核酸的荧光染料——菲啶溴红

菲啶溴红是近年来应用较为广泛的一种荧光染料,它与核酸有特异的结合能力,对体内核酸(DNA和RNA)的合成以及与核酸有关的多聚酶和转录酶都有抑制作用,并具有抗病毒活性和诱发突变的性能。最初菲啶溴红是一种抗锥虫药物,后来观察到它有抑制蛋白质合成的功能。1964年Le Pecq等人发现菲啶溴红与核酸结合后,其荧光强度显著增高,引起人们的重视。此后对菲啶溴红-核酸的物理化学性质进行了一     

石韦不同极性萃取物体外降血糖活性研究北大核心CSCD

为进一步明确石韦对α-葡萄糖苷酶和α-淀粉酶抑制作用机制,该研究以石韦95%乙醇提取物的不同极性萃取物为试材,阿卡波糖为阳性对照,采用pNPG法(p-nitrophenyl-α-D-glucopyranoside,pNPG)、DNS法(3,5-dinitro salicylic acid,DNS)考察石韦不同极性萃取物的α-葡萄糖苷酶和α-淀粉酶抑制活性,并采用酶促动力学方法与Lineweaver-Burk曲线分析最强活性萃取物对α-葡萄糖苷酶的抑制作用类型,以期为石韦的进一步开发利用提供科学依据。结果表明:石韦水萃取物对α-葡萄糖苷酶和α-淀粉酶的抑制作用最强,其IC_(50)值分别为(4.71±0.72)μg·mL^(-1)、IC_(50)=(48.40±0.32)μg·mL^(-1),并显著强于其他萃取物(P<0.05),且对α-葡萄糖苷酶抑制作用比阿卡波糖强,对α-淀粉酶抑制作用弱于阿卡波糖。阿卡波糖对α-葡萄糖苷酶和α-淀粉酶抑制作用的IC_(50)值分别为(2857.36±1.35)μg·mL^(-1)和(16.41±0.63)μg·mL^(-1)。酶促动力学显示水萃取物对α-葡萄糖苷酶为可逆性抑制,Lineweaver-Burk曲线显示为竞争性抑制。综上结果表明,石韦水萃取物具有较好的α-葡萄糖苷酶和α-淀粉酶抑制活性,是一种胃肠道副作用小、天然α-葡萄糖苷酶抑制剂来源。     

五种苦苣苔科植物α-葡萄糖苷酶抑制活性研究

利用体外α-葡萄糖苷酶抑制模型对5种苦苣苔科植物进行活性评价,并与阳性对照Acarbose进行比较,发现5种植物不同部位均有一定的α-葡萄糖苷酶抑制活性。其中,牛耳岩白菜石油醚部位的抑制活性最高(IC50=26.19μg/mL,活性均远大于阳性对照Acarbose(IC50=1081.27μg/mL)。不同植物比较,牛耳岩白菜的α-葡萄糖苷酶抑制活性最好,其3种不同溶剂提取物与Acarbose相比均有很高抑制活性;对牛耳岩白菜提取物的α-葡萄糖苷酶抑制动力学研究结果表明,石油醚和乙酸乙酯提取物对α-葡萄糖苷酶抑制作用属于非竞争性抑制类型,Ki值分别为4.24和40.04μg/mL。正丁醇提取物则属于竞争性抑制类型(Ki=205.48μg/mL)     

鸭乙型肝炎病毒复制复合体中内源性DNA聚合酶测定方法的建立及其应用

应用~3H-TTP作为放射性掺入底物,建立了鸭乙型肝炎病毒复制复合体(DHBVRCs)内源性DNA聚合酶的测定方法,研究了该酶的生化特性,试验了12种药物对该酶的抑制作用。结果该方法测定的内源性DNA聚合酶为DHBV特异的,其活性依赖于4种dNTP和Mg~ ++的存在。最适Mg~++浓度为20mM。NP-40可刺激其活性。放线菌素D仅能抑制其30％的活性,膦羧基甲酸钠(PFA)、膦羧基乙酸钠(PAA)和苏拉明可抑制此酶,它们的半数抑制浓度分别为8.5μM、860μM和9.25μM。而Ara-AMP等几种药物对该酶无抑制作用。应用放射性掺入电泳自显影法证明,PFA对DHBV RCs中依赖DNA和RNA的DNA聚合酶均有显著的抑制作用。     

