海南粗榧新碱衍生物HH07A对肿瘤细胞内DNA拓扑异构酶Ⅱ和蛋白激酶C活性的影响

HH07A 5.5 μmol·L^-1作用L1210细胞24 h后, 可使细胞的DNA拓扑异构酶Ⅱ活性下降; 在无细胞系统, HH07A 0.55 mmol·L^-1也能直接促进DNA拓扑异构酶Ⅱ引起的DNA链断裂. 经HH07A (L1210细胞5.5 μmol·L^-1, HL-60细胞8.25 μmol·L^-1)作用24 h后, L1210及HL-60细胞的胞浆蛋白激酶C(PKC)活性升高, 胞膜PKC活性下降, 而全细胞PKC活性变化不大. 在无细胞系统中, HH07A 1.1 mmol·L^-1能明显抑制PKC的活性.     

海南粗榧新碱衍生物HH07A的抗肿瘤作用

用细胞生长曲线测定法及软琼脂集落形成分析法研究了HH07A对几种肿瘤及正常细胞生长的影响。结果表明,1.5ug·ml^-1及3μg·ml^-1HH07A能分别明显抑制L1210和HL-60细胞的生长。3种肿瘤细胞对HH07A的敏感性依次为L1210>KB>HL-60,而正常小鼠粒系祖细胞GM-CPC对药物的敏感性则低于前三者,且HH07A3.5μg·ml^-1对HL-60细胞无分化诱导作用。HH07A对腹水型L1210白血病小鼠、S180小鼠均有较明显的治疗作用,使L1210荷瘤小鼠、S180荷瘤小鼠存活时间延长。也能抑制S180实体瘤的生长。     

异靛甲体外抑制DNA和RNA生物合成的抗癌作用机理

以同位素参入法及光谱法观察异靛甲对核酸合成的抑制作用和药物与DNA相互作用。结果表明,异靛甲对DNA,RNA合成有较强的抑制作用,其IC_(50)分别为9和15μmol/L,药物作用非常迅速。30μmol/L异靛甲作用15μmin后DNA,RNA合成被抑制95％以上。实验还表明,异靛甲不能损伤DNA模板,也不抑制DNA拓扑异构酶及DNA多聚酶Ⅰ,但能明显抑制T7 RNA多聚酶,在’100μmol/L浓度下对mRNA的合成抑制达70％以上。     

阿克拉霉素B抗单纯疱疹病毒Ⅱ型活性的研究

本文报道了阿克拉霉素B(ACM-B)对单纯疱疹病毒Ⅱ型(HSV-2)复制及其DNA聚合酶活性的影响。实验采用单层Vero细胞,空斑降低测定用来评价实验结果。加入ACM-B,明显地降低病毒空斑的形成。当ACM-B剂量大于0.3μm时,空斑数降低到零,表明ACM-B抑制HSV-2的复制。另外,ACM-B也抑制HSV-2诱导的DNA聚合酶活性。这提示,药物对HSV-2复制的抑制作用可能是由于对病毒DNA合成的抑制。吸收光谱分析证实,ACM-B能与活化DNA模板相互作用。ACM-B也抑制E.Coli DNA pol.Ⅰ及L1210DNA pol.α,证明ACM-B对HSV-2 DNApol.的抑制是非选择性的.ACM-B对HSV-2复制的抑制效应较ACM-A强,而细胞毒性效应也较后者为高。     

APHIDICOLIN对疱疹病毒诱导的DNA聚合酶的抑制作用

Aphidicolin抑制HSV-1、HSV-2、CMV及EBV诱导的DNA聚合酶的ID_(50)分别为0.7、2.7. 6.8及11μM.以HSV-1 DNA聚合酶对aphidicolin最敏感。抑制作用依赖于所使用的模板-引物。对HSV-1、HSA-2 DNA聚合酶抑制方式,当反应系统内模板-引物为饱和浓度时,aphidicolin对底物显示非竞争性抑制;当四种底物为饱和浓度时,aphidicolin对模板-引物表现为反竞争性抑制。Aphidicolin和Foscarnet在酶分子上的作用点可能不同。     

应用聚合酶链反应检测结核杆菌

聚合酶链反应是近年发展起来的一种核酸序列扩增技术,即在体外,以待检DNA为模板,用聚合酶催化一对引物间特定DNA片断的合成。聚合酶链反应直接检测临床标本中的结核杆菌,用于结核病快速诊断国内外均有报道,与结核病细菌学传统检查     

