风吹楠种子油萃取工艺研究及其脂肪酸成分分析

本文以氯仿、乙醚和正己烷-异丙醇(v/v=3∶2)三种不同溶剂对风吹楠种子油进行萃取,比较了不同溶剂对种子萃出率的影响,结果表明以乙醚为溶剂时萃出率最高;并考察了萃取时间、超声波以及萃取溶剂体积对萃出率的影响。最终优化的萃取工艺为:以乙醚为溶剂,液料比为10∶1(v/w),超声波(25℃,60 W)萃取2h,萃出率达到60.27 wt%。萃取的种子油经转酯化后,GC-MS分析其脂肪酸组分,结果表明风吹楠种子油脂中月桂酸(42.58%)和肉豆蔻酸(47.55%)占总脂肪酸的90.13%,不饱和脂肪酸不到3%。     

Development of quantitative metabolomics for <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Accurate, reliable and reproducible measurement of intracellular metabolite levels has become important for metabolic studies of microbial cell factories. A first critical step for metabolomic studies is the establishment of an adequate quenching and washing protocol, which ensures effective arrest of all metabolic activity and removal of extracellular metabolites, without causing leakage of metabolites from the cells. Five different procedures based on cold methanol quenching and cell separation by filtration were tested for metabolomics of Pichia pastoris regarding methanol content and temperature of the quenching solution as key parameters. Quantitative evaluation of these protocols was carried out through mass balance analysis, based on metabolite measurements in all sample fractions, those are whole broth, quenched and washed cells, culture filtrate and quenching and washing solution. Finally, the optimal method was used to study the time profiles of free amino acid and central carbon metabolism intermediates in glucose-limited chemostat cultures. Acceptable recoveries (>90%) were obtained for all quenching procedures tested. However, quenching at −27°C in 60% v/v methanol performed slightly better in terms of leakage minimization. We could demonstrate that five residence times under glucose limitation are enough to reach stable intracellular metabolite pools. Moreover, when comparing P. pastoris and S. cerevisiae metabolomes, under the same cultivation conditions, similar metabolite fingerprints were found in both yeasts, except for the lower glycolysis, where the levels of these metabolites in P. pastoris suggested an enzymatic capacity limitation in that part of the metabolism.     

Metabolic profiling of transgenic rice progeny using gas chromatography–mass spectrometry: the effects of gene insertion, tissue culture and breeding

The Bacillus thuringiensis ??-endotoxin and cowpea trypsin inhibitor genes have been introduced into the rice genome to improve its pest resistance via Agrobacterium-mediated transformation. A gas chromatography-mass spectrometry (GC?CMS) based metabolic profiling method was employed to determine the unpredictable metabolic changes resulting from the gene insertion and tissue culture separately. Descendants of the same transformant were obtained from different breeding programs, including both the transgenic and null-segregant progeny. The comparison of the transgenic and respective null-segregant plants enabled the evaluation of variations caused by transgenes; also the null-segregant plants were compared with the wild-type control to identify the influence of tissue culture. Based on the GC?CMS metabolic profiles, the principal component analysis and significant differences determined by Student??s t-test suggested that there were more metabolic changes from the tissue culture than those from the insertion of the transgenes. By comparing different breeding programs, it was clear that the progeny which was developed after several generations of backcross with the non-transformed rice as the recurrent parent, displayed fewer metabolic differences from the non-transformed parent. A GC?CMS based metabolic profiling study confirmed that backcrossing can help to reduce unwanted variations that occur during transformation processes.     

In vitro analyses of the anti-fibrotic effect of SPARC silencing in human Tenon's fibroblasts: comparisons with mitomycin C

Failure of glaucoma filtration surgery (GFS) is commonly attributed to scarring at the surgical site. The human Tenon's fibroblasts (HTFs) are considered the major cell type contributing to the fibrotic response. We previously showed that SPARC (secreted protein, acidic, rich in cysteine) knockout mice had improved surgical success in a murine model of GFS. To understand the mechanisms of SPARC deficiency in delaying subconjunctival fibrosis, we used the gene silencing approach to reduce SPARC expression in HTFs and examined parameters important for wound repair and fibrosis. Mitomycin C-treated HTFs were used for comparison. We demonstrate that SPARC-silenced HTFs showed normal proliferation and negligible cellular necrosis but were impaired in motility and collagen gel contraction. The expression of pro-fibrotic genes including collagen I, MMP-2, MMP-9, MMP-14, IL-8, MCP-1 and TGF-β(2) were also reduced. Importantly, TGF-β(2) failed to induce significant collagen I and fibronectin expressions in the SPARC-silenced HTFs. Together, these data demonstrate that SPARC knockdown in HTFs modulates fibroblast functions important for wound fibrosis and is therefore a promising strategy in the development of anti-scarring therapeutics.     

