首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 140 毫秒
1.
乙肝病毒核衣壳启动子内三链DNA的形成   总被引:2,自引:0,他引:2  
用电泳迁移分析方法研究了21nt脱氧寡核苷酸G3TG2TGT2G5TG2TGT与129bp的乙肝病毒核衣壳启动子片段内一位点结合形成的三链DNA的特异性及稳定性。在克隆有HBV基因组的质粒pCRP10的酶切产物中,CP1仅与含Cpdisplay structure  相似文献   

2.
用正丁醇抽提,硫酸铵分级沉淀,DEAE-纤维素和SephacrylS-200柱层析,从南方鲇(Silurus meridionalis Chen)肠粘膜中提取出碱性磷酸酶(AKP)。提纯倍数为39.50倍,比活为68.35μ/mg蛋白,提取酶液经PAGE和SDS-PAGE只呈现一条区带。该酶的分子量为132140,N末端氨基酸为门冬氨酸,最适pH为10.10,7.5>pH>11.5时不稳定,最适温度为40℃左右,对热不很稳定,以磷酸苯二钠为底物其K_m值为1.72×10~(-3)mol/L。Mg~(2+)、Mn~(2+)为该酶的激活剂,KH_2PO_4、L-CyS、ME、DFP、EDTA-Na_2为抑制剂。选用KH_2PO_4和DFP作抑制类型的判断,结果表明,KH_2PO_4属竞争性掏剂,其抑制常数为2.3mmol/L;DFP为非竞争性抑制剂,抑制常数为1.05mmol/L。  相似文献   

3.
LHRH—A2及无机离子促进草鱼脑垂体离体分泌GH的初步研究   总被引:1,自引:0,他引:1  
用培养瓶(皿)培养法研究LHRH-A2、Ca^2+、K^+对草鱼脑垂体器官分泌GH的影响。结果表明,1nmol/L、10nmol/L、100nmol/L和1000nmol/L的LHRH-A2都能显著促进GH的分泌;随着Ca^2+浓度(0.1、1、3mmol/L)的增高GH的分泌增强,在1mol/L和3mmol/L Ca^2+条件下的GH分泌水平显著高于在0.1mmol/L Ca^2+条件下的GH分  相似文献   

4.
目的和方法:采用双微电极电压钳(TEV)法研究细胞外Mn^2+对非洲爪蟾卵母细胞表达的内向整流钾通道(IRK1)的阻断作用。结果:细胞外Mn^2+浓度分别为1、1.25、2.5、5、10和20mmol/L,K^+浓度为90mmol/L,可见Mn^2+对IRK1的瞬间电流(旋加电压后2ms)具有Mn^2+浓度依赖性和电压依赖性阻断作用;细胞外加Mn^2+浓度较高时强;细胞外K^+浓度为90mmol/  相似文献   

5.
用BamHI和HindII将丙肝病毒C+E1DNA片段从其克隆载体pGEM3zf-HCV/C+E1上切下,经Taq酶补齐3’末端后插入到载体pSVL-T中,构建成丙肝病毒C+E1真核表达载体pSVL-HCV/C+E1。本实验中重组效率达64.7%(11/17),正向插入为50%(2/4)。  相似文献   

6.
采用3H-TdR参入法,测定碱性成纤维细胞生长因子(bFGF)、胰岛素和内皮素-1(ET-1)对体外培养的大鼠肾小球系膜细胞(MC)增殖的影响,以及胰岛素与bFGF或ET-1促MC增殖的协同作用。结果表明,不同浓度的bFGF(5-200ng/ml)和胰岛素(0.1-2.4U/ml)均显著升高MC的3H-TdR参入值(cpm值)(P<0.01)。ET-1对MC的cpm值的影响依剂量不同呈现两种不同的效应,在10-9-10-7mol/L时,随着浓度的升高,MC的cpm值明显升高(P<0.01),并以10-8mol/L作用最强;当升高到10-6mol/L时,MC的cpm值出现降低趋势。胰岛素与bFGF或低浓度ET-1(≤10-8mol/L)共同作用于MC时,MC的cpm值明显高于二者单独作用之和(P<0.01),与高浓度ET-1(>10-7mol/L)共同作用于MC时,MC的cpm值小于二者单独作用之和(P>0.05)。上述结果说明,胰岛素、bFGF和ET-1均能显著促进MC增殖;胰岛素与bFGF或低浓度的ET-1促MC增殖具有正协同作用,与高浓度ET-1呈现负协同作用。  相似文献   

