儿茶素单体EGCG酶法修饰研究及其产物抗氧化活性评价

利用Novozym 435脂肪酶在非水介质中催化儿茶素单体EGCG(表没食子儿茶素没食子酸酯)的酶促酰化反应,以增加EGCG的脂溶性。探讨了溶剂种类、水活度、加酶量、反应时间、反应温度、酰基供体等条件对酰化反应的影响。借助液质联用仪及红外光谱仪对合成产物进行鉴定,表明在叔戊醇体系中,脂肪酶可催化EGCG与丁酸乙烯酯的反应,酰化后EGCG主体结构不变,其分子中引入了四碳链的烷基。对修饰后EGCG抗氧化活性的评价表明:在相同的添加量下,酶修饰EGCG活性略低于未改性EGCG,但是清除DPPH·、O-·2自由基能力总体高于TBHQ、维生素C,清除·OH自由基能力低于TBHQ,高于维生素C。     

茶儿茶素抑制肿瘤血管生成的作用

对近几年茶儿茶素抑制肿瘤血管生成的研究进展进行了综述。从抑制血管内皮细胞生长、抑制细胞黏附分子表达和抑制基底膜降解三个部分讨论了其抗肿瘤血管生成的机制,并对儿茶素研究存在的问题和儿茶素开发前景进行了探讨。     

研制首个杀灭HIV的“仿生杀手”

“乙酰化修饰”——即在蛋白质分子链上嫁接上一个乙酰基分子,是蛋白质最主要的修饰方式之一。修饰后的蛋白质可以对细胞内的各类通路进行精确的调节与控制,完成对基因所发出的“指令”的执行过程。复旦大学生物医学研究院分子细胞生物学研究室的赵世民和雷群英等分别研究了乙酰化对蛋白质进行修饰以及对代谢通路进行调控的问题,开辟了代射研究的薪领域。他们鉴定出大量乙酰化化修饰蛋白质,并且首次发现乙酰化普遍能修饰代谢酶,调节代谢通路及代谢酶的活性。     

Maxam-Gilbert DNA序列分析实验方法(续)

碱基专一性的化学切断反应碱基专一性的部分切断反应包括以下三步:1.用具有碱基专一性的化学试剂对DNA分子中的某一种碱基进行部分修饰,控制修饰反应的条件,使DNA分子中每一个那样的碱基分别只在一部分DNA分子中被修饰;2.利用另一种化学试剂将被修饰的碱基从脱氧核糖     

抑癌蛋白p53乙酰化修饰的调控网络

p53是细胞内最重要的抑癌蛋白质之一;细胞对p53分子功能的调控主要通过一系列翻译后修饰(PTMs)完成。其中,乙酰化修饰既可在总体水平调控p53的转录活性,又可位点特异性地调控p53依赖的转录选择性,进而精确控制p53在细胞周期阻滞、凋亡、衰老、自噬和代谢等关键生物学过程中的作用。本综述以p53乙酰化修饰研究的时间脉络为轴,首先总结了发生在p53各结构域内乙酰化修饰的建立机制,包括催化p53位点特异性乙酰化发生的乙酰基转移酶,以及各位点乙酰化修饰对p53分子功能调节的机制。其次,本综述总结了参与去除p53乙酰化修饰的关键去乙酰基酶家族,以及这些因子参与调控p53分子功能的生物学意义。同时,本文综述了能够特异性读取p53乙酰化修饰状态的识别蛋白质,以及这些识别蛋白质与p53互作,进而协同调控下游靶基因转录的分子调控网络。此外,本文概述了p53乙酰化修饰与其它类型翻译后修饰之间的“交谈”,以及这些修饰之间通过时空特异互作方式影响p53功能的分子机制。最后,本文基于p53乙酰化修饰,对肿瘤分子医学的研究前景进行讨论与展望。     

