.6 g/g,与原粉和超微粉的水溶性指数30.22、30.65 g/g相比,酶解之后提高约两倍。在料液比1:4 g/mL、酶量为4400 U/g、反应时间为2.5 h时,吸光度值由未酶解小麦胚芽粉的0.501降低到0.348,酶解小麦胚芽粉中脂肪酶的活力达到最小值。经过复合蛋白酶酶解,小麦胚芽粉的水溶性指数大大提高,小麦胚芽粉的脂肪酶活力有所下降,且酶解对麦胚芽粉的色差影响不显著。在与奶粉的复配实验中,小麦胚芽粉原粉、超微粉以及酶解粉在添加量为30%时,均获得最高的感官评分。     

非糊化条件对小麦蛋白肽酶法制备的影响

为了探讨联合制备小麦蛋白肽和小麦淀粉的新方法,以小麦面粉为原料,以小麦蛋白肽的纯度、得率、水解度、还原糖质量分数为评价指标,研究了淀粉非糊化状态下不同处理条件对两种蛋白酶制备小麦蛋白肽的作用效果。结果表明：与浸泡前加酶相比,浸泡后加酶生产的蛋白肽纯度高,还原糖质量分数低,但两者得率相差较小。最佳浸泡条件为：温度50 ℃,时间2.5 h。此条件下,中性蛋白酶水解得到的小麦蛋白肽纯度为27%,得率为75%,水解度为17.6%,还原糖质量分数为5.9%;碱性蛋白酶水解得到的小麦蛋白肽纯度为25%,得率为85%,水解度为23.7%,还原糖质量分数为9%。预处理过程中,宜采用浸泡后加酶。可根据需要选择不同蛋白酶生产,但碱性蛋白酶中淀粉酶含量较高会产生相对多的淀粉糖。     

Role of α-Helical Structure in Organic Solvent-Activated Homodimer of Elastase Strain K

Recombinant elastase strain K overexpressed from E. coli KRX/pCon2(3) was purified to homogeneity by a combination of hydrophobic interaction chromatography and ion exchange chromatography, with a final yield of 48% and a 25-fold increase in specific activity. The purified protein had exhibited a first ever reported homodimer size of 65 kDa by SDS-PAGE and MALDI-TOF, a size which is totally distinct from that of typically reported 33 kDa monomer from P. aeruginosa. The organic solvent stability experiment had demonstrated a stability pattern which completely opposed the rules laid out in previous reports in which activity stability and enhancement were observed in hydrophilic organic solvents such as DMSO, methanol, ethanol and 1-propanol. The high stability and enhancement of the enzyme in hydrophilic solvents were explained from the view of alteration in secondary structures. Elastinolytic activation and stability were observed in 25 and 50% of methanol, respectively, despite slight reduction in α-helical structure caused upon the addition of the solvent. Further characterization experiments had postulated great stability and enhancement of elastase strain K in broad range of temperatures, pHs, metal ions, surfactants, denaturing agents and substrate specificity, indicating its potential application in detergent formulation.     

蛋白酶产生菌LN02发酵玉米黄粉蛋白的条件优化

通过单因素实验和正交实验初步优化了蛋白酶产生菌LN02发酵玉米黄粉蛋白的条件。结果表明,菌株LN02发酵玉米黄粉蛋白的最佳条件为：初始pH值5.5、装液量60 mL/250 mL三角瓶、发酵温度35℃、发酵时间5 d。     

TMPRSS4 as a Poor Prognostic Factor for Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is characterized by the lack of immunohistochemical staining for estrogen receptors (ER), progesterone receptors (PR), and lack of overexpression or amplification of human epidermal growth factor receptor 2 (HER2). Our aim was to investigate the expression of transmembrane protease, serine 4 (TMPRSS4) in TNBC patients and its possible relationship to the outcome of the disease. A total of 72 TNBC patients and 109 non-TNBC patients who were diagnosed between 2003 and 2008 were enrolled in this study. Immunohistochemistry was used to compare the expression pattern of TMPRSS4 in TNBC and non-TNBC groups, and the prognostic significance was assessed by Kaplan-Meier analysis and Cox proportional hazards regression in TNBC patients. The rate of high expression of TMPRSS4 was significantly higher in TNBC group than that in non-TNBC group. High expression of TMPRSS4 was significantly correlated with lymph node metastasis, histological grade, and tumor size. TNBC patients with high TMPRSS4 expression showed the poorer overall survival (OS) and disease-free survival (DFS) than those patients with low TMPRSS4 expression. In multivariate analysis, only lymph node metastasis and TMPRSS4 expression were the independent prognostic factors for OS and DFS in TNBC. Our study provides evidence that TMPRSS4 expression is associated with lymph node metastasis, tumor size, and histological grade in TNBC patients, and also is an independent prognostic factor for TNBC.     

Synthesis of a Long Acting HIV Protease Inhibitor via Metal or Enzymatic Reduction of the Appropriate Chloro Ketone and Selective Zinc Enolate Condensation with an Amino Epoxide

This paper describes a new convergent approach to the synthesis of an HIV protease inhibitor which was designed to be suitable in long acting formulations. Unique features in the synthesis include an asymmetric hydrogenation as well as enzymatic reduction of a key chloro ketone intermediate, to set the threo stereochemistry in the corresponding epoxide and the diastereoselective coupling of the latter with the zinc enolate of a suitable functionalized amide derivative.