SDS‐PAGE‐free protocol for comprehensive identification of cytochrome P450 enzymes and uridine diphosphoglucuronosyl transferases in human liver microsomes

Comprehensive identification of cytochrome P450 enzymes (CYPs) and uridine diphosphoglucuronosyl transferases (UGTs) in human liver microsomes (HLMs) was performed with an SDS‐PAGE‐free protocol. HLMs were solubilized with 5% v/v ionic liquid, 1‐butyl‐3‐methyl imidazolium tetrafluoroborate, followed by tryptic digestion, and 2D‐SCX‐RPLC‐ESI‐MS/MS (LTQ XL) analysis in triplicate. In total, 27 CYPs and 12 UGTs were confidently identified with average sequence coverage as 30.99 and 25.07%, average peptide number as 14 and 13, and average unique peptide number as 7 and 4, respectively. The highly similar isoforms of CYP3A, CYP2C, and CYP4F subfamilies could be unambiguously differentiated from each other, despite the fact that the sequence similarity of CYP2C9 and CYP2C19 is 91%. In addition, protein spectral count was used to approximately evaluate the relative abundance of identified CYPs and UGTs, and the results agreed with previous immunochemistry reports.     

UVRAG: At the crossroad of autophagy and genomic stability

UVRAG is a promoter of the autophagy pathway, and its deficiency may fuel the development of cancers. Intriguingly, our recent study has demonstrated that this protein also mediates the repair of damaged DNA and patrols centrosome stability, mechanisms that commonly prevent cancer progression, in a manner independent of its role in autophagy signaling. Given the central role of UVRAG in genomic stability and autophagic cleaning, it is speculated that UVRAG is a bona fide genome protector and that the decrease in UVRAG seen in some cancers may render these cells vulnerable to chromosomal damage, making UVRAG an appealing target for cancer therapy.     

太湖水华期间有毒和无毒微囊藻种群丰度的动态变化

采用荧光定量PCR技术分析太湖3个湖区(梅梁湾、贡湖湾和湖心)水体中有毒和无毒微囊藻基因型丰度及有毒微囊藻比例的季节变化(2010年4-9月),并与环境因子进行统计分析。结果表明,有毒微囊藻基因型丰度及所占比例存在季节和空间差异:从4-8月,有毒微囊藻基因型丰度及其比例呈逐渐增加趋势,到9月开始下降;梅梁湾水体中有毒微囊藻基因型丰度及其比例高于贡湖湾和湖心。梅梁湾、贡湖湾和湖心有毒微囊藻在微囊藻种群中的比例变化范围分别为(26.2±0.8)%-(64.3±2.2)%、(4.4±0.2)%-(22.1±1.8)%和(10.4±0.4)%-(20.6±1.5)%。相关分析结果表明,有毒微囊藻丰度、总微囊藻丰度和叶绿素a浓度呈极显著正相关(P<0.01),均与温度呈显著正相关(P<0.05);有毒微囊藻比例与磷浓度呈显著正相关(P<0.05),与温度呈极显著正相关(P<0.01)。研究结果表明,温度和磷浓度是决定太湖有毒微囊藻种群丰度及其比例的关键因子。     

河北省县域农田生态系统供给功能的健康评价

引入"生态系统服务功能"理论,构建县域农田生态系统供给功能健康评价指标——农田生产力稳定系数,该指标被定义为:一定时期内(若干年),县域自然生态系统第一性生产力波动系数与县域农田生态系统第一性生产力波动系数的平均比值;并构建了评价指标与人类福利指标(用县域人均耕地纯收入表征)关系模型,作为评价指标的阈值判定方法。通过河北省136个县的实证研究表明,农田生产力稳定系数对农田生产力稳定性和农田生产力高低具有综合性指示作用;人类社会对生态系统反馈影响的忍受界限对评价指标阈值的确定非常关键;1991—2000年和2001—2009年两个阶段中,河北省县域农田生产力稳定系数整体呈上升趋势,空间格局较为一致,农田生产力稳定系数较低、农田供给功能健康状况较差的县域集中分布在冀北生态脆弱地区;农田生态系统供给功能健康状况与县域贫困状况形成紧密的空间耦合关系。     

一次陆源降雨污水引起血红哈卡藻赤潮的成因

以2007年烟台四十里湾海域血红哈卡藻(Akashiwo sanguinea Hirasaka)赤潮为对象,研究赤潮消长与水环境因子的关系。研究发现大量陆源降雨污水输入后,海水盐度急剧下降、营养盐大幅增加,特别是活性磷酸盐浓度明显增加,促进了血红哈卡藻的生长繁殖并最终形成赤潮。赤潮发生前海区第一优势种为尖刺拟菱形藻(Pseudo-nitzschia pungens Halse),优势度0.47(0.42—0.52),多样性指数2.63(2.43—2.89);赤潮发生时血红哈卡藻密度范围1.05×105—4.10×106个/L,优势度0.92(0.83—0.99),多样性指数0.27(0.15—0.64);赤潮消退后中肋骨条藻(Skeletonema costatum Cleve)为第一优势种,优势度0.49(0.43—0.55),多样性指数2.46(2.19—2.84)。赤潮的发生、发展、消亡与化学需氧量(COD)、无机氮(DIN)、活性磷酸盐(DIP)、富营养化指数(E)呈显著正相关(P<0.05),与盐度呈显著负相关(P<0.01)。赤潮前、后该海域为贫营养、P限制、叶绿素a含量中等,赤潮期间该海域为富营养、P限制、叶绿素a含量高。通过影响力评定,活性磷酸盐、COD、盐度是此次赤潮发生的主要诱发因子,当活性磷酸盐含量低于0.3μmol/L时,硅藻逐渐取代甲藻,此次赤潮消散。