麦芽根中5′-磷酸二酯酶和5′-磷酸单酯酶的提取

建立一种高效快速的从麦芽根中提取5′-磷酸二酯酶和磷酸单酯酶的方法。通过将麦芽根粉碎后,取一定粒度的麦芽根加一定量的去离子水浸泡,过滤后得粗酶液。用紫外分光光度法测定5′-磷酸二酯酶和5′-磷酸单酯酶的酶活,研究粉碎粒度、料液比、抽提温度、抽提时间等因素对麦芽根中5′-磷酸二酯酶和5′-磷酸单酯酶提取的影响。将原料粉碎至100目,以去离子水为提取剂,于4℃提取20h,料液质量比为1:10,5′-磷酸二酯酶的酶活力可达160U/ml。该法简单、快速、准确、适应性强,为5′-磷酸二酯酶的提取与检测提供了有效的工具。     

5'-核苷酸的合成方法比较

5'-核苷酸在农业、食品和医药行业有着广泛的用途.比较了目前5'-核苷酸的工业和实验室合成方法,包括化学合成法,微生物发酵法,酶解法和酶催化法.     

高活力5′-磷酸二酯酶制备及水解RNA的研究

目的:获得高活力5′-磷酸二酯酶液,提高核酸RNA酶解效率。方法:采用超滤和盐析技术对从麦芽根浸提液中纯化5′-磷酸二酯酶工艺进行研究,采用单因子试验法优化酶解工艺条件。结果:浸提液依次经过5万Da超滤膜浓缩、40%饱和度硫酸铵盐析、5万Da超滤膜脱盐后,酶活力可达1 500U/ml;第1次超滤膜透过液可作为浸提液循环使用,酶活力是水浸提的1.15倍;第2次超滤膜透过液浓缩5倍后,可回收56.46%硫酸铵,浓缩母液可按1∶2比例循环使用;在底物浓度5.8%、酶用量8%、反应时间2h条件下,RNA酶解率可达95%。结论:初步建立了适合工业化规模的核苷酸生产新工艺。     

利用麦芽根抽提液降解RNA生产5′-核苷酸

国内核苷酸生产目前多采用液体深层发酵或固体培养桔青霉得到5′-磷酸二脂酶,以降解核酸成为5′-单核苷酸。啤酒厂有大量的副产物麦芽根,其中含有降解RNA的各种酶类。用水浸泡麦根后,将抽提液经过适当的热处理,用     

鸡8个功能基因5'-侧翼区与内含子的 SNP多样性比较

本研究旨在探讨鸡功能基因5'-侧翼区的单核苷酸多态性(SNP)水平.作者以自来航鸡、隐性白洛克鸡、杏花鸡、丝羽乌骨鸡、固始鸡、红色原鸡为材料,扩增了GH、DDBC1、RIKEN、RASGRP3、IGF1、THRSP、VIP及PRL共8个基因的5'-侧翼区DNA序列.扩增序列全长为8,399 bp,SNP的总数为161个,平均每52 bp出现一个SNP.8个功能基因5'-侧翼区核苷酸多样性的θ均值和π均值分别为0.00620±0.00110和0.00559±0.00100.比较检验表明,5'-侧翼区的SNP多样性显著低于内含子区域.5'-侧翼区是基因表达的一个重要调控区域,在分子进化和系统发生过程中承受着比内含子区域更大的选择压,其较低的SNP多样性是适应性较好的表现.Tajima检验与Fu和Li检验表明,与鸡繁殖性状显著关联的VIP基因和PRL基因很可能是人工选择或自然选择的目的基因.     

人肝刺激因子基因5'-侧翼序列克隆和结构分析

为探讨人肝刺激因子(human hepatic stimulator substance,hHSS)基因表达调控的分子机制,分离人了hHSS基因组DNA,克隆其5'-侧翼序列4 890 bp,与GenBank比对后,将hHSS基因定位于人16号染色体.通过逆转录PCR(RT-PCR)和5'RACE确定hHSS基因转录起始位点,为进一步研究hHSS基因启动子活性及转录调控特点提供了必要的实验依据.     

蛇毒的研究与利用——Ⅳ.浙江产蝮蛇(Agkistrodon halys Pallas)蛇毒的柱层析分离及磷酸二酯酶的大规模纯化

应用二乙胺基乙基葡聚糖A-50和盐浓度梯度洗脱的方法,对浙江产蝮蛇毒进行了柱层析分离,获得14个蛋白峰,测定了磷酸二酯酶、磷酸单酯酶、5'-核苷酸酶、蛋白水解酶、氨基酸酯酶、L-氨基酸氧化酶、磷脂酶A、出血毒素、抗凝血活酶组份以及血纤溶酶样物质在蛋白质峰中的分布。介绍了一种大规模制备磷酸二酯酶的简便、经济的纯化方法,对用这种方法制备的酶制剂的某些主要质量标准进行了分析鉴定并与国外同类产品进行了比较,该酶用于人工合成核糖和脱氧核糖寡核苷酸的顺序分析获得了较为满意的结果。     