PEG修饰对PLL-EGF基因载体靶向结合作用的影响

聚阳离子基因载体系统由于安全性好和便于设计等优点,近年来在基因治疗中的应用发展迅速.在进行基因药物的体内靶向输送时,目前国际上主要通过在基因输送系统中修饰聚乙二醇(PEG)和靶向分子来提高体内输送的稳定性和靶向性.PEG的修饰可能会遮蔽靶向分子的功能呈现,因此建立定量分析方法评价PEG修饰对靶向结合作用的影响非常重要.将连接有表皮生长因子(EGF)的聚赖氨酸(PLL)基因载体作为研究模型,建立BIAcore检测方法,比较PLL-EGF,PEG7000修饰的PLL-EGF,PEG20000修饰的PLL-EGF对表皮生长因子受体(EGFR)的结合和解离速率,评价PEG修饰对PLL-EGF靶向功能呈现的影响.结果表明,PEG7000的修饰降低了EGF和EGFR之间的结合速率,提高了解离速率,整体减弱了靶向分子的靶向结合能力.PEG20000的修饰进一步减弱靶向分子功能的呈现.因此在进行靶向型聚阳离子基因输送系统设计时,考察PEG修饰对靶向结合能力的影响程度非常重要.该研究结果也对其他基因载体系统的设计提供必要的参考.     

海藻多糖生物活性及分子修饰

简要介绍了近年来有关海藻多糖抗病毒、抗肿瘤、免疫促进、免疫抑制等生理活性的最新研究 ,重点介绍了不同种类海藻多糖的不同生理活性机理的研究进展。对多糖与生理活性之间的构效关系进行了阐述 ,在构效关系基础上进行多糖分子结构修饰是提高海藻多糖生理活性、降低毒副作用的有效手段。进一步介绍了目前多糖分子修饰常用方法 ,并对修饰后分子的生理活性改变进行了阐述。     

修饰SOD及未修饰SOD的稳定性比较研究

测定了分子修饰ＳＯＤ的酶活力及其在不同温度下的稳定性,与未修饰的ＳＯＤ进行了比较,对在不同温度下测定的结果进行数据处理,经推算得出分子修饰ＳＯＤ在２５℃条件下,保存９５％的酶活力达３．５年,保存９０％的酶活力达７．２年,未修饰ＳＯＤ在２５℃条件下,保存９５％的酶活力为１０３ｄ,保存９０％的酶活力为２１３ｄ。     

新型蛋白质修饰剂的合成及修饰牛血红蛋白的初步研究

以L-谷氨酸和己二酸为原料合成了一种新型的四官能团蛋白质修饰剂,并用核磁共振和红外光谱对其结构进行了表征。然后以其为修饰剂,对牛血红蛋白的化学修饰进行了初步的研究,并通过高效液相色谱、聚丙烯酰胺凝胶电泳和血氧分析仪对交联牛血红蛋白的分子量和携氧性能进行了表征。结果表明,该修饰剂可以使牛血红蛋白同时在分子内和分子间发生化学交联,并较好地保持携氧能力（P50:21.7mmHg,Hill系数:2.01）,因此在众多用于开发人工血液代用品的化学修饰剂中该修饰剂具有良好的应用前景。     

遗传修饰技术在绵羊分子设计育种中的应用

利用遗传修饰技术可以使动物在遗传水平发生改变,实现在个体内表达外源基因或对其内源基因的功能造成影响。在动物育种中,可以利用遗传修饰技术在分子水平进行设计并实现品种的快速改良。从传统的遗传修饰技术、病毒载体、精子载体等介导的遗传修饰技术到新型人工核酸酶介导的基因编辑技术,尤其是CRISPR/Cas9为代表的人工核酸酶的运用使得基因编辑动物的制备变得更加高效快捷,并迅速在多个物种中得到应用。这些方法也已经拓展到了绵羊(Ovis aries)遗传育种领域。利用遗传修饰技术在绵羊中进行分子育种比传统的育种方式具有更大的优势,可以使用多种策略直接对性状进行快速改良,并且可以加快育种进程。本文详细介绍了遗传修饰技术在绵羊中的研究历程,探讨了通过遗传修饰技术进行绵羊分子设计育种的可能性,并提出以上技术和方法在绵羊育种中面临的问题和挑战。     

The dynamics of phenolic compounds and soluble sugars in the leaves of the silver birch (<Emphasis Type="Italic">Betula pendula</Emphasis> Roth) after defoliation and their significance in entomological plant resistance