7.
细胞松弛素B促微丝解聚对DNA合成的作用   总被引:2,自引:0,他引:2  
利用微丝(MF)解聚药物细胞松弛素B(CB)处理G0期小鼠C3H10T1/2成纤维细胞,对G0至S期DNA合成,胸腺嘧啶核苷激酶(TK)活性、TK基因表达、钙调素(CaM)水平和一些细胞周期早期基因的表达进行了观察。G0期细胞经3mg/LCB处理2h,促MF解聚增强了血清对S期细胞TK活性、TK基因表达和DNA合成的刺激作用,并促进细胞提前进入S期。血清刺激G0期细胞进入晚G1期和S期时,CaM  相似文献   

8.
本文以蒙古沙土鼠双颈总动脉结扎(BCAO)前脑缺血模型Ca2+/CaMPKⅡ活性变化为指标,研究了以氯胺酮(KT)、右美沙芬(DM)、苄丙咯(BP)及硝苯吡啶(ND)为代表的配体门控Ca2+通道(LGCC)及电压门控Ca2+通道(VGCC)两类Ca2+通道拮抗剂对缺血性脑损伤的保护作用。结果如下:(1)脑缺血后,胞浆型及颗粒型Ca2+/CaMPKⅡ活性均明显下降;(2)缺血前单独用药,KT、DM、BP及ND对酶活性均具有剂量依赖性的保护作用;(3)与单独用药相比,联用异类药,KT+BP、KT+ND及DM+BP的保护作用显著增高,而联用同类药,BP+ND及KT+DM的保护作用无明显增高。结果提示:脑缺血中,LGCC及VGCC两类Ca2+通道均参与了缺血性脑损伤过程;两类Ca2+通道的单一拮抗对缺血性脑损伤均有保护作用,而联合拮抗效果更佳。  相似文献   

9.
采用膜片钳技术以全细胞方式在小鼠腹腔渗出巨噬细胞(PEM)中记录到一种不完全失活的外向K+电流(Io),该电流在膜电位正于-10mV时激活,对K+具有高度特异性,其半值电导电位V1/2为79.5mV,在膜电位正于30mV时,该电流失活,在60-120mV的膜电位范围内,其失活时间常数τi与膜电位无关.随着胞外K+离子浓度([K+]o)升高,该电流的失活过程减慢。在生理电压范围内(-80-0mV),该电流缺乏稳态失活,且其失活不具有频率依赖性。胞外4-AP(3mmol/L)、Ba2+(3mmol/L)及TEA(5mmol/L)可抑制该电流,抑制率分别为85%,66%及31%。胞外Zn2+(1mmol/L)可影响该电流活性,对该电流的抑制具有电压依赖性  相似文献   

10.
灵芝多糖对人脐血LAK细胞活性的影响   总被引:2,自引:0,他引:2  
徐新  曹容华 《生物技术》1994,4(2):33-35,23
本文研究了灵芝多糖(GLP)对人脐血LAK(CB—LAK)细胞活性的影响,结果发现,单独GLP能刺激人脐血单个核细胞(CBMC)增殖,但不能诱导LAK活性,当与50u/mlrIL—2伍用时,可增殖CB—LAK细胞诱导活性,不同剂量GLP(0.5—100μg/ml)影响作用不同,以10μg/ml浓度最好.在不同浓度rIL—2(10—100u/ml)诱导CB—LAK细胞过程中加入GLP(10μg/ml),可明显提高细胞增殖能力,减少rIL—2用量。GLP亦能促进效应阶段CB—LAK细胞对Raji肿瘤靶细胞的杀伤作用(P<0.001)。由此看出,GLP具有增强CB—LAK细胞活性的作用,是一很好的生物反应调节剂(BRM),有必要进行深入的研究。  相似文献   

