Regulation of intracellular decorin via proteasome degradation in rat mesangial cells

Decorin (DCN) is a member of small leucine‐rich proteoglycan family that neutralizes the bioactivity of transforming growth factor‐beta1 (TGF‐β1). It has been proven to be a promising anti‐fibrotic agent to treat glomerulonephritis. But the underlining mechanism for regulating and degrading intracellular DCN is still not fully understood. In this study, we investigated the roles of ubiquitination in the regulation of cytoplasmic DCN metabolism in rat mesangial cells (MC) by immunoprecipitation and Western blot. The results showed that a proportion of cytoplasmic DCN was ubiquitinated in normal MC and was enhanced in N‐glycosylation inhibitor (tunicamycin)‐treated MC. After being treated with the proteasome inhibitor MG132, ubiquitinated DCN accumulated and displayed a prolonged half‐life, accompanied by decreased TGF‐β1 expression and reduced collagen IV mRNA level in MC. This study demonstrated that the stability and function of cytoplasmic DCN can be regulated by ubiquitin‐proteasome system (UPS) in MC, which implies that regulating the ubiquitination and degradation of DCN might be a novel approach for modulating MC bioactivity. J. Cell. Biochem. 111: 1010–1019, 2010. © 2010 Wiley‐Liss, Inc.     

辛伐他汀和非诺贝特对HepG2细胞载脂蛋白M表达的影响

目的:观察辛伐他汀和非诺贝特及两者联合对HepG2细胞载脂蛋白M表达的影响。方法:分别以不同浓度的辛伐他汀(0、1、5、10、25μmol/L)和非诺贝特(0、50、100mmol/L)及辛伐他汀(5.0μmol/L)+非诺贝特(50mmol/L)干预HepG2细胞24h。提取各组细胞总RNA和蛋白质,分别采用实时RT-PCR和WesternBlot检测apoM的mRNA和蛋白的表达。结果:辛伐他汀和非诺贝特均呈剂量依赖性上调载脂蛋白M基因和蛋白的表达(P<0.05)。联合用药比单药更能显著上调载脂蛋白M的表达(P<0.05)。结论:他汀类和贝特类药物均可上调载脂蛋白M表达,两药联合的作用更为显著。     

利用LabVIEW构建血管生物反应器数据采集系统

对组织工程血管的参数监测在血管的培养过程中有着重要的意义。传统的仪表硬件系统能对反应器部分参数进行测量,但仍存在一些不足之处,本文通过分析确定血管生物反应器的监控参数,并利用虚拟仪器技术及LabVIEW软件开发平台构建了血管组织工程参数监测系统,该系统在实际应用中表明:系统简捷、运行稳定,能够监测血管的体外培养并达到了预定的精度要求。Labview的     

Molecular Engineering of Fungal GH5 and GH26 Beta-(1,4)-Mannanases toward Improvement of Enzyme Activity

Microbial mannanases are biotechnologically important enzymes since they target the hydrolysis of hemicellulosic polysaccharides of softwood biomass into simple molecules like manno-oligosaccharides and mannose. In this study, we have implemented a strategy of molecular engineering in the yeast Yarrowia lipolytica to improve the specific activity of two fungal endo-mannanases, PaMan5A and PaMan26A, which belong to the glycoside hydrolase (GH) families GH5 and GH26, respectively. Following random mutagenesis and two steps of high-throughput enzymatic screening, we identified several PaMan5A and PaMan26A mutants that displayed improved kinetic constants for the hydrolysis of galactomannan. Examination of the three-dimensional structures of PaMan5A and PaMan26A revealed which of the mutated residues are potentially important for enzyme function. Among them, the PaMan5A-G311S single mutant, which displayed an impressive 8.2-fold increase in k_cat/K_M due to a significant decrease of K_M, is located within the core of the enzyme. The PaMan5A-K139R/Y223H double mutant revealed modification of hydrolysis products probably in relation to an amino-acid substitution located nearby one of the positive subsites. The PaMan26A-P140L/D416G double mutant yielded a 30% increase in k_cat/K_M compared to the parental enzyme. It displayed a mutation in the linker region (P140L) that may confer more flexibility to the linker and another mutation (D416G) located at the entrance of the catalytic cleft that may promote the entrance of the substrate into the active site. Taken together, these results show that the directed evolution strategy implemented in this study was very pertinent since a straightforward round of random mutagenesis yielded significantly improved variants, in terms of catalytic efiiciency (k_cat/K_M).     