橡实不同极性萃取物对α-淀粉酶和α-葡萄糖苷酶抑制作用研究CSCD

探索橡实不同极性萃取物对α-淀粉酶和α-葡萄糖苷酶的抑制作用。采用40%乙醇和纤维素酶组合提取,石油醚、乙酸乙酯依次萃取分离,获得橡实粗提物、石油醚相、乙酸乙酯相、萃余水相。通过测定酶抑制作用,研究各极性萃取物的体外降糖活性,并利用高效液相色谱(HPLC)和分子对接实验验证各提取物可能的活性成分。结果表明:橡实不同极性萃取物对α-淀粉酶和α-葡萄糖苷酶均有明显的抑制作用,其中乙酸乙酯相抑制效果最佳,其对α-淀粉酶和α-葡萄糖苷酶IC_(50)分别为1.590±0.073 mg/g、3.927±0.019(×10^(-3) mg/g);通过HPLC含量测定发现乙酸乙酯相中6种活性成含量最高,鞣花酸含量为500.75±6.93 mg/g,高于其他组分10~50倍;分子对接结果表明只有鞣花酸与α-淀粉酶和α-葡萄糖苷酶有良好的结合活性。以上结果表明,鞣花酸可能是橡实乙酸乙酯萃取物抑制α-淀粉酶和α-葡萄糖苷酶的主要活性成分。     

桦褐孔菌活性物质的提取工艺及体外抗糖尿病活性

对桦褐孔菌活性物质的提取工艺及其体外抗糖尿病活性进行研究。桦褐孔菌子实体用乙醇浸提后,乙醇粗提物用不同有机溶剂萃取,醇提残渣再以热水浸提,用标准曲线法测定活性组分中活性成分的含量,并检测活性物质对羟基自由基(·OH)、超氧阴离子自由基(O2-·)的清除效果以及对关键糖代谢酶α-淀粉酶、α-葡萄糖苷酶的抑制作用。结果表明:4种活性组分(乙酸乙酯相、正丁醇相、水相和粗多糖)对羟基自由基(·OH)和超氧阴离子自由基(O2-·)都有较强的清除作用,其中乙酸乙酯组分的活性最高,乙醇粗提物萃取组分对α-淀粉酶有抑制活性,而粗多糖对α-葡萄糖苷酶有抑制作用;桦褐孔菌具有抗氧化和抗糖尿病活性,其活性与活性物质种类及其含量具有相关性。     

植物苯丙氨酸解氨酶的研究 Ⅰ.植物激素对甘薯块根苯丙氨酸解氨酶和肉桂酸4-羟化酶活性变化及其伴随性的影响

切伤能诱导甘薯块根切片苯丙氨酸解氨酶(PAL)和肉桂酸4-羟化酶(CA4H)活性增高。生长素(IAA)、激动素(BAP)和乙烯(乙烯利)能刺激甘薯块根切片的PAL和CA4H活性,刺激效应乙烯利>IAA>BAP。赤霉素(GA_3)无效。红光对PAL和CA4H无诱导作用,但能增强BAP的效应。放线菌素D和环己亚胺都能抑制PAL和CA4H活性增高,但二者在时相上有差异。在各种情况下,这二种酶的动态变化总是相伴随的,这种伴随性开始于对放线菌素D敏感的步骤,随后转移到对环己亚胺敏感的步骤。     

雷帕霉素对二种鳞翅目昆虫细胞自噬和凋亡的影响

以2种鳞翅目昆虫细胞为材料,采用雷帕霉素进行处理,初步研究自噬作用与昆虫细胞凋亡的关系。结果表明:雷帕霉素能够提高家蚕细胞系BMN-e细胞的自噬水平,并能诱导BMN-e细胞发生凋亡;自噬抑制剂3-甲基腺嘌呤能抑制雷帕霉素诱导的BMN-e细胞凋亡。相反,雷帕霉素虽能诱导斜纹夜蛾细胞系SL-HP细胞的自噬水平提高,但不能诱导斜纹夜蛾Spodoptera litura(Fabricius)细胞发生凋亡;雷帕霉素的预处理能抑制放线菌素D诱导的斜纹夜蛾细胞系SL-HP细胞发生凋亡;自噬抑制剂3-甲基腺嘌呤对放线菌素D诱导的细胞凋亡没有影响。因此家蚕Bombyx mori细胞自噬水平的提高与细胞凋亡具有正相关性,而斜纹夜蛾细胞自噬水平的提高与细胞凋亡不相关,相反还对细胞凋亡的诱导具有一定的抑制作用。     