11.
在IRIS Indigo2(SGI公司)工作站上,利用InsightⅡ/MSI软件包,以TAT三链DNA为模板,采用同源模建的方法,分别建立起两个含21nt的脱氧寡核苷酸CP1(G3TG2TGT2G5TG2TGT)和CP3(TGTG2TG5T2GTG2TG3)的三维结构.采用分子力学方法进行能量优化,将得到的能量最低结构作为分子的优势构象.研究结果显示,CP1的能量低于CP3的能量,即前者的结构较后者稳定.从而证明了CP1与乙肝病毒(HBV)的核心启动子(Cp)片段之间能稳定地形成三链DNA,并能特异性地抑制DNA结合蛋白与Cp片段的结合.这些结果表明,三链DNA的形成有可能抑制DNA的转录.  相似文献   

12.
The binding of a 19-mer guanosine-rich oligodeoxyribonucleotide, TG3TG4TG4TG3T (ODN 1), to a complementary polypurine DNA target was investigated by DNase I footprinting and restriction endonuclease protection assays. Monovalent cations inhibited intermolecular purine-purine-pyrimidine triple-helical DNA formation, with K+ and Rb+ being most effective, followed by NH4+ and Na+. Li+ and Cs+ had little to no effect. Similar results were observed with the G/A-rich oligonucleotide AG3AG4AG4AG3AGCT. Kinetic studies indicated that monovalent cations interfered with oligonucleotide-duplex DNA association but did not significantly promote triplex dissociation. The observed order of monovalent cation inhibition of triplex formation is reminiscent of their effect on tetraplex formation with G/T-rich oligonucleotides. However, using electrophoretic mobility shift assays we found that the oligonucleotide ODN 1 did not appear to form a four-stranded species under conditions promoting tetraplex formation. Taken together, our data suggest that processes other than the self-association of oligonucleotides into tetraplexes might be involved in the inhibitory effect of monovalent cations on purine-pyrimidine-purine triplex formation.  相似文献   

13.
酸雨作用下的森林冠层盐基离子(Ca2+,Mg2+,K+)淋洗   总被引:14,自引:1,他引:13  
在韶山针阔叶混交林中设立了10个30 m×30 m的样方,对1年中各个季节的森林截留沉降、降雨后树冠层总滤出量、盐基离子滤出量以及树冠层对H+和NH4+的摄入量进行了分析和估算.韶山森林湿沉降成分中以Ca2+为主,Mg2+,K+含量较低.树冠层盐基离子总滤出量中Ca2+最高,达到155.34 mmo1 m-2a-1,Mg2+最低,为30.74mmol m-2a-1,K+居中,为84.13 mmol m-2a-1.Ca2+的大量滤出表明它是树冠层缓冲降水酸度的主要介质,同时也表明酸雨对韶山森林的潜在危害,其在总滤出量中的比重的季节变化是夏(58.4%)>春(54.1%)>冬(51.4%)>秋(32.5%).盐基离子的滤出量以冬→春→夏→秋依次递减,但是树冠层季节摄入NH4的量在30-100mmo1 m-2而对H+的摄入量则在30-180 mmol m-2.  相似文献   