小麦叶锈病新抗源筛选

小麦叶锈病是小麦生产的主要病害之一,发病严重时往往导致大幅度减产。叶锈菌生理小种的变异易导致抗病基因抗性的丧失,因此不断获得新抗源对小麦抗病育种至关重要。小麦近缘植物中含有丰富的小麦育种所需的抗病基因。本研究从小麦-近缘植物双二倍体、附加系、代换系或易位系等创新种质中筛选出小麦叶锈病新抗源,为利用这些新抗源打下基础。苗期对116份供试材料人工接种美国堪萨斯州流行的小麦叶锈菌混合生理小种 (Lrcomp) ,其中部分材料人工接种09-9-1441-1等5个中国当前流行的叶锈菌生理小种进行抗性鉴定,筛选获得新抗源。116份种质中,31份免疫、近免疫或高抗Lrcomp。含有希尔斯山羊草、尾状山羊草、拟斯卑尔脱山羊草、两芒山羊草、卵穗山羊草、沙融山羊草、柱穗山羊草、顶芒山羊草、小伞山羊草、偏凸山羊草、中间偃麦草、茸毛偃麦草、长穗偃麦草、粗穗披碱草、栽培黑麦、非洲黑麦、提莫菲维染色质的部分种质免疫或高抗Lrcomp,而含二角山羊草、无芒山羊草、沙生冰草、多年生簇毛麦和一年生簇毛麦染色质的种质表现中感至高感Lrcomp。希尔斯山羊草4S染色体、尾状山羊草C#1和D#1染色体和两芒山羊草、顶芒山羊草中可能含有未被报道的抗Lrcomp的新基因,值得进一步向小麦转育。小麦-粗穗披碱草1HtS.1BL罗伯逊易位系对Lrcomp及 09-9-1441-1和09-9-1426-1等5个中国当前流行叶锈菌生理小种近免疫,值得利用染色体工程等方法获得小片段抗病易位系应用于我国小麦抗叶锈育种。     

异戊烯基焦磷酸转运对雷公藤甲素生物合成的影响

雷公藤甲素是一种具有显著抗炎、抗肿瘤和免疫抑制活性的天然产物,市场需求量大,临床应用前景广阔。文中以雷公藤悬浮细胞为实验材料,通过对不同培养时期(7 d、14 d)的细胞外源性添加D,L-甘油醛(DLG)以阻断异戊烯基焦磷酸(IPP)转运,分析诱导前后的细胞活性及生物量、雷公藤甲素累积量及其生物合成上游途径关键酶基因的变化规律,探究IPP转运在雷公藤甲素生物合成过程中的时空特点及其影响机制。通过实验结果可知:IPP转运参与雷公藤甲素的生物合成;在细胞培养早期,IPP转运主要经由质体(MEP途径)转运到细胞质(MVA途径)中,而细胞培养晚期则相反;阻断IPP转运,可反馈调节雷公藤甲素生物合成上游途径关键酶基因的表达,进而影响雷公藤甲素的累积。上述研究填补了雷公藤甲素生物合成过程中IPP转运特点和机制方面的空白,为雷公藤甲素合成生物学关键技术的开发奠定了理论基础,具有重要的现实意义。     

植物耐热相关基因研究进展

植物受到高温胁迫时,会激活某些特定基因的表达,从而增强植物的耐热性。近年来,随着生物技术的不断发展,植物耐热相关基因被相继克隆并转化植物体。本文对植物耐热的分子机制、相关基因的克隆、耐热性基因工程研究进展进行了综述,并提出了植物耐热基因工程的研究方向。     

水稻种子花色苷形成及其对稻谷储藏特性的影响

花色苷是有色稻种皮和颖壳里呈现颜色的主要物质,属于类黄酮类物质,具有抗氧化性。本研究围绕水稻种子形成过程中花色苷含量的变化以及其对稻谷储藏特性变化的影响,探讨花色苷在稻谷储藏中延长种子寿命的作用。结果显示:选取的3个品种中,水稻种子的花色苷形成主要在发育初期,随着颖果体积的增大,颖果花色苷的含量会逐渐降低,而颖壳的花色苷含量变化不显著,将种子进行人工老化后,仍只有颖果的花色苷含量出现下降的趋势,表明种子在老化过程中颖壳的花色苷不参与抗老化的各项代谢反应。老化种子SOD活性分析显示,花色苷含量低的中籼9311可能是通过SOD酶活性增加,削减高温高湿胁迫对种子衰老的影响;由稻谷感染霉菌情况显示在3个水稻品种中,花色苷含量高的品种的抗霉菌能力要稍优于花色苷含量低的品种。但综合来看,花色苷含量高低并不是决定种子寿命的主要因素。