多芳基取代蝶啶类化合物的合成及其抗肿瘤活性多芳基取代蝶啶类化合物的合成及其抗肿瘤活性

目的研究多芳基取代蝶啶类化合物的合成及其抗肿瘤活性。方法由4,6-二氨基-5-亚硝基嘧啶衍生物与对-氨基苯乙腈衍生物通过环化合成目的物;采用MTT法测定目的物的抗肿瘤活性。结果设计、合成了9个新化合物(I～III),其结构经元素分析、IR,¹HNMR和MS等确定。化合物I～III的体外抗肿瘤活性较明显。结论化合物I～III显示了一定的体外抗肿瘤活性,但它们与小牛胸腺DNA之间并无明显作用,提示可能是通过抑制二氢叶酸还原酶(DHFR)或其他叶酸依赖性酶而起抗肿瘤作用。     

海南粗榧新碱衍生物HH07A对体外L1210细胞的杀伤作用

体外培养的小鼠L1210细胞被HH07A2μg·ml^-1作用24h后,与对照组细胞相比,其细胞数不再增长,有丝分裂数及集落形成率下降,细胞形态及细胞周期动力学均发生一定的变化。且HH07A大剂量短期作用抑制Ll210细胞集落形成的效率高于低剂量持续作用。     

苯对小鼠骨髓细胞DNA代谢的影响

本实验用DNA合成前体~3H-TdR 体外细胞培养掺入法,研究了苯中毒CFW小鼠骨髓细胞DNA合成代谢的变化。结果证明苯染毒小鼠的骨髓细胞DNA合成代谢发生明显变化,与外周血细胞的变化密切相关。作者还测定了苯染毒小鼠DNA聚合酶的活性,结果表明苯对DNA聚合酶的活性没有影响,研究证明苯对DNA合成代谢的影响并不是由于本对DNA 聚合酶活性的影响所致。     

胆红素对腹水型肝癌细胞生长及DNA合成的光敏作用(英文)

目的:观察胆红素光敏反应对腹水型肝癌(Hep A)细胞DNA的影响及其机理。方法:细胞经1.0×10~5lx照光10min后加脱氧[~3H]胸苷,测DNA的合成。细胞用0.5％台盼兰染色后计数。结果:胆红素光敏反应使细胞死亡率增加;DNA合成明显受到抑制(P<0.01);且随浓度的增加和照光时间的延长而加剧。在自然光照下,照光组与避光组DNA合成没有明显区别(P>0.05)。结论:胆红素光敏反应对Hep A细胞有明显的杀伤作用;自然光照组不产生光敏反应;光敏反应的产生与~1O_2和H_2O_2密切相关,而与OH·和O_2~-无关。     

The effects of Penicillium roqueforti toxin on the activity of rat hepatic DNA polymerases

PR toxin, a mycotoxin from cultures of Penicillium roqueforti, inhibited the in vitro activities of rat liver DNA polymerase alpha, beta, and gamma irrespectively of the nature of template-primer used. The concentration required for 50% inhibition of DNA polymerase alpha was 5-6 X 10(-6) M, while those for DNA polymerase beta and gamma were several times higher. By using DNA polymerase beta as a model, and based on the enzyme and template-primer concentration effects and also from the kinetic analysis on PR toxin inhibition, we concluded that two action mechanisms of PR toxin inhibition on in vitro DNA synthesis are operative. Inhibition of the in vitro DNA synthesis directed by DNA template was mediated primarily through alteration of the enzyme itself, whereas in the DNA synthesis reaction directed by RNA template DNA primer, the impairment of template or primer function due to PR toxin treatment probably had occurred. The inhibition of DNA polymerase by PR toxin persisted even after exhaustive dialysis. Addition of PR toxin to an ongoing reaction also inhibited DNA synthesis. Inactivation of DNA polymerase activity of PR toxin likely involved some essential amino acid residues other than sulfhydryl groups.     

海南粗榧新碱衍生物HH07A在大鼠体内的代谢转化研究

用GC-MS联用技术对海南粗榧新碱衍生物HH07A在大鼠体内代谢转化进行了研究。尿样经XAD-2固相提取、酶水解、浓缩并硅烷化,用GC-MS联用仪分离鉴定尿中HH07A及其4个代谢物,推断4个代谢物结构及其体内代谢途经。     

Mode of action of acyclovir triphosphate on herpesviral and cellular DNA polymerases