14.
A differential effect is found of various bivalent cations (Ba2+, Ca2+, Mg2+, Cd2+, Co2+, Mn2+, Ni2+, Zn2+ and Hg2+) on stability of intermolecular Py-Pu-Pu triplex with different sequence of base triads. Ca2+, Mg2+, Cd2+, Co2+, Mn2+, Ni2+ and Zn2+ do stabilize the d(C)n d(G)n d(G)n triplex whereas Ba2+ and Hg2+ do not. Ba2+, Ca2+, Mg2+ and Hg2+ destabilize the d(TC)n d(GA)n d(AG)n triplex whereas Cd2+, Co2+, Mn2+, Ni2+ and Zn2+ stabilize it. The complexes we observe are rather stable because they do not dissociate during time of gel electrophoresis in the co-migration experiments. Chemical probing experiments with dimethyl sulfate as a probe indicate that an arbitrary homopurine-homopyrimidine sequence forms triplex with corresponding purine oligonucleotide in the presence of Mn2+ or Zn2+, but not Mg2+. In the complex the purine oligonucleotide has antiparallel orientation with respect to the purine strand of the duplex. Specifically, we have shown the formation of the Py-Pu-Pu triplex in a fragment of human papilloma virus HPV-16 in the presence of Mn2+.  相似文献   

15.
In this work we selected double-stranded DNA sequences capable of forming stable triplexes at 20 or 50 degrees C with corresponding 13mer purine oligonucleotides. This selection was obtained by a double aptamer approach where both the starting sequences of the oligonucleotides and the target DNA duplex were random. The results of selection were confirmed by a cold exchange method and the influence of the position of a 'mismatch' on the stability of the triplex was documented in several cases. The selected sequences obey two rules: (i) they have a high G content; (ii) for a given G content the stability of the resulting triplex is higher if the G residues lie in stretches. The computer simulation of the Mg2+, Na+and Cl-environment around three triplexes by a density scaled Monte Carlo method provides an interpretation of the experimental observations. The Mg2+cations are statistically close to the G N7 and relatively far from the A N7. The presence of an A repels the Mg2+from adjacent G residues. Therefore, the triplexes are stabilized when the Mg2+can form a continuous spine on G N7.  相似文献   

16.
We have studied the membrane-bound tyrosine protein kinases (TPK) in cultivated aortic smooth muscle cells (ASMC) from the rabbit, and found that the cytosolic (membranous)TPK and the nuclear (membranous)TPK are two distinctive types of isozymes as judged by criteria on dynamics: (1) using synthetic poly(Glu. Ala. Tyr)n(6:3:1) as a substrate, the relative extents of stimulation by Mn2+ (450%) was more effective than that by Mg2+ (100%) on cytosolic TPK, but Mn2+ was less effective (100%) than Mg2+ (130%) on nuclear TPK: (2) the concentrations of Mn2+ and Mg2+ giving maximal stimulation on cytosolic TPK were 15 mmol/L and 50 mmol/L, whereas on nuclear TPK were 1 mmol/L and 5 mmol/L, respectively. These results indicate that the attitudes toward Mn2+ and Mg2+ can distinguish the cytosolic TPK from the nuclear one. In nucleus, with the entry of ASMC from G0 to G1 stage, TPK activity increased rapidly and reached the peak (12 times G0 activity) at the early G1 stage (3 hrs from G0), then decreased dramatically to basic line, and remained at a lower level thereafter. In cytosol, the TPK activity decreased with the entry of ASMC from G0 to G1 stage and reached its lowest point at the middle of G1 stage (6 hrs from G0), and then increased with a transient peak at the late G1 stage (9 hrs). It went down to G0 level before DNA synthesis.  相似文献   

17.
Nucleic acids containing tracts of contiguous guanines tend to self-associate into four-stranded (quadruplex) structures, based on reciprocal non-Watson-Crick (G*G*G*G) hydrogen bonds. The quadruplex structure is induced/stabilized by monovalent cations, particularly potassium. Using circular dichroism, we have determined that the induction/stabilization of quadruplex structure by K+is specifically counteracted by low concentrations of Mn2+(4-10 mM), Co2+(0.3-2 mM) or Ni2+(0.3-0.8 mM). G-Tract-containing single strands are also capable of sequence-specific non-Watson-Crick interaction with d(G. C)-tract-containing (target) sequences within double-stranded DNA. The assembly of these G*G.C-based triple helical structures is supported by magnesium, but is potently inhibited by potassium due to sequestration of the G-tract single strand into quadruplex structure. We have used DNase I protection assays to demonstrate that competition between quadruplex self-association and triplex assembly is altered in the presence of Mn2+, Co2+or Ni2+. By specifically counteracting the induction/stabilization of quadruplex structure by potassium, these divalent transition metal cations allow triplex formation in the presence of K+and shift the position of equilibrium so that a very high proportion of triplex target sites are bound. Thus, variation of the cation environment can differentially promote the assembly of multistranded nucleic acid structural alternatives.  相似文献   

