铁离子引起的DNA电荷逆转和凝聚

在生物体中,DNA会凝聚成紧密结构,对于遗传信息的储存和转录有重要意义。对于DNA的凝聚以及伴随的电荷逆转已经有了比较深入的研究,这些研究大多集中于一些水解性较弱的离子,而对于那些易水解对于溶液pH影响较大的金属离子（例如铁离子）的研究较少。本文系统地研究了铁离子引起的DNA电荷逆转和凝聚,通过与DNA在三氯六氨络合钴凝聚的对比,探索了铁离子引起的DNA的电荷逆转和凝聚的可能机制。     

丙二醇和吐温-80对人淋巴细胞的SCE的诱发及DNA损伤

本文研究了医药工业中常用的两种助溶剂丙二醇和吐温-80对人淋细胞姊妹染色单体互换(SCE)的诱发作用及对 DNA 的损伤作用.丙二醇(4.13mg/mL)对 SCE 无明显的诱发作用,对 DNA 复制模板也无损伤.吐温-80能诱发 SCE 频率增高,剂量与效应呈线性相关.当浓度为0.001～0.2mg/mL 时与对照组相比差异极显著(p<0.01).使 SCE 频率增加到对照组的2倍的浓度为0.12mg/mL.浓度为1mg/mL 时严重抑制细胞增殖和分裂.它也能损伤 DNA 复制模板.因此,用吐温-80作助溶剂时应该慎重.     

壳聚糖与DNA相互作用的单分子方法研究

利用原子力显微镜与磁镊技术研究不同浓度的壳聚糖与多种DNA分子的作用,对其凝聚形态及力谱曲线进行观察。在恒力情况下,壳聚糖与DNA的电荷比k分别为3和4时,从DNA的力谱曲线上可以看见0.2~0.5μm左右的跳跃阶梯及线性增长区域,原因可能是壳聚糖-DNA聚合物中出现了环状、棒状及球状结构;利用原子力显微镜观察壳聚糖与λ-DNA及bpr322-DNA作用后的凝聚形态,发现随着壳聚糖与DNA电荷比k的增加,球状与棒状聚合物的比例明显减小,而且凝聚物的尺寸也整体减小。最后根据实验结果,建立了壳聚糖导致DNA构象变化的直观模型。     

用AFM对质粒DNA在云母表面的自组装研究

通过原子力显微镜(AFM)对在云母表面上自然挥干的不同质量浓度的质粒DNA分子进行扫描，原子力显微镜图像显示：当质量浓度为0．1μg／mL时，DNA分子呈现出线性结构，随着质量浓度加大至1μg／mL，DNA分子相互缠绕为网状结构，质量浓度为10μg/mL时，呈现出具有典型的聚合形态，当质量浓度进一步加大到100μg／mL，则呈现出明显的树状脉络结构．通过原子力显微镜对质粒DNA分子的形态表征，可以看出质粒DNA分子具有典型的自组装的特性．     

茶多酚诱导卵巢癌COC1细胞凋亡的研究

卵巢癌是女性生殖系统恶性肿瘤之一,而茶多酚具有抗肿瘤和抗氧化的作用,作者研究茶多酚对体外培养的卵巢癌COC1细胞凋亡的影响.采用不同浓度茶多酚处理卵巢癌COC1细胞,24h后分别采用AO/PI双染法和琼脂糖凝胶电泳法(DNA Ladder),观察和分析茶多酚对COC1细胞凋亡的影响.结果显示,低浓度茶多酚(≤500μg/mL)处理24h后,出现早起凋亡现象;而高浓度的茶多酚(2 000μg/mL)后,出现了明显的晚期凋亡现象;过高浓度(4 000μg/mL)处理,细胞出现死亡现象.不同浓度的茶多酚处理后,细胞均可检测到DNA Ladder,且随着茶多酚浓度的增加,DNA裂解程度加深,电泳中DNA Ladder更明显.实验结果表明,不同浓度茶多酚处理均可以诱导卵巢癌细胞株COC1产生细胞凋亡,研究结果为筛选新型的抗癌药物提供参考.     

