寡聚脱氧核苷酸的结构与抗降解特性的研究

合成了４段具有不同高级结构或不同修饰的寡聚脱氧核苷酸,检查它们在２０％血清中的稳定性．发现：（１）寡核苷酸主要被血清中的３′外切核酸酶降解,未经修饰的线性寡核苷酸降解严重;（２）末端部分硫代修饰的寡核苷酸稳定性明显提高;（３）自身互补形成的配对结构可有效保护３′末端．具有４个以上（含４个）ＧＣ对的３′端发夹结构寡核苷酸,其抗核酸酶的能力几乎与硫代修饰的寡核苷酸相当．     

用甲基化的寡聚核苷酸作反义制剂

在含甲基磷酸根的寡核苷酸中,核苷酸之间含有非离子键,能抵抗细胞核酸酶的降解。一这种寡聚物能被培养的哺乳动物细胞完全吸收,它们能与细胞或病毒mRNA的启始密码或编码区或前体RNA的拼接位点有效的结合,特异性地抑制mRNA在细胞中表达。在甲基磷酸根寡核苷酸上衍生与靶mRNA共价交联的功能团,可以增强这种反义寡聚物的效率。这种寡聚核苷酸类似物是研究和控制基因表达的有用工具,并有希望开发成为治疗制剂。     

EGFR反义脱氧寡核苷酸及其硫代修饰片段抑制人肝癌细胞系BEL-7404细胞生长

本文旨在研究针对EGFR mRNA的反义寡核苷酸片段对BEL-7404细胞EGFR基因表达及其对细胞生长的影响。合成了互补于EGFR基因5′起始编码区的21聚脱氧寡核苷酸(ODNs)。实验结果显示,3.2μmol/L ODNs明显抑制BEL-7404细胞生长,[~3H]TdR掺入抑制试验显示其对细胞DNA合成的抑制作用表现剂量依赖性;密度扫描分析显示ODNs处理6、24h后,肝癌细胞EGFR mRNA分别下降10.5%和14.3%,ODNs处理4天后,细胞EGFR含量下降37.4%,同时观察了同一长度、针对同一靶基因区域的硫代磷酸型反义寡核苷酸对BEL-7404细胞的作用,表明3.2μmol/L ODNs在作用30h内,细胞DNA合成抑制率达62.1%,但此后渐下降,而SODNs在作用96h后达相应的抑制率(此后作用仍持续),提示S-ODNs在体外培养体系中有较好的稳定性。结果表明,针对EGFR基因的反义脱氧寡核苷酸片段可通过部分封闭EGFR基因表达而抑制BEL-7404细胞的生长,而硫代修饰型比未修饰型反义核酸片段具有更好的稳定性。     

端粒酶RNA组分的反义寡核苷酸对BEL-7404人肝癌细胞端粒酶活性的抑制和细胞凋亡的诱导

端粒酶在四株人肝癌细胞中均有表达,而在正常人肝组织标本中却为阴性.在终浓度为1μmol/L时,针对端粒酶RNA组分的反义寡核苷酸对BEL7404人肝癌细胞端粒酶活性具明显抑制作用,而正义和错义寡聚物则对该细胞端粒酶活性几乎没有影响.随着反义物浓度的降低,抑制作用亦随之减弱.用终浓度为5μmol/L的反义物处理培养的BEL-7404人肝癌细胞96 h后,细胞形态发生变化.原位末端标记法(TLUNEL)显示阳性标记细胞,流式细胞仪分析表明反义物作用后细胞凋亡率达到42.58%.     