The dynamics of phenolic compounds, flavonols, catechines, tannins, and soluble sugars in the leaves of the silver birch Betula pendula Roth after strong (75%) and total (100%) artificial defoliation was studied. It was shown that the flavonol content in the leaves did not change after strong and total defoliation, while the amount of tannins did not change during the first 15 days but increased later on 1 and 2 years after 75% and 100% defoliation. The catechine content did not change during the first 15 days and increased later on 1 year after strong and total defoliation; however, it returned to the level of control plants 2 years after both types of defoliations. The amount of soluble sugars in the leaves increased 2 days after 75% defoliation; however, their content conformed to that in control plants after 10 days and it remained later 1 and 2 years after the damage. The amount of soluble sugars in the leaves also did not change 1 year after 100% defoliation.     

三硝基苯磺酸法测定聚乙二醇化重组人生长激素的平均修饰度

目的:建立测定三种不同结构聚乙二醇化重组人生长激素(PEG-rhGH)平均修饰度的三硝基苯磺酸法(TNBS)。方法:采用基质辅助激光解吸附飞行时间质谱(MALDI-TOF)测定PEG-rhGH的分子量,求得理论平均修饰度。既而通过正交试验、游离PEG干扰等方法学考察,建立TNBS法。结果:从质谱法可知PEG-rhGH的理论修饰度为10%,TNBS法测得三种PEG-rhGH的修饰度分别为为10%、12%及10%,与理论值相符。结论:TNBS操作简单,不需要专门的仪器,可作为测定PEG-rhGH平均修饰度的方法。     

Experimental evolution, loss-of-function mutations, and "the first rule of adaptive evolution"

Adaptive evolution can cause a species to gain, lose, or modify a function; therefore, it is of basic interest to determine whether any of these modes dominates the evolutionary process under particular circumstances. Because mutation occurs at the molecular level, it is necessary to examine the molecular changes produced by the underlying mutation in order to assess whether a given adaptation is best considered as a gain, loss, or modification of function. Although that was once impossible, the advance of molecular biology in the past half century has made it feasible. In this paper, I review molecular changes underlying some adaptations, with a particular emphasis on evolutionary experiments with microbes conducted over the past four decades. I show that by far the most common adaptive changes seen in those examples are due to the loss or modification of a pre-existing molecular function, and I discuss the possible reasons for the prominence of such mutations.     

三硝基苯磺酸法测定聚乙二醇化重组人生长激素的平均修饰度

目的：建立测定三种不同结构聚乙二醇化重组人生长激素（PEG-rhGH）平均修饰度的三硝基苯磺酸法（TNBS）。方法：采用基质辅助激光解吸附飞行时间质谱（MALDI-TOF）测定PEG-rhGH的分子量,求得理论平均修饰度。既而通过正交试验、游离PEG干扰等方法学考察,建立TNBS法。结果：从质谱法可知PEG-rhGH的理论修饰度为10%,TNBS法测得三种PEG-rhGH的修饰度分别为为10%、12%及10%,与理论值相符。结论：TNBS操作简单,不需要专门的仪器,可作为测定PEG-rhGH平均修饰度的方法。     

Comparison of the effects of chemical and isotopic dilution on the dissociation of bound labeled ligands

The dissociation of a labeled ligand from a binding structure to which it is reversibly attached can be promoted either by dilution or by chase. The kinetics of the dissociation brought up by dilution can be modified or not by the presence of various concentrations of cold ligand, according to the molecular mechanism of interaction. Analog computer simulation leads to the following results: (i) no cooperativity, monomolecular dissociation: no modification; (ii) positive or negative cooperativity (sequential models): acceleration, no modification, or slowing down (according to the kinetic constants); (iii) positive cooperativity (concerted model): no modification; (iv) two-step interaction: no modification if both interaction steps take place in the same phase, otherwise acceleration; and, (v) bimolecular association and dissociation: acceleration. This methodology could be used in order to characterize the molecular properties of various binding structures in the field of drug and hormone receptors as well as in enzymology.     