PMA与PCR结合的细菌活细胞检测方法

基于PCR的分子生物技术在检测过程中不能有效区分样品中细菌的死活状态,这会导致对细菌数量的错误估计.文中用叠氮溴化丙锭(PMA)对样品基因组提取进行前处理,使PMA与样品中死细胞的DNA分子共价交联,并抑制该DNA分子的PCR扩增.结果表明:当样品中PMA质量浓度大于3μg/mL、曝光时间大于3 min时,PMA可抑制细胞膜破裂的E.coli死细胞DNA的PCR扩增;PMA质量浓度高于50μg/mL时对活细胞DNA的PCR扩增有一定影响;当浊度小于10NTU时,PMA能有效地抑制死细胞DNA的PCR扩增,而当浊度大于100NTU时,PMA失去效果.     

不同浓度血红蛋白对小鼠骨髓瘤细胞体外培养的影响

用含不同浓度的牛源性血红蛋白细胞培养液培养小鼠骨髓瘤细胞,通过细胞计数绘制生长曲线,计算最大增殖浓度和倍增时间,并进行比较分析.结果表明:当培养液的血红蛋白浓度在1~5 mg/dL时小鼠骨髓瘤细胞生长较好,细胞最大增殖浓度在1.2×106个/mL以上,倍增时间小于20 h,当培养液的血红蛋白浓度为6 mg/dL时细胞生长抑制,细胞最大增殖浓度和倍增时间为6.7×105个/mL和21.6 h.由此可见,培养液中血红蛋白浓度≤5 mg/dL时不影响细胞生长,达到6 mg/dL时抑制细胞生长.     

MTT法测定百合多糖细胞毒性的研究

目的：研究百合多糖的细胞毒性。方法：水煮醇沉法提取百合多糖,MTT法测定百合多糖的细胞毒性。结果：当百合多糖浓度为1mg/mL时,细胞存活率为64.2%;葡萄糖浓度为1mg/mL时,细胞存活率为76.2%。结论：百合多糖具有较低细胞毒性。     

肿瘤细胞ECA109和PC12对二甲基亚枫耐受剂量的分析

通过观察不同剂量二甲基亚砜(dimethyl sulfoxide,DMSO)作用下ECA109和PC12细胞形态和生长状态的变化,确定该溶剂对上述两种肿瘤细胞的相对安全剂量。按照一定浓度梯度(V/V)将DMSO加入培养液,干预后的24 h、48 h和72 h于倒置相差显微镜下观察AO/EB染色前后细胞的形态特征;同时用四甲基偶氮唑蓝(MTT)显色法检测细胞的生存率并计算半数抑制浓度(50%inhibiting concentration,IC50)。结果:ECA109细胞和PC12细胞对DMSO的耐受性明显不同,DMSO的浓度为8 mL/L时,24 h内ECA109细胞生长正常,形态完整,活力与对照组无差别,浓度为10 mL/L时,细胞生存率可下降10%。对PC12而言,DMSO浓度不高于14 mL/L时,对细胞的形态和生长几乎没有影响;浓度提高到20 mL/L时,24 h时细胞生存率可下降27%。结论:PC12细胞对DMSO的耐受性高于ECA109;ECA109细胞24 h内的应用终浓度不应超过10 mL/L,PC12细胞DMSO的应用浓度可提高到14 mL/L;干预时间如需延长,DMSO的加入剂量应相应降低。     

佛手水煎剂对RAW264.7癌细胞增殖的影响

为了观察佛手水煎剂对RAW 264.7癌细胞增殖的影响,用生药量0.625,1.250,2.500和5.000 mg/mL的佛手水煎剂处理RAW 264.7癌细胞,采用四甲基偶氮唑蓝(MTT)法测定细胞活力、台盼蓝排斥法测定细胞存活率、吖啶橙/溴化乙锭(AO/EB)荧光染色法检测细胞凋亡与坏死.结果表明:与对照组比较,0.625,1.250和2.500 mg/mL佛手水煎剂处理后,RAW 264.7癌细胞固缩,细胞核染色质凝聚、片断化,有凋亡小体出现,表现出典型的细胞凋亡特征;5.000 mg/mL佛手水煎剂处理后,RAW 264.7癌细胞肿胀,细胞膜破裂,呈现坏死症状,RAW 264.7癌细胞的增殖明显受到抑制(P＜0.01),半数抑制浓度(IC50)为2.073 mg/mL.说明佛手水煎剂能诱导癌细胞凋亡,抑制癌细胞增殖.     