p~(53)基因在U 937细胞生长和分化过程中的调节作用

本文报道了p~(53)基因对人白血病细胞系U 937细胞生长和分化的调节作用。重组人GM-CSF(rhGM-CSF)可诱导U 937细胞向成熟巨噬细胞分化,这反映在分化后的细胞表达有巨噬细胞许多表型特征和功能活性。在这一分化过程中同时伴随着p~(53)基因的表达增加和U 937细胞生长受抑。进一步,用反义脱氧寡聚核苷酸抑制试验特异性地抑制p~(53)基因表达,结果发现p~(53)反义脱氧寡聚核苷酸可以明显抑制rhGM-CSF诱导U 937细胞向成熟巨噬细胞分化,同时也明显解除rhGM-CSF介导细胞分化过程中的细胞生长抑制作用。这些结果说明p~(53)基因在U 937细胞的生长和分化过程中可能起偶联调控作用。     

修饰性肽核酸的细胞转运

肽核酸是人工合成的寡核苷酸类似物,以N-(2-氨乙基)甘氨酸结构单元替代DNA分子中的戊糖-磷酸结构。与天然核酸相比,肽核酸可以更高效地与DNA或RNA特异性杂交,在分子生物学和基因药物领域具有良好的应用前景。但是,肽核酸骨架呈电中性,难以高效穿过细胞膜,这成为工程应用的最大障碍。为了改善肽核酸的细胞转运性能,对肽核酸进行化学修饰是近年来的研究热点。结合近十年来文献报道和本实验室的工作,对肽核酸的骨架修饰和配合物结合修饰两类增强细胞转运的修饰方法进行综述,并对修饰性肽核酸细胞转运研究中存在的问题以及未来的研究趋势及其应用提出了见解。     

碱基上的氨臂对T4多核苷酸激酶催化的影响

寡聚核苷酸内部和5′末端含有的氨臂修饰胸苷酸,能够明显地影响T₄多核苷酸激酶催化的[γ- ³²P]ATP的转移反应.其转移效率为未修饰寡聚核苷酸的50%和2%.这种修饰的寡聚核苷酸对T₄ RNA连接酶催化的反应没有影响.     

基因组编辑技术中供体DNA类型及选择

基因组编辑技术能够实现基因组的精确修饰和改造,是后基因组时代研究基因功能和遗传信息的主要手段。传统的基因打靶技术通过低效率的细胞自发同源重组实现目的基因的定点修饰。真核细胞中DNA双链断裂介导的同源重组效率远高于自发同源重组,利用人工核酸内切酶特异性地在基因组靶序列处引入双链断裂,通过提供适当形式的、含有一定长度同源臂的供体DNA,能够实现相对高效的基因组靶向编辑。本文系统总结了环状质粒、线性化质粒、聚合酶链式反应产物及单链寡聚脱氧核苷酸4种类型的供体DNA在基因组精确编辑研究中的应用及候选原则,以期为以后相关研究中供体DNA的选择、设计提供参考和借鉴。     

基因组编辑技术中供体DNA类型及选择北大核心CSCD

基因组编辑技术能够实现基因组的精确修饰和改造,是后基因组时代研究基因功能和遗传信息的主要手段。传统的基因打靶技术通过低效率的细胞自发同源重组实现目的基因的定点修饰。真核细胞中DNA双链断裂介导的同源重组效率远高于自发同源重组,利用人工核酸内切酶特异性地在基因组靶序列处引入双链断裂,通过提供适当形式的、含有一定长度同源臂的供体DNA,能够实现相对高效的基因组靶向编辑。本文系统总结了环状质粒、线性化质粒、聚合酶链式反应产物及单链寡聚脱氧核苷酸4种类型的供体DNA在基因组精确编辑研究中的应用及候选原则,以期为以后相关研究中供体DNA的选择、设计提供参考和借鉴。     

