融合基因umcel5N-CBM的构建、表达及融合酶性质分析

BM,并实现了融合基因在大肠杆菌BL21(DE3)pLysS中的表达.研究结果表明,融合酶Umcel5N-CBM与结晶纤维素(avicel)以及滤纸粉末的结合能力比原始酶Umcel5N提高了约一倍,但未显示出降解结晶纤维素的新活性,说明在结晶纤维素的降解过程中,纤维素酶的催化功能域起到关键作用.     

DNA methylation changes in photoperiod-thermo-sensitive male sterile rice PA64S under two different conditions

Epigenetic modification can occur at a high frequency in crop plants and might generate phenotypic variation without changes in DNA sequences. DNA methylation is an important epigenetic modification that may contribute to environmentally-induced phenotypic variations by regulating gene expression. Rice Photoperiod-Thermo-Sensitive Genic Male Sterile (PTGMS) lines can transform from sterility to fertility under lower temperatures and short-day (SD) conditions during anther development. So far, little is known about the DNA methylation variation of PTGMS throughout the genome in rice. In this study, we investigated DNA cytosine methylation alterations in the young panicles of PTGMS line PA64S under two different conditions using methylation sensitive amplified polymorphism (MSAP) method. Compared with the DNA methylation level of PA64S under lower temperatures and SD conditions (fertility), higher methylation was observed in PA64S (sterility). The sequences of 25 differentially amplified fragments were successfully obtained and annotated. Three methylated fragments, which are homologous to D2, NAD7 and psaA, were confirmed by bisulfite sequencing and their expression levels were also evaluated by qPCR. Real time quantitative PCR analysis revealed that five of the six selected methylated genes were downregulated in PA64S (sterility). These results suggested that DNA methylation may be involved in the sterility–fertility transition of PA64S under two different environmental conditions.     

The effect of turmeric extracts on inflammatory mediator production.

Major compounds of several commonly used botanicals, including turmeric, have been purported to have anti-inflammatory actions. In order to test the anti-inflammatory activity of compounds isolated from rhizomes of Curcuma longa L. (Zingiberaceae), we have established an in vitro test system. HL-60 cells were differentiated and exposed to lipopolysaccharide (LPS) from Escherichia coli (1 microg/ml) in the presence or absence of botanical compounds for 24 h. Supernatants were collected and analyzed for the production of tumor necrosis factor alpha (TNF-alpha) and prostaglandin E2 (PGE2) using standard ELISA assays. Water-soluble extracts were not cytotoxic and did not exhibit biological activity. Organic extracts of turmeric were cytotoxic only at concentrations above 50 microg/ml. Crude organic extracts of turmeric were capable of inhibiting LPS-induced TNF-alpha (IC50 value = 15.2 microg/ml) and PGE2 (IC50 value = 0.92 microg/ml) production. Purified curcumin was more active than either demethoxy- or bisdemethoxycurcumin. Fractions and subfractions of turmeric extracts collected via preparative HPLC had differing biological activity, ranging from no activity to IC50 values of < 1 microg/ml. For some fractions, subfractionation resulted in a loss of activity, indicating interaction of the compounds within the fraction to produce an anti-inflammatory effect. A combination of several of the fractions that contain the turmeric oils was more effective than the curcuminoids at inhibiting PGE2. While curcumin inhibited COX-2 expression, turmeric oils had no effect on levels of COX-2 mRNA.     

艾滋病病毒蛋白酶高效表达载体及体外活性检测系统的构建

艾滋病是本世纪80年代初发现的一种烈性传染病,5年病死率为100％,致病因子为人免疫缺陷病毒,该病毒的蛋白酶在病毒复制和成熟中具有决定性的意义。由于目前国内外尚未获得艾滋病病毒蛋白酶的高效表达的重组子及活性检测系统,限制了它的研究与应用。本文利用PCR技术修饰了艾滋病病毒蛋白酶的基因,使其具有便于克隆及表达用的限制酶切位点及转录终止码,井在其C末端设置了一个可用于检验该酶活性的特殊序列。DNA序列分析揭示上述突变策略成功,将修饰后的艾滋病病毒蛋白酶基因克隆入大肠杆菌表达系统,并获得高效表达(>30％),Western-Bolt鉴定结果表明所表达的蛋白为艾滋病病毒所特有,并具有较好的生物活性。     

藕莲有性繁殖栽培

根据莲(Nelumbo nucifera)生物学特性,既可用藕作种进行无性繁殖,也可用种子播种进行有性繁殖。藕莲在我国有数千年的栽培历史,但素以无性系栽培。尚未见到有性繁殖栽培的报道。作者自1980年起进行了有性繁殖栽培试验。       