不同价态的阴离子在DNA凝聚效应中的抑制作用

为了定量地研究阴离子对DNA凝聚和电荷中和过程的抑制作用，利用动态光散射和原子力显微镜研究了不同价态阴离子对阳离子介导的DNA凝聚效应的影响。在动态光散射实验中，阴离子可以使阳离子作用后的DNA的电泳迁移率变小，即对DNA的电荷中和有抑制作用，而且阴离子价态越高，对电泳迁移率的抑制作用越强。改变阳离子种类但控制相同的阳离子浓度，发现阴离子对DNA的电荷中和有同样的抑制作用及趋势，同时用原子力显微镜观察DNA的凝聚形态变化也得出一致的结果。在相同阳离子浓度下，高价阴离子相对低价态阴离子，对DNA凝聚的抑制作用更强，使DNA的凝聚形态变得更加松散，进一步证实了阴离子对阳离子导致的DNA凝聚有减弱作用。     

不同价态的阴离子在DNA凝聚效应中的抑制作用

为了定量地研究阴离子对DNA凝聚和电荷中和过程的抑制作用,利用动态光散射和原子力显微镜研究了不同价态阴离子对阳离子介导的DNA凝聚效应的影响。在动态光散射实验中,阴离子可以使阳离子作用后的DNA的电泳迁移率变小,即对DNA的电荷中和有抑制作用,而且阴离子价态越高,对电泳迁移率的抑制作用越强。改变阳离子种类但控制相同的阳离子浓度,发现阴离子对DNA的电荷中和有同样的抑制作用及趋势,同时用原子力显微镜观察DNA的凝聚形态变化也得出一致的结果。在相同阳离子浓度下,高价阴离子相对低价态阴离子,对DNA凝聚的抑制作用更强,使DNA的凝聚形态变得更加松散,进一步证实了阴离子对阳离子导致的DNA凝聚有减弱作用。     

盐类对光解水放氢反应的影响

报道ＮａＣｌ，ＫＣｌ，ＭｇＣｌ２和ＣａＣｌ２对光解水放氢影响的某些结果．在所研究的ＴｉＯ２，Ｋ２ＰｔＣｌ６，ＥＤＴＡ酸（或还原铁粉）的反应系统中，ＭｇＣｌ２，ＣａＣｌ２对产氢的影响大于ＮａＣｌ和ＫＣｌ，ＮａＣｌ对产氢的影响大于ＫＣｌ．与没有盐类存在时相比，加入盐类可使放氢增至２倍以上．海水中的Ｋ＋，Ｎａ＋，Ｃａ＋＋和Ｍｇ＋＋浓度均落在对反应速度变化较敏感的浓度区域，而且用ＮａＣｌ，ＫＣｌ，ＭｇＣｌ２和ＣａＣｌ２４种盐类模拟海水，也可以使放氢量提高至２倍以上，提示这类反应可能在生物进化中的重要地位．     

Study of the interaction of DNA and histones by spin-stretching and droplet evaporation

Molecular combing is a powerful and simple method for aligning DNA molecules onto a surface. Using this technique combined with fluorescence microscopy, DNA-histone complexes are stretched on a hydrophobic polymethyl methacrylate (PMMA) surface and observed directly. We have developed a new method to stretch single DNA-histone complexes, termed spin-stretching. The results show that the histones markedly enhance DNA binding to the PMMA surface. DNA winds around the histones and therefore decreases in length. The number of histones that bind to each DNA molecule is found to correlate with the histone concentration. The combed DNA-histone complexes are found to depend on two factors: the binding force on the surface and the centrifugal force at its local position. Na+ ions should compete with histones for binding to DNA; however, the observed competitive binding effect of Na+ ions at low concentrations was negligible.     

Single molecule DNA compaction by purified histones

The compaction of single DNA molecules by purified histones is studied using magnetic tweezers. The compaction rate increases rapidly when the histone concentration is increased from 0.002 to 0.2 mmol/L, and saturates when the concentration is beyond 0.2 mmol/L. The time course of compaction is exponential at low histone concentrations. It becomes sigmoidal at high concentrations. Cooperativity between the histones bound to DNA is proposed to be responsible for the transition. The histones are loaded onto DNA randomly at low concentrations. They tend to bind DNA cooperatively at high con-centrations because the structural torsions of DNA induced by the bound histones become overlapping so that the binding of one histone facilitates the binding of others. Under very large forces, the com- pacted histone-DNA complex can be disrupted in a discrete manner with a step size of -60 nm. But the histones cannot be completely stripped off DNA, as is revealed by the lowered B-S transition plateau of the histone-bound DNA.     

