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1.
双倍体酵母细胞D7经单核能为11.4MeV/u的An和U离子辐照后,测定了细胞随剂量的存活率和突变率。获得细胞对Au和U离子的失活截面分别为2.54μm2和1.92μm2。在存活率为37%的条件下,Au、U离子的RBE分别为0.28和0.19。在突变实验中,研究了DNA断链后的重组与倒位,它们对Au和U离子的截面为:8.3×10-2μm2[σm-rec(Au)],9.5×10-5μm2[σm-rec(U)]和6.1×10-4μm2[σm-rev(Au)]和3.8×10-5μm2[σm-rev(U)].最后,对所获结果进行了讨论。  相似文献   

2.
应用蛋白dotblot技术检测了低氧内皮细胞条件培养液(HECCM)和常氧内皮细胞条件培养液(NECCM)内PDGF相对含量,并利用[3H]-TdR掺入法和流式细胞术观察了HECCM和NECCM及加入特异PDGF抗体对肺动脉平滑肌细胞(PASMC)生长的影响。结果表明,HECCM中的PDGF含量明显高于NECCM;HECCM能明显增强PASMC内DNA合成,促进PASMC从Go/G1期进入S期;当预先加入PDGF-B链抗体时,则会明显地抑制HECCM对PASMC的DNA合成,阻止PASMC从Go/G1期进入S期。结果提示,低氧时PASMC增殖与肺动脉内皮细胞分泌释放PDGF增加有关  相似文献   

3.
采用聚合酶链反应(PCR)技术和DNA体外重组方法,克隆出579bp的丙型肝炎病毒(HCV)NS4b基因片段,插入到原核高效表达载体pET-28a中,构建重组质粒pET/NS4b,转化大肠杆菌BL21(DE3)菌株,经IPTG诱导培养后,获得了目的蛋白的高效表达。SDS-PAGE分析显示在30kD处有一条表达的目的蛋白区带。通过固定化金属配体亲和层析(IMAC)纯化目的的蛋白,ELESA检测结果表  相似文献   

4.
有序差异显示:一种基因表达谱系统比较法   总被引:2,自引:0,他引:2  
系统研究具有同一基因组的各种细胞群之间基因的差异表达谱十分重要。目前,研究基因差异表达的技术大致有mRNA差异显示[1]、RDA[3]、SSH[4、5]和cDNA阵列[6]等。近几年,还发展了一些研究基因差异表达谱系统的技术,如RLCS(restrictionland-markcDNAscanning)[8]、GEF(geneexpres-sionfingerprinting)[2]和RNA指纹法[9]等。然而,这些技术或较为复杂,或灵敏度偏低。本文拟介绍一种有效的基因表达谱系统比较法——有序差…  相似文献   

5.
短杆菌肽S(GS)是一个十肽分子(环-(L-Val-L-Orn-L-Leu-D-Phe-L-Pro)2),是由两个β转角和两个β片层结构所构成[1]。GS的功能是通过破坏葛兰氏阴性菌的膜结构来完成其抗菌活性。迄今为止,人们对GS与膜结合特点的研究结果还不一致。Dateman等人利用2H-NMR,31P-NMR和DSC等技术研究了GS与DPPC多层脂膜的相互作用,指出肽仅仅作用于膜表面[2];而Higashijima等[3]及张凤立等用2D-NMR的研究结果[4]表明,GS的疏水部分应插入膜内。蛋白质及多肽的H/D交换动力学受其分子内氢键,分子空间结构的致密度及其周边环境如膜环境[5]的影响。我们利用衰减全反射红外光谱(ATR-FTIR)对与脂膜结合前后的短杆菌肽S的H/D交换动力学进行了研究,实验结果提示GS插入了脂膜双层。  相似文献   

6.
本文采用Ca~2+指示剂的分光光谱法测定巨噬细胞(Mφ)内Ca~2+浓度([Ca~2+]i)、APAAP桥联酶标法检测Mφ膜上Ⅰa抗原的表达,研究肌醇磷脂代谢中第二信使分子甘油二酯(DG)在去甲肾上腺素(NE)促进MφⅠa表达效应中的作用,以进一步探讨NE效应的跨膜信息传递机制。结果表明:蛋白激酶C(PKC)抑制剂4αPDD(25μg/ml)虽不影响NE(10 ̄-8mol/L)升高Mφ[Ca ̄2+]i的效应,却显著减弱了NE促进MφⅠa抗原表达的效应;而PKC激动剂PMA(10nmol/L)本身促进MφⅠa抗原表达的作用不明显,也不能进一步增强NE促进MφⅠa抗原表达的效应。结果提示:DG激活的PKC系统也参与了NE促进MφⅠa抗原表达的信息传递过程,并与另一第二信使分子肌醇-1,4,5-三磷酸(IP_3)介导的Ca ̄2+途径协同发挥作用。  相似文献   