链霉菌无痕敲除方法研究进展

链霉菌是革兰氏阳性放线菌,能产生大量具有重要价值的天然代谢产物.随着基因测序技术、分子生物学和合成生物学的不断发展,人类对链霉菌家族有了更深的了解,从分子水平对其基因组进行改造的手段也越来越多.通过对链霉菌基因组合理、高效的精简,将提高链霉菌代谢产物的产量和质量,降低底物原料的消耗量.无痕敲除就是开展此项研究工作的重要手段.文章综述了近年来在链霉菌中广泛使用的分子操作方法并重点对链霉菌无痕敲除方法进行了总结.     

In vivo acetylation identified at lysine 70 of human lens alphaA-crystallin.

Posttranslational modification of protein lysyl residues that change the net charge of the molecule may alter the protein conformation. Such modifications are of particular significance among lens proteins, because conformational changes are associated with the development of cataract. A previously unidentified acetylated form of alphaA-crystallin has been isolated from the water-soluble portion of human lenses. The alphaA-crystallins were fractionated by anion exchange HPLC into seven peaks, each containing more than one form of alphaA-crystallin. The previously reported deamidated and phosphorylated forms were identified by their molecular masses, determined by electrospray ionization mass spectrometry. In addition to these modifications, approximately 5% of alphaA-crystallin had a modification that decreased the charge by one and increased the molecular mass by 42 u. This modification, identified as acetylation, was located uniquely at Lys 70. Like any modification that alters the surface charge, acetylation may affect protein conformation and intermolecular interactions, thereby altering the solubility or chaperone properties of alphaA-crystallin. Acetylation of lysine 70 is potentially significant since it is located in a region that has been implicated in the chaperone activity of alphaA-crystallin.     

人线粒体tRNA修饰碱基与遗传性脑肌病

人的多种遗传疾病与线粒体tRNA基因突变有关,这些突变导致疾病发生的分子机理是当前研究的热点.通过研究线粒体tRNA分子上的碱基修饰情况,人们发现了一类特殊的带有牛磺酸衍生物基团的修饰,这类修饰主要位于线粒体tRNALys和线粒体tRNALeu(UUR)反密码子第一位摆动(wobble)位点的碱基上.最近的研究表明,位于这两种线粒体tRNA基因上的多种突变与遗传性脑肌病相关,包括A8344G,A3243G,T3271C等等,它们可以导致tRNA上相应摆动位点的碱基修饰缺失.无论是在体外培养的带有相应突变的细胞内,还是在来源于脑肌病病人的组织中,科学家都发现了相同的线粒体tRNA碱基修饰缺陷.通过分子手术证实,此类碱基修饰对于维持这两种tRNA的反密码子与mRNA上相应密码子的相互识别至关重要,缺失了这种修饰的tRNA将无法识别一些对应的密码子.通过进一步的实验,人们还鉴定出负责催化此类碱基修饰的酶.这些研究不但揭示了线粒体遗传性脑肌病相关突变的致病机理,也将为研究基因治疗提供可能的新手段.     

Dynamic O-GlcNAc modification of nucleocytoplasmic proteins in response to stress. A survival response of mammalian cells

Cellular response to environmental, physiological, or chemical stress is key to survival following injury or disease. Here we describe a unique signaling mechanism by which cells detect and respond to stress in order to survive. A wide variety of stress stimuli rapidly increase nucleocytoplasmic protein modification by O-linked beta-N-acetylglucosamine (O-GlcNAc), an essential post-translational modification of Ser and Thr residues of metazoans. Blocking this post-translational modification, or reducing it, renders cells more sensitive to stress and results in decreased cell survival; and increasing O-GlcNAc levels protects cells. O-GlcNAc regulates both the rates and extent of the stress-induced induction of heat shock proteins, providing a molecular basis for these findings.     

A frequency-controlled random mutagenesis method for GC-rich genes

A novel random mutagenesis strategy was developed by combining sodium bisulfite modification with polymerase chain reaction (PCR). This method introduced the predominant substitution of GC to AT, meaning that it was more suitable for mutagenesis of GC-rich genes and helped to decrease the GC content of target DNA. Mutation efficiency correlated with modification time and different mutation frequency could easily be obtained by controlling modification time. The results indicated that this method could yield a desired and adequate frequency of random mutation to the DNA of interest, especially GC-rich genes, and provided a powerful tool for directed molecular evolution.