白唇竹叶青蛇毒5’-核苷酸酶的分离纯化及性质

用DEAE-SephadexA-25、Sephadex-G-100和CM-SephadexC-50三步柱层析分离法,从白唇竹叶青(Trimeresurus albolabris)蛇毒中分离纯化出具有5'-核苷酸酶活性的组分.SDS-聚内烯酰胺凝胶电泳测定其分子量为48.03kDa,HPLC柱层析图谱为单一峰.该组分是一个糖蛋白,以一磷酸腺苷(AMP)为底物时,其酶活力为330.33 μg Pi/(min·mg);而以二磷酸腺苷(ADP)为底物时,其酶活力为123.56μg Pi/(min·mg).金属离子zn2 、Fe3 和Cu2 对5'-核苷酸酶活性有显著的抑制作用,EDTA可完全抑制其酶活性.该酶的最适pH为9,最适温度为50℃.该组分还具有抑制由ADP诱导的血小板聚集的生物功能.     

ggpps 5'-侧翼序列的克隆及其启动子功能验证

目的:研究牻牛儿基牻牛儿基焦磷酸合成酶(geranylgeranyl diphosphate synthase,GGPPs)基因启动子的活性;方法:从曼地亚红豆杉细胞中克隆ggpps基因5'-侧翼序列,并将该侧翼序列代替pBI121质粒上的CaMV35S启动子,以Gus基因作为报告基因构建植物表达载体,并进一步导入农杆菌LBA4404中获得阳性转化子,然后用叶盘转化法验证该侧翼序列的启动子活性;结果:本研究从曼地亚红豆杉细胞中成功克隆了ggpps基因的5'-侧翼序列,并且验证了该侧翼序列具有启动子活性;结论:ggpps基因的5'-侧翼序列的测序结果表明本实验成功克隆了该侧翼序列,启动子功能验证结果表明ggpps 5'-侧翼序列具有启动子活性,这些结果为进一步的通过缺失法进行ggpps基因启动子功能研究奠定了基础.     

响应面法优化红花籽粕5-羟色胺衍生物提取工艺

利用单因素实验及响应面法优化红花籽粕5-羟色胺衍生物的提取工艺。通过单因素试验考察提取溶剂、原料粒度、提取时间、提取温度、液料比对红花籽粕5-羟色胺衍生物提取得率的影响,确定各因素的适宜水平。在此基础上,以5-羟色胺衍生物提取得率为响应值进行Box-Behnken中心组合试验设计,并建立二次回归方程,得到红花籽粕5-羟色胺衍生物的最佳提取工艺条件:提取溶剂为无水乙醇、原料粒度为32目、提取时间为2.4 h、提取温度为89℃、液料比为19∶1,此条件下,红花籽粕5-羟色胺衍生物的提取得率可达0.34%。     

5'-Fluoro-5'-deoxyaristeromycin

5'-Fluoro-5'-deoxyaristeromycin (2) has been prepared via a Mitsunobu coupling of (1S,2S,3R,4S)-2,3-(cyclopentylidenedioxy)-4-fluoromethylcyclopentan-1-ol with N6-bis-boc protected adenine. This procedure is adaptable to preparing a number of 5'-fluoro-5'-deoxycarbocyclic nucleoside analogs with diversity in the heterocyclic base. Antiviral analysis found promising activity for 2 toward measles but no other viruses. No cytotoxicity was observed for 2.     

Enzyme-catalyzed redox reactions with the flavin analogues 5-deazariboflavin, 5-deazariboflavin 5'-phosphte, and 5-deazariboflavin 5'-diphosphate, 5' leads to 5'-adenosine ester.

The ability of 5-deazaisoalloxazines to substitute for the isoalloxazine (flavin) coenzyme has been examined with several flavoenzymes. Without exception, the deazaflavin is recognized at the active site and undergoes a redox change in the presence of the specific enzyme substrate. Thus, deazariboflavin is reduced catalytically by NADH in the presence of the Beneckea harveyi NAD(P)H:(flavin) oxidoreductase, the reaction proceeding to an equilibrium with an equilibrium constant near unity. This implies an E0 of -0.310 V for the deazariboflavindihydrodeazariboflavin couple, much lower than that for isoalloxazines. With this enzyme, both riboflavin and deazariboflavin show the same stereospecificity with respect to the pyridine nucleotide, and despite a large difference in Vmax for the two, both have the same rate-determining step (hydrogen transfer). Direct transfer of the hydrogen is seen between the nicotinamide and deazariboflavin in both reaction directions. DeazaFMN reconstituted yeast NADPH: (acceptor) oxidoreductase (Old Yellow Enzyme), and deazaFAD reconstituted D-amino acid:O2 oxidoreductase and Aspergillus niger D-glucose O2 oxidoreductase are all reduced by substrate at approximately 10(-5) the rate of holoenzyme; none are reoxidized by oxygen or any of the tested artificial electron acceptors, though deazaFADH-bound to D-amino acid:O2 oxidoreductase is rapidly oxidized by the imino acid product. Direct hydrogen transfer from substrate to deazaflavin has been demonstrated for both deazaFAD-reconstituted oxidases. These data implicate deazaflavins as a unique probe of flavin catalysis, in that any mechanism for the flavin catalysis must account for the deazaflavin reactivity as well.     