7.
叶绿体类囊体膜脂-膜蛋白的相互作用   总被引:2,自引:0,他引:2  
叶绿体类囊体膜脂-膜蛋白的相互作用陈志强,许春辉,匡廷云(中国科学院植物研究所,北京100044)THELIPID-PROTEININTERACTIONINCHLOROPLASTTHYLAKOIDMEMDRANE¥ChenZhi-qiang;XuCh...  相似文献   

8.
昆虫谷胱甘肽S-转移酶分离纯化的新方法   总被引:4,自引:0,他引:4  
谷胱甘肽S-转移酶(glutathioneS-transferases,GST)是一类具有多种生理功能的同功酶.从蜡螟幼虫(Galeriamelonela)的提取液中分离纯化谷胱甘肽S-转移酶的基本方法如下:首先将冷冻的蜡螟幼虫在磷酸缓冲液中匀桨,经10000g和100000g分级离心;取上清液通过QAE-SephadexA-25离子交换柱层析除去部分色素和杂蛋白;然后采用谷胱甘肽-琼脂糖凝胶亲和层析(GSH-QT4),四溴酚酞二磺酸盐-琼脂糖凝胶亲和层析(BSP-QT4),铜离子-琼脂糖凝胶螯合层析(Cu2+-QT4)及PBE94-Sepharose(PBE94)聚焦层析等层析技术进一步分离纯化.将上述方法获得的色谱峰以CDNB和DCNB为底物检测生物活性.具有生物活性部分的蛋白质,通过SDS-PAGE测定其分子量.实验结果表明,采用GSH-QT4亲和层析法获得的活性峰,在SDS-PAGE图谱上呈现出两条带,分子量为24kD,24.5kD左右;Cu2+-QT6螯合层析法分离的活性峰,呈现出一条带,分子量为24kD左右;PBE94-聚焦层析法分离获得三个活性峰:第一色谱峰,呈现出一条带,分子量为23kD左右  相似文献   

9.
高效毛细管电泳在DNA序列分析中的应用   总被引:1,自引:0,他引:1  
种康 《植物学通报》1995,12(1):60-62
高效毛细管电泳在DNA序列分析中的应用种康(兰州大学化学系,兰州730000)APPLICATIONOFHIGH-PERFORMANCECAPILLARYELECTCTORHORESISONDNASEQUENCING¥ChongKang(Depart...  相似文献   

10.
李宝民  刘振声 《病毒学报》1996,12(4):330-334
将0.6kbLMP1前段基因反向插入逆转录病毒载体(pZIP)中,构建成pZIP-反义LMP1载体,再转入PA317包装细胞中,用G418筛选性克隆,获得产生1.7×10^5(CFU/ml)反杂交实验证实pZIP-反义LMP1载体基车整合到CNE2细胞的DNA中,并且有载体基因RNA的表达,观察反义LMP1基罟对CN#2细胞生长的影响,发现反义LMP1基因可降低细胞生长速度,减弱CNE2细胞在这果  相似文献   

11.
Dalian EB 《Biofizika》2002,47(2):253-258
The interaction of meso-tetra(4-N-oxyethylpyridyl)porphyrin and its 3-N-substituted analogue with DNA was studied by UV spectrophotometry and circular dichroism methods. It was shown that the position of the side group on the pyridyl ring substantially affects the mechanism of interaction with DNA. 4-N-oxyethylpyridyl porphyrins more preferably intercalate into the DNA structure but worse realize the external binding than 3N-porphyrins.  相似文献   