贴梗海棠α-葡萄糖苷酶抑制活性研究

对不同月份采摘的贴梗海棠叶α-葡萄糖苷酶抑制活性进行研究,发现3月和7月采集的贴梗海棠叶不同提取部位均具有一定的α-葡萄糖苷酶抑制活性。其中,7月贴梗海棠叶中乙酸乙酯部位(IC50=47.27μg/mL)的α-葡萄糖苷酶抑制活性最好,远高于阳性对照阿卡波糖(IC50=1213.38μg/mL)。7月贴梗海棠叶中乙酸乙酯部位和正丁醇部位的α-葡萄糖苷酶抑制活性均高于3月叶中相应部位,而石油醚部位低于3月叶的石油醚部位,且都高于阳性对照阿卡波糖。此外,各提取物对α-葡萄糖苷酶的抑制作用均具有剂量依赖性。     

胎羊3β,20α-羟类甾醇氧化还原酶活性位点的组氨酸残基的鉴定

为进一步研究胎羊3β,20α-羟类甾醇氧化还原酶活性中心的特性,我们合成了放射性的16α-溴乙酸基孕酮和5α-二氢睾酮-17-溴乙酸酯作为亲和标记试剂。二者都是以不可逆的方式,活性位点定向地竞争性抑制酶的活性。当酶的浓度为1μmol/L,抑制剂的浓度为100μmol/L时,使酶失去一半活性所需时间(t_0.5)分别为75 min(16α-BAP)和480 min(5α-DTB)。当在反应混合液中有底物孕酮或5α-二氮睾酮存在时,可降低抑制剂对酶活性的抑制速度。把标记的酶用盐酸水解,用氨基酸分析仪进行分析,最后确定,位于酶的活性中心的被标记氨基酸为组氨酸,它可能在氧化还原反应过程中发挥着重要作用。     

Poly(ADP-ribosyl)ation of terminal deoxynucleotidyl transferase in vitro

The activity of purified bovine thymus terminal deoxynucleotidyl transferase was markedly inhibited when the enzyme was incubated in a poly(ADP-ribose)-synthesizing system containing purified bovine thymus poly(ADP-ribose) polymerase, NAD+, Mg2+ and DNA. All of these four components were indispensable for the inhibition. The inhibitors of poly(ADP-ribose) polymerase counteracted the observed inhibition of the transferase. Under a Mg2+-depleted and acceptor-dependent ADP-ribosylating reaction condition [Tanaka, Y., Hashida, T., Yoshihara, H. and Yoshihara, K. (1979) J. Biol. Chem. 254, 12433-12438], the addition of terminal transferase to the reaction mixture stimulated the enzyme reaction in a dose-dependent manner, suggesting that the transferase is functioning as an acceptor for ADP-ribose. Electrophoretic analyses of the reaction products clearly indicated that the transferase molecule itself was oligo (ADP-ribosyl)ated. When the product was further incubated in the Mg2+-fortified reaction mixture, the activity of terminal transferase markedly decreased with increase in the apparent molecular size of the enzyme, indicating that an extensive elongation of poly(ADP-ribose) bound to the transferase is essential for the observed inhibition. Free poly(ADP-ribose) and the polymer bound to poly(ADP-ribose) polymerase were ineffective on the activity of the transferase. All of these results indicate that the observed inhibition of terminal transferase is caused by the poly(ADP-ribosyl)ation of the transferase itself.     