18.
Competition between triplex formation with double-stranded DNA and oligonucleotide self-association was investigated in 23mer GA and GT oligonucleotides containing d(GA)5 or d(GT)5 repeats. Whereas triplex formation with GT oligonucleotides was diminished when temperature increased from 4 to 37 degrees C, triplex formation with GA oligonucleotides was enhanced when temperature increased within the same range due to the presence of competing intermolecular GA oligonucleotide self-structure. This self-structure was determined to be a homoduplex stabilized by the internal GA repeats. UV spectroscopy of these homoduplexes demonstrated a single sharp transition with rapid kinetics (Tm = 38.5-43.5 degrees C over strand concentrations of 0.5-4 microM, respectively, with transition enthalpy, delta H = -89 +/- 7 kcal/mol) in 10 mM MgCl2, 100 mM NaCl, pH 7.0. Homoduplex formation was strongly stabilized by multivalent cations (spermine > Mg2+ = Ca2+) and destabilized by low concentrations of monovalent cations (K+ = Li+ = Na+) in the presence of divalent cations. However, unlike GA or GT oligonucleotide-containing triplexes, the homoduplex formed even in the absence of multivalent cations, stabilized by only moderate concentrations of monovalent cations (Li+ > Na+ > K+). Through the development of multiple equilibrium states and the resulting depletion of free oligonucleotide, it was found that the presence of competing self-structure could decrease triplex formation under a variety of experimental conditions.  相似文献   

19.
We report here that the 64-base pair (bp) guanine-rich polypurine:polypyrimidine tract derived from the right end of the rat long interspersed DNA element is reactive in a supercoil-dependent manner with a variety of chemical probes of non-B DNA structure. At pH 5.0 in the presence of Mg2+, part of the sequence (position 10-40) forms the following two types of triplexes: a G.G.C triplex, and an unusual C.G.C triplex. The latter structure is much more prevalent than the former and is unusual in that the resultant free purine strand forms a hairpin loop. In the absence of Mg2+ the G.G.C triplex disappears and the amount of C.G.C triplex is diminished, and at pH 7.5 in the presence or absence of Mg2+, little or no triplex is observed. Deletion of the 24-bp region just 3' of the triplex-forming region greatly reduces the amount of triplex formed. In this region, which includes an 18-bp polypurine:polypyrimidine sequence, both strands exhibit a moderate symmetric reactivity with the chemical probes tested, independent of pH and Mg2+. The implications of this structurally complex region for the properties of the rat L1 element are discussed.  相似文献   

20.
利用亲和层析方法纯化了小麦草酸氧化酶G和ψG,并对其生化特性进行了初步分析.G和ψG的最适pH为3.5,在60℃以下较稳定.当草酸浓度大于0.2mmol/L时,G和ψG的活性受到抑制.G和ψG的Km值分别为0.084和0.053mmol/L.0.1 mmol/L的EDTA、NH4 、Cl-、Mn2 、Mg2 、Na 和K 对G和ψG的活性没有影响,0.1 mmol/L的Zn2 、Cu2 、Fez 、Al3 、CO32-、NO3-和SO42-抑制G和ψG的活性.0.1 mmol/L的H2PO4-和HPO42-仅抑制G的活性.0.1 mmol/L的核黄素、FMN和FAD抑制ψG活性,G的活性则不受FAD和FMN的影响.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号