12.
The interaction of transition metal complexes of cationic porphyrins bearing five membered rings, meso-tetrakis(1,2-dimethylpyrazolium-4-yl)porphyrin (MPzP, M=Mn(III), Ni(II), Cu(II) or Zn(II)), with calf thymus DNA (ctDNA) has been studied. Metalloporphyrins NiPzP and CuPzP are intercalated into the 5'GC3' step of ctDNA. MnPzP is bound edge-on at the 5'TA3' step of the minor groove of ctDNA, while ZnPzP is bound face-on at the 5'TA3' step of the major groove of ctDNA. The binding constants of the metalloporphyrins to ctDNA range from 1.05x10(5) to 2.66x10(6) M(-1) and are comparable to those of other reported cationic porphyrins. The binding process of the metallopyrazoliumylporphyrins to ctDNA is endothermic and entropically driven. These results have revealed that the kind of central metal ions of metalloporphyrins influences the binding characteristics of the porphyrin to DNA.  相似文献   

13.
Interactions of meso-tetra-(4-N-oxyethylpyridyl) porphyrin (TOEPyP(4)), its 3-N analog (TOEPyP(3)) and their Co, Cu, Ni, Zn metallocomplexes with duplex DNA have been investigated by uv/visible absorbance and circular dichrosim spectroscopies. Results reveal the interactions of these complexes with duplex DNA are of two types. (1) External binding of duplex DNA by metalloporphyrins containing Zn and Co, and (2) Binding of duplex DNA both externally and internally (by intercalation) by porphyrins not containing metals, and metalloporphyrins containing Cu and Ni. Results indicate that (4N-oxyethylpyridyl) porphyrins intercalate more preferably in the structure of duplex DNA and have weaker external binding than 3N-porphyrins.  相似文献   

14.
The complex formation of porphyrins with DNA leads to changes of stability of DNA. In the present study we investigated binding properties and the thermodynamic parameters of a water-soluble, cationic planar Cu(II)-containing meso-tetrakis(4-N-butyl-pyridiniumyl)porphyrin [CuTButPyP4] and nonplanar Co(II)-containing meso-tetrakis(4-N-butyl-pyridiniumyl)porphyrin [CoButPyP4] with calf thymus DNA in the presence of divalent manganese ions. For displaying the changes of thermodynamic parameters (Tm and ΔT) the melting curves of DNA-porphyrin complexes in the presence of Mn2+ ions have been obtained. The enthalpy (ΔH) of helix-coil transition has been also evaluated. It was shown that the binding of ions to DNA proceeds in two stages depending on the manganese/DNA phosphates molar ratio [Mn]/[P]. At the first stage (0.001 < [Mn]/[P] < 1), the interaction of manganese ions with DNA phosphates occurs, causing an additional screening of their negative charge and the stabilization of the double helix. As a result, the best conditions for intercalation of CuTButPyP4 or of peripheral rings of CoButPyP4 occur. The significant increase of Tm, but less changes of ΔT were observed. At the second stage (1 < [Mn]/[P] < 4), the ions interact with both the phosphates and the nitrogen bases of DNA. At this stage, it is possible for the manganese ion to coordinate simultaneously to the oxygen atom of the phosphate and the neighboring base of DNA. At a higher [Mn]/[P] ratio, the destabilization of the double helix begins, and partial breakage of the hydrogen bonds between the nitrogen bases occurs. Respectively the destabilization of DNA in the presence of both porphyrins takes place.  相似文献   

15.
Nový J  Urbanová M 《Biopolymers》2007,85(4):349-358
The interactions of two different porphyrins, without axial ligands-5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin-Cu(II) tetrachloride (Cu(II)TMPyP) and with bulky meso substituents-5,10,15,20-tetrakis(N,N,N-trimethylanilinium-4-yl)porphyrin tetrachloride (TMAP), with (dG-dC)10 and (dA-dT)10 were studied by combination of vibrational circular dichroism (VCD) and electronic circular dichroism (ECD) spectroscopy at different [oligonucleotide]/[porphyrin] ratios, where [oligonucleotide] and [porphyrin] are the concentrations of oligonucleotide per base-pair and porphyrin, respectively. The combination of VCD and ECD spectroscopy enables us to identify the types of interactions, and to specify the sites of interactions: The intercalative binding mode of Cu(II)TMPyP with (dG-dC)(10), which has been well described, was characterized by a new VCD "marker" and it was shown that the interaction of Cu(II)TMPyP with (dA-dT)10 via external binding to the phosphate backbone and major groove binding caused transition from the B to the non-B conformer. TMAP interacted with the major groove of (dG-dC)10, was semi-intercalated into (dA-dT)10, and caused significant variation in the structure of both oligonucleotides at the higher concentration of porphyrin. The spectroscopic techniques used in this study revealed that porphyrin binding with AT sequences caused substantial variation of the DNA structure. It was shown that VCD spectroscopy is an effective tool for the conformational studies of nucleic acid-porphyrin complexes in solution.  相似文献   