Delayed inhibition of DNA synthesis in mouse jejunum by low doses of actinomycin D

Actinomycin D (0.008 μg/gm of body weight) injected intraperitoneally every two hours, produced a prompt 50% inhibition of RNA synthesis in the jejunum of mice, and a delayed inhibition of DNA synthesis, that reached its maximum inhibition (68% of control values) 4.5 hours after the first injection of actinomycin D. Autoradiographic studies indicated that this low level of actinomycin D inhibited a step in the G₁ phase of the cell cycle, preventing the initiation, but not affecting the continuation, of DNA biosynthesis. The activity of DNA polymerase was not affected under these conditions. The results are substantially similar to those previously obtained with Ehrlich ascites cells growing in the peritoneal cavity of mice and can be interpreted as indicating that in the G₁ phase of dividing cells there is an actinomycin sensitive step whose inhibition prevents the entrance of cells into the DNA-synthesis phase.     

The mechanism of inhibition of bacteriophage T7 RNA synthesis by acridines, diacridines and actinomycin D.

A homologous series of diacridines, as well as 9-amino acridine, were assayed for their ability to interfere with the synthesis of RNA (bands U-VI) by bacteriophage T7 DNA-dependent RNA polymerase transcribing T7 DNA in vitro; their action was compared to that of actinomycin D. It was found that, in contrast to actinomycin D which inhibits chain elongation, the acridines tested inhibited chain initiation only; no evidence for inhibition of chain elongation was noted. No clear-cut differentiation between single and double intercalators on the mechanism of inhibition of RNA synthesis could be determined, except that the latter are more potent inhibitors. However, it appears that diacridines connected with a diethyldiamine and a butyldiamine chain are less inhibitory to the synthesis of the RNA of Bands III and IV. The results furthermore indicate that the estimation of the number average molecular weight alone, without identification of the product RNA, is a potentially misleading method of determining the mode of action of these drugs.     

Regulation of DNA metabolic enzymes upon induction of preB cell development and V(D)J recombination: up-regulation of DNA polymerase delta.

Withdrawal of interleukin-7 from cultured murine preB lymphocytes induces cell differentiation including V(D)J immunoglobulin gene rearrangements and cell cycle arrest. Advanced steps of the V(D)J recombination reaction involve processing of coding ends by several largely unidentified DNA metabolic enzymes. We have analyzed expression and activity of DNA polymerases alpha, beta, delta and epsilon, proliferating cell nuclear antigen (PCNA), topoisomerases I and II, terminal deoxynucleotidyl transferase (TdT) and DNA ligases I, III and IV upon induction of preB cell differentiation. Despite the immediate arrest of cell proliferation, DNA polymerase delta protein levels remained unchanged for approximately 2 days and its activity was up-regulated several-fold, while PCNA was continuously present. Activity of DNA polymerases alpha,beta and epsilon decreased. Expression and activity of DNA ligase I were drastically reduced, while those of DNA ligases III and IV remained virtually constant. No changes in DNA topoisomerases I or II expression and activity occurred and TdT expression was moderately increased early after induction. Our results render DNA polymerase delta a likely candidate acting in DNA synthesis related to V(D)J recombination in lymphocytes.     

Interaction of granaticin B with the transcription system ofBacillus subtilis

The interaction of granaticin B, a quinone antibiotic produced by Streptomyces granaticolor, with some biologically important bivalent metal ions, DNA and ATP was demonstrated spectrophotometrically. The activity of isolated RNA polymerase was higher when the DNA of phage SP 50 served as template than with DNA isolated from Bacillus subtilis. Granaticin B inhibited in vitro RNA synthesis, similarly to certain other antibiotics (the inhibition was three times lower than that caused by actinomycin D or streptolydigin and slightly higher than that by epsilon-pyrromycinone). The inhibitory effect was higher when the Mg2+ concentration in the reaction mixture was decreased. The inhibition was then proportional to the concentration of the DNA template. DNA-dependent RNA synthesis is thus inhibited in vitro by granaticin B but this does not appear to be the only site of action of this antibiotic in vivo.     

Comparison of the effects of zinc deprivation and actinomycin D on ribonucleic acid synthesis by stimulated lymphocytes.