Amino substituted derivatives of 5'-amino-5'-deoxy-5'-noraristeromycin

The potent antiviral potential of 5'-amino-5'-deoxy-5'-noraristeromycin (2) is limited by associated toxicity. To seek derivatives of 2 that circumvent this undesirable property, three amino substituted derivatives (acetyl, 3; formyl, 4; and methyl, 5) of 2 have been prepared in 4-7 steps from the same intermediate, (1S,4R)-4-(6-chloropurin-9-yl)cyclopent-2-en-1-ol (6). Key steps involved an improved Pd(0)-catalyzed allylic azidation and a novel Pd(0)-catalyzed allylic amidation. The three target compounds were evaluated against a large number of viruses and found to be inactive except for a very weak effect of 5 on human cytomegalovirus, varicella zoster virus, and Epstein-Barr virus. There was also no noteworthy cytotoxicity associated with the new derivatives. Thus, these results indicate variation of the cyclopentyl amine of 2 does not offer a means to improve upon its antiviral potential.     

Independence of brachymesophalangia-5 from brachymesophalangia-5 with cone mid-5

Analysis of hand radiographs of juvenile siblings of juvenile propositi indicates that brachymesophalangia-5 alone (without cones) is separately inherited without apparent sex bias while brachymesophalangia-5 with the cone-epiphysis of mid-5 and the cone-epiphysis of mid-5 alone are both apparently inherited as a complex and with a marked excess of females over males.     

Mechanism of inactivation of S-adenosyl-L-homocysteine hydrolase by 5'-deoxy-5'-S-allenylthioadenosine and 5'-deoxy-5'-S-propynylthioadenosine

5'-Deoxy-5'-S-allenylthioadenosine 1 and 5'-deoxy-5'-S-propnylthioadenosine 2, derived from adenosine, were prepared. 1 and 2 caused irreversible inactivation of AdoHcy hydrolase. ESI mass spectra analysis of the inactivated enzyme demonstrated that 1 and 2 were type II "mechanism-based" inhibitors.     

Stat5及JAK-Stat5通路

信号转导与转录因子（Stats）家族是一个由7个成员组成的转录调控家族,其中Stat5由于在多种细胞因子刺激时均可起到重要作用而引起了广泛的关注。Stat5具有两种不同类型,即Stat5a和Stat5b,它们均可被多种细胞因子所激活,启动JAK-Stat5信号通路,从而调节相应基因的表达。在两种Stat5的激活中,特殊位点的丝氨酸和酪氨酸的磷酸化对Stat5的激活起重要的作用。对信号通路严格的控制对于生物体来说具有重要的意义,在JAK-Stat5信号通路中,以SOCS3为代表的SOCS家族对JAK-Stat5的负反馈调节具有重要作用。     

Enzymatic synthesis of 5-azacytidine 5'-triphosphate from 5-azacytidine.

5-Azacytidine 5′-monophosphate (5-aza-CMP) was synthesized enzymatically from 5-azacytidine (5-aza-C) in a reaction catalyzed by uridine-cytidine kinase. In a second step, 5-azacytidine 5′-triphosphate (5-aza-CTP) was synthesized enzymatically from 5-aza-CMP using CMP kinase and nucleoside diphosphokinase. Due to the chemical instability of the triazide ring of 5-azacytosine at neutral and alkaline pH, the enzymatic synthesis and purification of the nucleotides by ion exchange chromatography were performed at acid pH. The enzymatically synthesized 5-aza-CTP had an ultraviolet absorbance spectrum at pH 5.5 similar to the spectrum of 5-aza-C. In the DNA-dependent RNA polymerase reaction, 5-aza-CTP inhibited the incorporation of [³H]CTP, but [³H]UTP, into RNA.     

Enzymatic conversion of deoxycytidine 5'-monophosphate to 5-methyldeoxycytidine 5'-triphosphate

A simple procedure for the synthesis of chiral acetic acids has been developed. The key step is an enzymatic exchange reaction which introduces ³H from ³H-labeled water into ethane 1,2-diol. The method involves no resolution of racemic intermediates and the products are of high specific radioactivity and optical purity.