寡核苷酸药物及寡核苷酸制备技术研究进展

寡核苷酸是生物医学和生命科学研究中调节基因表达的基本工具,并被开发为基因靶向治疗药物,用于治疗病毒、肿瘤和遗传病。寡核苷酸药物主要包括反义寡核苷酸、小干扰RNA、核酶、脱氧核酶、反基因、Cp G寡核苷酸、转录因子诱饵和核酸适配体等。天然的寡核苷酸在体内很容易被降解,特异性低,且有毒副作用。因此,药物寡核苷酸通常带有特定的修饰基团,如硫代磷酸二酯键、氟代、甲基以及锁核酸等,以增强寡核苷酸在体内的稳定性,提高特异性,并降低其毒副作用。目前,寡核苷酸主要采用化学方法合成,但化学合成的寡核苷酸初产物纯度低,而纯化十分困难。大规模核酸合成仪和纯化设备十分昂贵,因而大量合成和纯化寡核苷酸的成本高昂,大大限制了寡核苷酸药物的研究和应用。尽管已经涌现了多种多样的核酸扩增和检测方法,但用于扩增寡核苷酸的方法极少,且均不适合大量制备寡核苷酸。一种新的基于热循环的寡核苷酸扩增方法,称为"聚合酶-内切酶扩增反应"(Polymerase-endonuclease amplification reaction,PEAR),能够使寡核苷酸等小分子核酸在双酶催化下,利用独特的"滑动-切割机制"进行自我复制,并实现指数扩增。PEAR反应简单、高效、稳定。该方法已成功制备硫代和氟代修饰的寡核苷酸,与化学合成法相比,该技术不依赖于大规模DNA合成仪,降低了生产成本,适合大量生产高纯度的寡核苷酸,将有助于推动寡核苷酸药物的研究和应用。     

The role of pH and ion transport in oligosaccharide-induced proteinase inhibitor accumulation in tomato plants

Abstract. The induction of proteinase inhibitor (PrI) activity in young tomato plants by wounding and oligosaccharides has been shown to be prevented by pretreatment of the plants with phenolic acids such as aspirin. Aspirin applied over a wide range of concentrations is able to inhibit PrI induction by pectic fragments. The possibility that other weak acids may also inhibit PrI induction was investigated. Isobutyric acid and trimethylacetic acid were shown to be less effective as inhibitors than aspirin, and weak bases were without effect. However, it was demonstrated that various agents known to influence ATPase activity and intracellular pH were able to inhibit PrI induction, and in particular the striking effect of low concentrations of fusicoccin on PrI induction was noted.     

Inhibition of calmodulin-dependent and independent cardiac sarcoplasmic reticulum activities by R24571

R24571 a derivative of the antimycotic miconazole, appears to be 5 to 8 times more potent than trifluoperazine in its ability to inhibit the calmodulin-dependent phosphorylation of cardiac sarcoplasmic reticulum vesicles. The cAMP-dependent protein kinase mediated phosphorylation of cardiac sarcoplasmic reticulum was not affected by R24571. Sarcoplasmic reticulum Ca-dependent ATPase phosphoprotein intermediate formation was inhibited by R24571 concentrations that were 20 to 30 times greater than those required to inhibit calmodulin-dependent phosphorylation. However, both Ca-dependent and independent ATPase activities, as well as calcium uptake, were inhibited by R24571 concentrations that were similar to, or less than, those concentrations required to inhibit calmodulin-dependent sarcoplasmic reticulum phosphorylation. These results indicate the caution that should be exercised in using this new compound in assessing the possible involvement of calmodulin in other membrane processes.     