The effect of 5'-triphosphate of acyclovir (ACV) on DNA polymerases of two human herpes-viruses, herpes simplex virus type-1 (HSV-1) and Epstein-Barr virus (EBV) as well as human cellular DNA polymerases alpha and beta has been examined. Of the enzymes tested, HSV-1 DNA polymerase was the most sensitive to inhibition by acyclovir triphosphate (ACVTP). The EBV DNA polymerase and DNA polymerase beta were less sensitive. ACVTP inhibition was competitive with dGTP with Ki values of 0.03, 0.15, 9.8 and 11.9 microM for HSV-1 DNA polymerase, DNA polymerase alpha, EBV DNA polymerase and DNA polymerase beta, respectively. Substituting a synthetic primer template (dG) approximately 15 x (dC)n for activated DNA template did not alter the pattern of inhibition. In a time course experiment, addition of ACVTP instead of dGTP did not increase DNA synthesis and it appeared to act as a chain terminator in DNA replication catalyzed by either HSV-1 DNA polymerase or DNA polymerase alpha. Although EBV DNA polymerase was less sensitive to ACVTP inhibition, the nucleoside analog itself was inhibitory to EB virus production by P3HR1 cell line as determined by a reduction in the percentage of cells expressing virus capsid antigen (VCA). On day 4, ACV at 10 and 25 micrograms/ml reduced the cell growth by 10% and 32%, respectively, while it reduced the VCA-positive cells by 80% and 84%, respectively. These results indicate that inhibition of EBV DNA polymerase activity by ACVTP may not be the primary mechanism responsible for ACV inhibition of EBV replication.     

Role of a non-natural beta-C-nucleotide unit in DNA as a template for DNA and RNA syntheses and as a substrate for nucleolytic digestion.

A non-natural beta-C-nucleoside bearing a 3,4-dibenzyloxyphenyl group as a nucleobase (X) was synthesized and incorporated into a 34-mer oligomer with the sequence 5'-dTTTTTAAAAAAXATATAGCAGCGACATGTCACCG-3'. This synthetic oligonucleotide was examined for template activity in the enzymatic syntheses of DNA by the Klenow fragments of Escherichia coli DNA polymerase I and the recombinant DNA polymerase I, and in the synthesis of RNA by the E. coli RNA polymerase core enzyme. As a result, the template-directed polymerization of both DNA and RNA was precisely terminated at the position of X. The X-containing oligonucleotide was also tested for digestion by an exonuclease, Exo III nuclease (Exo III), and an endonuclease, Mung Bean nuclease (MB). The results indicate that the artificial nucleobase X acts as a terminator for digestion by Exo III, whereas the site X becomes susceptible to digestion by MB. These findings provide a useful tool for the size control of products in the synthesis and degradation of nucleic acids.     

全血DNA滤纸片中HIV1前病毒基因的PCR检测

建立套式引物聚合酶链式反应检测全血ＤＮＡ滤纸片中的ＨＩＶ１前病毒基因ｐｏ１ＤＮＡ片段。分别采集１２例ＨＩＶ１感染者的外周血约５０μｌ，经与ＥＤＴＡ－蛋白酶ｋ孵育后滴在无菌滤纸片上，３７℃下干燥，将滤纸片密封于塑料袋中，在４℃及室温下保存１周～６０周后，分别将滤纸片置０．５ｍｌ试管中直接进行ＨＩＶ１ｐｏ１基因的外侧引物ＰＣＲ检测，然后进行内侧引物的ＰＣＲ检测。结果表明，全血ＤＮＡ滤纸片在室温下保存２８周，在４℃下保存３４周仍可检出ＨＩＶ１目的基因。根据ＰＣＲ产物的琼脂糖凝胶电泳溴化乙啶染色带形并参比实验设立的标准对照可直接判断结果。该方法具有快速、特异、敏感的特点，敏感性可以达到检出１０个靶ＤＮＡ分子，可作为抗体确证试验的补充方法用于ＨＩＶ１感染的诊断     

A study of DNA bases sequence preference of aclarubicin B to bind DNA and inhibit DNA-dependent RNA synthesis in vitro

采用荧光淬灭法和体外无细胞ＲＮＡ合成系统研究了阿柔比星Ｂ结合ＤＮＡ和抑制ＤＮＡ依赖性ＲＮＡ合成的ＤＮＡ碱基顺序选择性。结果表明阿柔比星Ｂ与小牛胸腺ＤＮＡ，ｐｏｌｙ［ｄ（Ａ－Ｔ）］和ｐｏｌｙ［ｄ（Ｇ－Ｃ）］有明显结合；结合ＲＮＡ的活性小；与天然ＤＮＡ的结合力较与变性ＤＮＡ的结合力大。Ｓｃａｔｃｈａｒ分析显示阿柔比星Ｂ结合３种ＤＮＡ的亲和性依次为ｐｏｌｙ［ｄ（Ａ－Ｔ）］＞ｐｏｌｙ［ｄ（Ｇ－Ｃ）］＞小牛胸腺ＤＮＡ。同样，用大肠杆菌ＲＮＡ多聚酶和大鼠肝细胞核游离ＲＮＡ多聚酶实验均显示阿柔比星Ｂ的抑制力依次为ｐｏｌｙ［ｄ（Ａ－Ｔ）］＞ｐｏｌｙ［ｄ（Ｇ－Ｃ）］＞ｐｏｌｙ［ｄ（Ｉ－Ｃ）］。上述结果证明抑制ＲＮＡ合成的碱基顺序选择性与其结合ＤＮＡ碱基顺序的选择性有关