FRIL蛋白体外维持脐血造血干/祖细胞的作用及细胞周期调控基因的表达

为探讨新的豆类凝集素(Flt3 receptor-interacting lectin,FRIL)体外维持脐血CD34^ 细胞的作用以及维持过程中细胞周期调控基因HTm4及HTm4S mRNA的表达及意义,我们利用FRIL维持培养脐血CD34^ 细胞,对其增殖曲线、细胞周期及集落形成能力进行常规分析,并用半定量RT—PCR法分别测定FRIL体外维持不同时间后脐血CD34^ 细胞中周期调控基因HTm4及HTm4S mRNA的表达变化。结果显示,FRIL培养的CD34^ 造血干／祖细胞的增殖趋势平缓,整个培养期间细胞增殖倍数不超过起始的3倍：14d之前,FRIL培养细胞的高增殖潜能集落形成细胞(HPP—CFC)形成集落数与FL组无差别,其后则维持高于FL的情况。细胞周期分析则显示,在28d的培养过程内,利用FRIL培养的细胞始终有80％以上维持在G0期;而周期调控基因HTm4及HTm4S在刚分离的脐血CD34^ 细胞中的表达水平较高;但培养1d后,几乎检测不到HTm4基因的表达;培养3～14d,该基因的表达回升并持续维持在高水平。而HTm4S基因的表达在第7d达最高水平,其余时间基本呈稳定表达。转染HTm4和HTm4S,亚细胞定位结果显示HTm4主要定位于核周围,而HTm4S则定位于整个胞浆,由此可能导致它们功能的区别。以上结果提示,长期培养体现出FRIL在维持造血干／祖细胞多能性上的优势;细胞周期调控基因HTm4及其新剪接子参与了FRIL体外长期维持脐血造血干／祖细胞处于静息状态的过程。     

Ubiquitin-proteasome pathway modulates mouse oocyte meiotic maturation and fertilization via regulation of MAPK cascade and cyclin B1 degradation

Degradation of proteins mediated by ubiquitin-proteasome pathway (UPP) plays important roles in the regulation of eukaryotic cell cycle. In this study, the functional roles and regulatory mechanisms of UPP in mouse oocyte meiotic maturation, fertilization, and early embryonic cleavage were studied by drug-treatment, Western blot, antibody microinjection, and confocal microscopy. The meiotic resumption of both cumulus-enclosed oocytes and denuded oocytes was stimulated by two potent, reversible, and cell-permeable proteasome inhibitors, ALLN and MG-132. The metaphase I spindle assembly was prevented, and the distribution of ubiquitin, cyclin B1, and polo-like kinase 1 (Plk1) was also distorted. When UPP was inhibited, mitogen-activated protein kinase (MAPK)/p90rsk phosphorylation was not affected, but the cyclin B1 degradation that occurs during normal metaphase-anaphase transition was not observed. During oocyte activation, the emission of second polar body (PB2) and the pronuclear formation were inhibited by ALLN or MG-132. In oocytes microinjected with ubiquitin antibodies, PB2 emission and pronuclear formation were also inhibited after in vitro fertilization. The expression of cyclin B1 and the phosphorylation of MAPK/p90rsk could still be detected in ALLN or MG-132-treated oocytes even at 8 h after parthenogenetic activation or insemination, which may account for the inhibition of PB2 emission and pronuclear formation. We also for the first time investigated the subcellular localization of ubiquitin protein at different stages of oocyte and early embryo development. Ubiquitin protein was accumulated in the germinal vesicle (GV), the region between the separating homologous chromosomes, the midbody, the pronuclei, and the region between the separating sister chromatids. In conclusion, our results suggest that the UPP plays important roles in oocyte meiosis resumption, spindle assembly, polar body emission, and pronuclear formation, probably by regulating cyclin B1 degradation and MAPK/p90rsk phosphorylation.     

Theoretical electrical conductivity of hydrogen-bonded benzamide-derived molecules and single DNA bases

A benzamide molecule is used as a “reader” molecule to form hydrogen bonds with five single DNA bases, i.e., four normal single DNA bases A,T,C,G and one for 5methylC. The whole molecule is then attached to the gold surface so that a meta-molecule junction is formed. We calculate the transmission function and conductance for the five metal–molecule systems, with the implementation of density functional theory-based non-equilibrium Green function method. Our results show that each DNA base exhibits a unique conductance and most of them are on the pS level. The distinguishable conductance of each DNA base provides a way for the fast sequencing of DNA. We also investigate the dependence of conductivity of such a metal–molecule system on the hydrogen bond length between the “reader” molecule and DNA base, which shows that conductance follows an exponential decay as the hydrogen bond length increases, i.e., the conductivity is highly sensitive to the change in hydrogen bond length.     

Inhibition of IFN-gamma receptor signaling by hepatitis C virus non-structural protein NS4B]

The function of NS4B is incompletely understood. The aim of the study is to understand the influence of NS4B on anti-viral response. After cell line stably expressing NS4B established, the influence of IFN-alpha of different concentration on VSV was studied using plaque assay; cell expression profiling caused by NS4B was studied using DNA microarray, and the IFNGR1 fluorescence intensity was analyzed. Our data showed that HCV-NS4B could suppress immuno-associated gene expression, in particular, IFN-gamma receptor signal transduction-related genes. Taken together, NS4B could play some roles in HCV resistance to IFN therapy.