The selectivity of protein kinase inhibitors: a further update

The specificities of 65 compounds reported to be relatively specific inhibitors of protein kinases have been profiled against a panel of 70-80 protein kinases. On the basis of this information, the effects of compounds that we have studied in cells and other data in the literature, we recommend the use of the following small-molecule inhibitors: SB 203580/SB202190 and BIRB 0796 to be used in parallel to assess the physiological roles of p38 MAPK (mitogen-activated protein kinase) isoforms, PI-103 and wortmannin to be used in parallel to inhibit phosphatidylinositol (phosphoinositide) 3-kinases, PP1 or PP2 to be used in parallel with Src-I1 (Src inhibitor-1) to inhibit Src family members; PD 184352 or PD 0325901 to inhibit MKK1 (MAPK kinase-1) or MKK1 plus MKK5, Akt-I-1/2 to inhibit the activation of PKB (protein kinase B/Akt), rapamycin to inhibit TORC1 [mTOR (mammalian target of rapamycin)-raptor (regulatory associated protein of mTOR) complex], CT 99021 to inhibit GSK3 (glycogen synthase kinase 3), BI-D1870 and SL0101 or FMK (fluoromethylketone) to be used in parallel to inhibit RSK (ribosomal S6 kinase), D4476 to inhibit CK1 (casein kinase 1), VX680 to inhibit Aurora kinases, and roscovitine as a pan-CDK (cyclin-dependent kinase) inhibitor. We have also identified harmine as a potent and specific inhibitor of DYRK1A (dual-specificity tyrosine-phosphorylated and -regulated kinase 1A) in vitro. The results have further emphasized the need for considerable caution in using small-molecule inhibitors of protein kinases to assess the physiological roles of these enzymes. Despite being used widely, many of the compounds that we analysed were too non-specific for useful conclusions to be made, other than to exclude the involvement of particular protein kinases in cellular processes.     

Anti-apoptotic potential of insect cellular and viral IAPs in mammalian cells

IAPs were identified as baculoviral proteins that could inhibit the apoptotic response of insect cells to infection. Of the viral IAPs, OpIAP and CpIAP can inhibit apoptosis, whereas AcIAP cannot. OpIAP and some mammalian homologues can inhibit mammalian cell death. Two mammalian IAPs bind to TNFRII associated factors (TRAFs), but the significance of this is unclear. Here we show that Drosophila cellular IAPs and two baculoviral IAPs (OpIAP and CpIAP) can inhibit mammalian cell death induced by overexpression of Caspases 1 and 2. IAPs must act on conserved components of the apoptotic mechanism, but as none of these IAPs could bind TRAF proteins, TRAFs are not likely to be important for IAP mediated apoptosis inhibition. As OpIAP protected against death induced by ligation of TNF receptor family members, but not by factor nor serum withdrawal from dependent cells, it can inhibit certain apoptotic pathways without affecting others.     

Evidence against a possible involvement of the serine, and thiol proteases in the exocytotic mechanism of catecholamine secretion in cultured bovine adrenal medullary cells

The effects of protease inhibitors on the secretion of catecholamines were studied in cultured bovine adrenal medullary cells. Although the inhibitors of serine proteases could inhibit the carbamylcholine-induced secretion, they failed to inhibit the secretion evoked by either high K+ or A23187. The thiol protease inhibitor had no effect on the secretion. These results therefore seem to indicate that the serine protease inhibitors may inhibit the receptor-mediated secretion probably through their effects on the plasma membrane, thus suggesting that a possible involvement of the serine, and thiol proteases in exocytosis may be unlikely.     

Inhibitors of TLR-9 act on multiple cell subsets in mouse and man in vitro and prevent death in vivo from systemic inflammation

In parallel with the discovery of the immunostimulatory activities of CpG-containing oligodeoxynucleotides, several groups have reported specific DNA sequences that could inhibit activation by CpG-containing oligodeoxynucleotides in mouse models. We show that these inhibitory sequences, termed IRS, inhibit TLR-9-mediated activation in human as well as mouse cells. This inhibitory activity includes proliferation and IL-6 production by B cells, and IFN-alpha and IL-12 production by plasmacytoid dendritic cells. Our studies of multiple cell types in both mice and humans show the optimal IRS to contain a GGGG motif within the sequence, and the activity to require a phosphorothioate backbone. Although the GGGG motif readily itself leads to formation of a tetrameric oligodeoxynucleotide structure, inhibitory activity resides exclusively in the single-stranded form. When coinjected with a CpG oligodeoxynucleotide in vivo, IRS were shown to inhibit inflammation through a reduction in serum cytokine responses. IRS do not need to be injected at the same site to inhibit, demonstrating that rapid, systemic inhibition of TLR-9 can be readily achieved. IRS can also inhibit a complex pathological response to ISS, as shown by protection from death after massive systemic inflammation induced by a CpG-containing oligodeoxynucleotides.     

