氟化钠与小牛胸腺DNA直接结合作用的研究

目的 探讨氟化钠与 DNA的直接结合作用。方法 应用自动程序升温紫外分光光度计和高效液相 L C- VP离子色谱分析仪对氟化钠与小牛胸腺 DNA的直接结合作用进行了研究。结果 6 .3,4 83.0 ,987.0mg/L 氟化钠对 5 .0 ,10 .0 ,2 2 .5 mg/L 小牛胸腺 DNA在 2 6 0 nm的吸收光谱未造成任何影响 ,在 2 0 5 nm处吸收峰虽有随氟化钠浓度升高而逐渐降低的倾向 ,但结果的可重复性较差 ;1.0 mg/L 的溴化乙锭可使 10 .0 mg/L 浓度的小牛胸腺 DNA融点温度显著升高 ,而 2 10 .0 m g/L 的氟化钠对其无明显影响 ;不同浓度小牛胸腺 DNA与氟化钠作用后 ,冰乙醇变性洗涤 ,经高效液相离子色谱分析仪检测 ,DNA水溶液中无氟离子检出。结论 氟化钠未与小牛胸腺 DNA发生稳定结合。

联吡啶衍生物芳基钌配合物的合成及其与DNA、蛋白质相互作用

以2种配体4,4′-dimethyl-2,2′-bipyridine(L1)和4′-methyl-(2,2′-bipyridine)-4-carbaldehyde oxime(L2),分别与芳基钌二聚体[RuCl_2(η~6-p-cymene)]2合成了2种新型单核配合物[Ru(η~6-p-cymene)(L1)Cl]Cl(1)和[Ru(η6-p-cymene)(L2)Cl]Cl(2)。应用元素分析、ESI-MS和~1H NMR对配合物的组成和结构进行表征,通过紫外光谱法和荧光光谱法研究了配合物的水解及其与CT-DNA和血清蛋白的结合性质,并且进行了细胞毒性研究。结果表明,在水溶液中配合物1比2在动力学上更稳定(k:0.383 h^(-1)(1)、1.458 h^(-1)(2));配合物均通过嵌入作用与双链DNA结合,但2有较强的结合能力(Kb:7.8×10~3L·mol^(-1)(1)、1.86×10~4L·mol^(-1)(2))。配合物均能与蛋白质发生相互作用,引起蛋白静态猝灭,但1作用较强(KA:1.04×10~5L·mol^(-1)(1)、8.62×10~4L·mol^(-1)(2))。配合物与蛋白的较强结合能力,可能是其细胞毒性不高的原因。

Ten1p promotes the telomeric DNA-binding activity of Cdc13p： implication for its function in telomere length regulation

In Saccharomyces cerevisiae, the essential gene CDC13 encodes a telomeric single-stranded DNA-binding protein that interacts with Stnlp and Tenlp genetically and physically, and is required for telomere end protection and telomere length control. The molecular mechanism by which Tenl participates in telomere length regulation and chromosome end protection remains elusive. In this work, we observed a weak interaction of Cdc13p and Tenlp in a gelfiltration analysis using purified recombinant Cdc13p and Tenlp. Tenlp itself exhibits a weak DNA-binding activity, but enhances the telomeric TG1-3 DNA-binding ability of Cdc13p. Cdc13p is communoprecipitated with Tenlp. In the mutant ten1-55 or ten1-66 cells, the impaired interaction between Tenlp and Cdc13p results in much longer telomeres, as well as a decreased association of Cdc13p with telomeric DNA. Consistently, the Ten1-55 and Ten1-66 mutant proteins fail to stimulate the telomeric DNA-binding activity of Cdc13p in vitro. These results suggest that Tenlp enhances the telomeric DNA-binding activity of Cdc13p to negatively regulate telomere length.

Synthesis, Crystal Structure and Interactions with Different G-quadruplex DNA and ctDNA of Zn-CAP

In this study, one mononuclear zinc（II） complex with 1,2-bis CAP （（5-chlorosalicylidene amino）-phenylene）： C22C13N2035Znl5 H0125 （Zn-CAP） was synthesized. The binding properties of Zn-CAP with G-quadruplex DNA and ctDNA （calf thymus DNA） were examined by fluorescence, CD （circular dichroism） spectroscopic and FRET （fluorescence resonance energy transfer） assay. In the fluorescence emission spectral analysis, the addition of three series of G-quadruplex DNA （G4-HTG21, G4-Pu27 and G4-c-kit-l） into the Zn-CAP solution induced moderate or add hypochromicity with total quenching ratios of 10.73%, 15.07% and 8.59% in the presence of K＋ were achieved, respectively. While the addition of ctDNA under same condition only caused 7.08% quenching on the fluorescence emission of Zn-CAP. In the CD spectral analysis, the interaction with Zn-CAP could induce significant spectral changes on the CD absorption of G4-HTG21, G4-Pu27 and G4-c-kit-1, with 106.00%, 93.06%, 113.47% increment at 232 nm absorption, along with a 81.11%, 92.80%, 83.72% decrement at 295 nm or 270 nm absorption, which demonstrated that the antiparallel structure of G-quadruplex DNA is more stable in the presence of Zn-CAP. Comparatively, the addition of Zn-CAP could induce significant spectral changes on the CD absorption of double helix ctDNA, with 64.17% decrement on the positive peak absorption, along with a 90.91% increment on the negative peak absorption. On the other hand, in the FRET-melting assay analysis, it was clear that Zn-CAP at 0.5 equivalences could raise the melting temperature of G-quadruplex （F2 IT or FPul 8T） by 3.45℃ and 15.85℃, indicating an obvious stabilization effect of Zn-CAP on G-quadruplex in Pu27. All the results indicated that Zn-CAP exhibited higher binding affinity and binding intensity to G-quadruplex DNA than ctDNA, especially G-quadruplex Pu27.

Wortmannin对视网膜缺血再灌注后HIF-1α表达的影响

目的:探讨磷酰肌醇-3激酶(phosphatidylinositol-3 kinase,PI3K)途径在大鼠视网膜缺血再灌注后对低氧诱导因子(HIF-1α)表达的影响。方法:将磷酰肌醇-3激酶(PI3K)特异抑制剂Wortmannin(WT)注入大鼠玻璃体内,抑制PI3K活性为A组,玻璃体内注射等量林格液作为B组,空白对照组为C组,采用前房灌注生理盐水升高大鼠眼压到110mm Hg(1 kPa=7.5 mm Hg)持续1 h的方法制作实验性视网膜缺血再灌注损伤模型,于再灌注后0、2、6、8、12、24、48 h取材,行免疫组织化学法染色观察HIF-1α在视网膜细胞的阳性表达;并应用RT-PCR来检测HIF-1αmRNA的表达。结果:视网膜缺血再灌注6、8、12、24、48 h,视网膜神经节细胞层及内核层细胞出现HIF-1α阳性表达且表达逐渐增加,12 h达到高峰,然后逐渐降低,预先玻璃体内注射PI-3K特异抑制剂Wortmannin(WT)的大鼠视网膜HIF-1α表达明显减弱,亦在12 h达到高峰,然后逐渐降低。结论:缺血再灌注后大鼠视网膜HIF-1α的表达是经过PI3K途径来实现,阻断PI3K途径,可以明显减少HIF-1α的表达,但从实验结果来看PI3K途径被阻断后并没有完全阻断HIF-1α的表达,由此猜测HIF-1α的表达可能存在多种途径。