1. EDTA inhibited incorporation of [3H]uridine into RNA of lymphocytes, but did not decrease uptake into the cold-acid-soluble fraction of the cells. The inhibition by EDTA was largely reversible by simultaneous addition of Zn2+. 2. Low concentrations pf actinomycin D (3 ng/ml) added at the time of stimulation of the cells inhibited [3H]uridine incorporation into RNA, but concentrations of 50-100 ng/ml were required to produce the same degree of inhibition if addition of actinomycin D was delayed until just before the incorporation was measured. This difference in sensitivity did not reg within the cells. 3. When added immediately before phytohaemagglutinin, actinomycin D (3 ng/ml) and EDTA produced similar time-courses of inhibition of uridine incorporation. 4. Uridine incorporation at 32h was inhibited when actinomycin D (3 ng/ml) or EDTA was added just before stimulation of the cells, but was only slightly affected when they were added at 32h. At intermediate times the incorporation of uridine remained sensitive to addition of EDTA for longer than it was sensitive to actinomycin D. 5. Polyacrylamide-gel separation of RNA synthesized in EDTA-treated cultures in the presence or absence of added Zn2+ showed that lower availability of Zn2+ resulted in a decreased rate of transfer of radioactivity from 32S to 28S rRNA and decreased survival of 28S rRNA relative to 18S rRNA. 6. Close similarities have been shown to exist between the effects of EDTA and low concentrations of actinomycin D. Not all the effects of EDTA could be explained by postulating that Zn2+ was a constituent of RNA polymerase I, nor were the effects of actinomycin D readily explained by previously suggested mechanisms of action of this antibiotic.     

Lesion bypass by human DNA polymerase mu reveals a template-dependent, sequence-independent nucleotidyl transferase activity

DNA polymerase mu (pol mu), which is related to terminal deoxynucleotidyl transferase and DNA polymerase beta, is thought to be involved in non-homologous end joining and V(D)J recombination. Pol mu is induced by ionizing radiation and exhibits low fidelity. Analysis of translesion replication by purified human pol mu revealed that it bypasses a synthetic abasic site with high efficiency, using primarily a misalignment mechanism. It can also replicate across two tandem abasic sites, using the same mechanism. Pol mu extends primers whose 3'-terminal nucleotides are located opposite the abasic site. Most remarkably, this extension occurs via a mode of nucleotidyl transferase activity, which does not depend on the sequence of the template. This is not due to simple terminal nucleotidyl transferase activity, because pol mu is unable to add dNTPs to an oligo(dT)29 primer or to a blunt end duplex oligonucleotide under standard conditions. Thus, pol mu is a dual mode DNA-synthesizing enzyme, which can act as either a classical DNA polymerase or as a non-canonical, template-dependent, but sequence-independent nucleotidyl transferase. To our knowledge, this is the first report on a DNA-synthesizing enzyme with such properties. These activities may be required for its function in non-homologous end joining in the processing of DNA ends prior to ligation.     

DNA elongation by the human DNA polymerase lambda polymerase and terminal transferase activities are differentially coordinated by proliferating cell nuclear antigen and replication protein A

DNA polymerase lambda contains template-dependent (DNA polymerase) and template-independent (terminal transferase) activities. In this study we enzymologically characterized the terminal transferase activity of polymerase lambda (pol lambda-tdt). Pol lambda-tdt activity was strongly influenced by the nature of the 3'-terminal sequence of the DNA substrate, and it required a single-stranded (ss) DNA 3'-overhang of about 9-12 nucleotides for optimal activity. The strong preference observed for pyrimidine versus purine nucleotide incorporation was found to be due, at least partially, to a steric block imposed by the residue Tyr-505 in the active site of pol lambda. Pol lambda-tdt was found to be able to elongate a 3'-ssDNA end by two alternative mechanisms: first, a template-independent one resulting in addition of 1 or 2 nucleotides, and second, a template-dependent one where a homopolymeric tract as short as 3 nucleotides at the 3'-end could be used as a template to direct DNA polymerization by a looping back mechanism. Furthermore repetitive cycles of DNA synthesis resulted in the expansion of such a short homopolymeric terminal sequence. Most importantly we found that the proliferating cell nuclear antigen was able to selectively block the looping back mechanism while stimulating the single terminal nucleotide addition. Finally replication protein A completely suppressed the transferase activity of pol lambda while stimulating the polymerase activity, suggesting that proliferating cell nuclear antigen and replication protein A can coordinate the polymerase and the terminal transferase activities of pol lambda.