Mink parvoviruses and interferons: in vitro studies.

Although interferons can inhibit the replication of a number of viruses, little is known about their ability to inhibit parvovirus replication. Therefore, in vitro experiments were done to determine if Aleutian disease virus and mink enteritis virus, two autonomously replicating mink parvoviruses, induced interferon, were sensitive to the effects of interferon, or inhibited the production of interferon. The results indicated that these parvoviruses neither induced nor were sensitive to the effects of interferon. Furthermore, preexisting parvovirus infections did not inhibit poly(I).poly(C)-induced interferon production. This independence from the interferon system may, therefore, be a general property of the autonomously replicating parvoviruses.     

Participation of proteasomes in Xenopus embryogenesis

We examined the effects of various protease substrates on Xenopus laevis embryogenesis. Thirty-three peptidyl-MCA substrates were added to the culture medium in which Xenopus embryos were developing. Five of the 33 substrates were found to inhibit embryogenesis at the early gastrula stage or much earlier ones. These results suggest that proteases that hydrolyze these substrates are involved in embryonic development. We found that the developmental stage of embryos is crucial for these substrates to inhibit their development. We purified a protease that hydrolyzes Pyr-Arg-Thr-Lys-Arg-MCA, a substrate that inhibits embryogenesis, from Xenopus embryos. This protease turned out to be a component of proteasomes. We found that 4 of the 5 substrates that inhibit embryogenesis are among the proteasome substrates. Thus, we concluded that proteasomes play a crucial role in the development of Xenopus embryos. Possibly, various catalytic subunits in proteasomes function independently, in stage-specific manners.     

A possible mechanism for cadmium-induced hypertension in rats

The mechanism of cadmium-induced hypertension was explored by measuring noradrenaline metabolism. Cadmium $\text{in vitro}$ was shown to inhibit both monoamine oxidase and catechol-O-methyltransferase, the two enzymes which inactivate the neurotransmitters noradrenaline and adrenaline. However, rats which were injected or fed (via the drinking water) with cadmium showed that, among the tissues surveyed, these two enzymes were inhibited significantly only in the aorta. $\text{In vitro}$, cadmium was found to inhibit noradrenaline binding to membranes from the heart, lung, and kidney, while stimulating binding to aortic membranes, which suggests that the effects may be specific. These results suggest that, in the aorta, cadmium may inhibit the two catabolic enzymes of noradrenaline, while at the same time stimulating noradrenaline-binding. Thus the effects of noradrenaline on vascular smooth muscle would be increased as well as prolonged.     

Suppressor cells for the afferent phase of contact sensitivity to picryl chloride: inhibition of DNA synthesis induced by T cells from mice injected with picryl sulfonic acid.

Previous reports have shown that picryl sulfonic acid (PSA) induces suppressor T cells that inhibit the effector phase of contact sensitivity, whereeas its DNP counterpart, dinitrobenzenesulfonate (DNBS) induces cells that inhibit the afferent phase of sensitization. Accordingly, cells from mice injected with DNBS, but not PSA, could be shown to inhibit the DNA synthesis in the lymph nodes that occurs during sensitization. It is now shown that PSA does induce T cells that suppress DNA synthesis but this can only be detected with enriched T cells or by using a regimen of PSA injection different frm previously used to induce suppressor cells for the effector phase. The T cells did not affect responses to oxazolone or dinitrofluorobenzene (DNFB) and were distinguishable from suppressors of the efferent phase in that they could be produced in adult thymectomized but not cyclophosphamide-treated mice. T cells from mice injected with DNBS that inhibited DNA synthesis to DNFB had the same properties.