16.
Factors influencing the binding of tetracationic porphyrin derivatives to DNA have been comprehensively evaluated by equilibrium dialysis, stopped-flow kinetics, etc., for mesotetrakis (4-N-methylpyridiniumyl)porphyrin [TMpyP (4)]. Technical difficulties have previously precluded a comprehensive study of metalloporphyrins. Since electrostatic interactions with the DNA and metal derivatization of the porphyrins have important consequences, we have investigated in greater detail two isomers of TMpyP (4) (meso-tetrakis(3-N-methylpyridiniumyl)porphyrin, [TMpyP (3)] and meso-tetrakis(2-N-methylpyridiniumyl)porphyrin [TMpyP (2)]) in which the position of the charged centers has been varied. A comprehensive study of the Cu(II) derivatives, e.g., CuTMpyP (4), was possible since the difficulties encountered previously with Ni(II) compounds were not a problem with Cu(II) porphyrins [J. A. Strickland, L. G. Marzilli, M. K. Gay, and W. D. Wilson (1988) Biochemistry 27, 8870-8878]. At 25 degrees C, the apparent equilibrium constants [Kobs] decreased with increasing [Na+] for all porphyrins. The Kobs values were comparable for TMpyP (4) and TMpyP (3) binding to either polyd(G-C).polyd(G-C) [poly[d(G-C)2]] or poly[d(A-T)].poly[d(A-T)] [poly[d(A-T)2]]. For the copper(II) porphyrins, the Kobs values were about fivefold greater. The Kobs value for CuTMpyP (2) binding to poly[d(G-C)2] was too small to measure under typical salt conditions; however, Kobs for binding to poly[d(A-T)2] was about two orders of magnitude smaller than those found for CuTMpyP (4) or CuTMpyP (3). Application of the condensation theory for polyelectrolytes suggests about three charge interactions when CuTMpyP (4), CuTMpyP (3), and TMpyP (3) bind to poly[d(G-C)2] or poly[d(A-T)2], a result comparable to that reported for TMpyP (4). At 20 degrees C and 0.115 M [Na+], incorporation of copper decreased the rates of dissociation from poly[d(A-T)2] by a 100-fold compared to those reported for TMpyP (4) but had little effect on the rates of dissociation from poly[d(G-C)2]. Also, movement of the H3CN+ group from the fourth to the third position of the pyridinium ring enhanced the rates of dissociation from poly[d(A-T)2] but decreased the rates of dissociation from poly[d(G-C)2]. From polyelectrolyte theory, the [Na+] dependence of the dissociation rates from poly[d(G-C)2] is consistent with intercalative binding, while that for poly[d(A-T)2] is consistent with an outside binding model. For calf thymus [CT] DNA at 20 degrees C, a greater decrease in the AT than in the GC imino 1H-nmr signal was observed upon addition of CuTMpyP (2), suggesting selective outside binding to the AT regions.(ABSTRACT TRUNCATED AT 400 WORDS)  相似文献   

17.
Abstract

Interactions of meso-tetra-(4-N-oxyethylpyridyl) porphyrin (TOEPyP(4)), its 3-N analog (TOEPyP(3)) and their Co, Cu, Ni, Zn metallocomplexes with duplex DNA have been investigated by uv/visible absorbance and circular dichrosim spectroscopies. Results reveal the interactions of these complexes with duplex DNA are of two types. (1) External binding of duplex DNA by metalloporphyrins containing Zn and Co, and (2) Binding of duplex DNA both externally and internally (by intercalation) by porphyrins not containing metals, and metalloporphyrins containing Cu and Ni. Results indicate that (4N-oxyethylpyridyl) porphyrins intercalate more preferably in the structure of duplex DNA and have weaker external binding than 3N-porphyrins.  相似文献   