Lysozyme refolding at high concentration by dilution and size—exclusion chromatography

This study of renaturation by dilution and size exclusion chromatography (SEC) addition of urea to improve yield as well as the initial and final protein concentrations showed that although urea decreased the rate of lysozyme refolding, it could suppress protein aggregation to sustain the pathway of correct refolding at high protein concentration; and that there existed an optimum urea concentration in renaturation buffer. Under the above conditions, lysozyme was successfully refolded from initial concentration of up to 40 mg/mL by dilution and 100 mg/mL by SEC, with the yield of the former being more than 40% and that of the latter being 34.8%. Especially, under the condition of 30 min interval time, i.e. tau > 2(t(R2) - t(R1)), the efficiency was increased by 25% and the renaturation buffer could be recycled for SEC refolding in continuous operation of downstream process.     

DNA triplet repeats mediate heterochromatin-protein-1-sensitive variegated gene silencing

Gene repression is crucial to the maintenance of differentiated cell types in multicellular organisms, whereas aberrant silencing can lead to disease. The organization of DNA into chromatin and heterochromatin is implicated in gene silencing. In chromatin, DNA wraps around histones, creating nucleosomes. Further condensation of chromatin, associated with large blocks of repetitive DNA sequences, is known as heterochromatin. Position effect variegation (PEV) occurs when a gene is located abnormally close to heterochromatin, silencing the affected gene in a proportion of cells. Here we show that the relatively short triplet-repeat expansions found in myotonic dystrophy and Friedreich's ataxia confer variegation of expression on a linked transgene in mice. Silencing was correlated with a decrease in promoter accessibility and was enhanced by the classical PEV modifier heterochromatin protein 1 (HP1). Notably, triplet-repeat-associated variegation was not restricted to classical heterochromatic regions but occurred irrespective of chromosomal location. Because the phenomenon described here shares important features with PEV, the mechanisms underlying heterochromatin-mediated silencing might have a role in gene regulation at many sites throughout the mammalian genome and modulate the extent of gene silencing and hence severity in several triplet-repeat diseases.     

肠道益生菌体外发酵山药低聚糖产短链脂肪酸的研究

短链脂肪酸具有促进肠道及人体健康的有益功能,采用体外模拟肠道内环境的方法,分析嗜酸乳杆菌、嗜热链球菌、保加利亚乳杆菌和植物乳杆菌、青春双歧杆菌、动物双歧杆菌等肠道益生菌发酵山药低聚糖产生短链脂肪酸的种类和含量。结果发现:山药低聚糖在模拟结肠环境中被乳酸菌作为碳源利用,产生对人体健康有积极作用的乳酸、乙酸及丙酸等短链脂肪酸,且产生短链脂肪酸的浓度与菌种及发酵时间相关。在48h发酵过程中,乳酸在植物乳杆菌48h发酵液中含量最高,达(13.800±0.243)mg/mL;乙酸在动物双歧杆菌48h发酵液中含量最高,达(1.850±0.003)mg/mL;丙酸则在动物双歧杆菌8h发酵液中含量最高,为(0.082±0.001)mg/mL。研究结果提示,可通过改善膳食结构调节肠道益生菌产生短链脂肪酸,维持肠道与人体健康。     

Monitoring the binding of metal cations and histones to DNA in real time using fluorescence assays

The binding of cations (Na +,K +,Mg 2+,Ca 2+,Mn 2+) and histones to DNA can be studied using fluorescence assays.Here,we measured the fluorescence intensity and fluorescence anisotropy of DNA and DNA-histone complexes in the presence of cations.We demonstrate that when different cations are added into a DNA solution,the fluorescence intensities of the stained DNA are reduced by different amounts.Compared with divalent cations,monovalent cations had a weaker effect on fluorescence intensity and fluorescence anisotropy.Divalent (Mn 2+,Mg 2+,Ca 2+) cations markedly enhanced the fluorescence anisotropy of DNA.The binding modes of monovalent and divalent cations to DNA may be different.Divalent cations can change the structure of DNA molecules,or promote the assembly of DNA strands.The addition of histones causes DNA condensation,which mostly occurs during the first few seconds.Cation binding to DNA is abrupt,and is much faster than that of histones.