18.
Interactions of porphyrins with nucleic acids   总被引:24,自引:0,他引:24  
The interactions of nucleic acids with water-soluble porphyrins and metalloporphyrins have been investigated by stopped-flow and temperature-jump techniques. Both natural DNA (calf thymus) and synthetic homopolymers [poly(dG-dC) and poly(dA-dT)] have been employed. The porphyrins studied belong to the tetrakis(4-N-methylpyridyl)porphine (H2TMpyP-4) series and can be divided into two groups: (i) those which have no axial ligands when bound to nucleic acids [e.g., Ni(II), Cu(II), and the nonmetallic derivatives] and (ii) those which maintain axial ligands upon binding [e.g., Mn(III), Fe(III), Co(III), and Zn(II) derivatives]. The reaction of both axially and nonaxially liganded porphyrins at AT sites is too rapid to be measured by the kinetic methods utilized, whereas at GC sites the interaction of the nonaxially liganded porphyrins is in the millisecond time range and can be monitored by both stopped-flow and temperature-jump techniques. These results corroborate previous static studies, utilizing visible spectroscopy and circular dichroism, which indicate that the formation of an intercalated complex occurs only at GC base pair sites with porphyrins which do not possess axial ligands. With all the porphyrins investigated, the complexes formed at AT sites are envisioned as being of an "external" type involving some degree of overlap between the porphyrin and the bases of the duplex. In relaxation experiments of poly-(dG-dC) with H2TMpyP-4, a large, reproducible effect is observed which can be analyzed as a single exponential. Rate constants for association and dissociation of the H2TMpyP-4/poly(dG-dC) complex are 3.7 X 10(5) M-1 s-1 and 1.8 s-1, respectively. Relaxation studies of mixtures of poly(dA-dT) and poly(dG-dC) with H2TMpyP-4 indicate that the transfer of the porphyrin from one homopolymer to another occurs via a mechanism involving dissociation rather than direct transfer.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

19.
Thirty-three porphyrins or metalloporphyrins corresponding to the general formula [meso-[N-methyl-4(or 3 or 2)-pyridiniumyl]n(aryl)4-nporphyrin]M (M = H2, CuII, or ClFeIII), with n = 2-4, have been synthesized and characterized by UV-visible and 1H NMR spectroscopy and mass spectrometry. These porphyrins differ not only in the number (2-4) and position of their cationic charges but also in the steric requirements to reach even temporarily a completely planar geometry. In particular, they contain 0, 1, 2, 3, or 4 meso-aryl substituents not able to rotate. Interaction of these porphyrins or metalloporphyrins with calf thymus DNA has been studied and their apparent affinity binding constants have been determined by use of a competition method with ethidium bromide which was applicable not only for all the free base porphyrins but also for their copper(II) or iron(III) complexes. Whatever their mode of binding may be, their apparent affinity binding constants were relatively high (Kapp between 1.2 x 10(7) and 5 x 10(4) M-1 under our conditions), and a linear decrease of log Kapp with the number of porphyrin charges was observed. Studies of porphyrin-DNA interactions by UV and fluorescence spectroscopy, viscosimetry, and fluorescence energy transfer experiments showed that not only the tetracationic meso-tetrakis[N-methyl-4(or 3)-pyridiniumyl]porphyrins, which both involved four freely rotating meso-aryl groups, but also the corresponding tri- and dicationic porphyrins were able to intercalate into calf thymus DNA. Moreover, the cis dicationic meso-bis(N-methyl-2-pyridiniumyl)diphenylporphyrin, which involved only two freely rotating meso-aryl groups in a cis position, was also able to intercalate. The other meso-(N-methyl-2-pyridiniumyl)n(phenyl)4-nporphyrins, which involved either zero, one, or two trans freely rotating meso-aryl groups, could not intercalate into DNA. These results show that only half of the porphyrin ring is necessary for intercalation to occur.  相似文献   

20.
Porphyrins carrying four charged sidechains, e.g., meso-tetrakis[4-N-methylpyridiniumyl]- and meso-tetrakis[4-N-(2-hydroxyethyl)pyridiniumyl]-porphyrin, bound and intercalated similarly into DNA as measured by helix stabilization and DNA unwinding studies in the presence of DNA topoisomerase I. Despite their different bulky sidechains, these complexes gave essentially identical DNase I footprinting patterns. In contrast, tetrasubstituted porphyrins carrying three phenyl rings and a single positively charged pyridiniumyl sidechain did not intercalate and exhibited little affinity for DNA. Thus, the presence of charged sidechains on the porphyrin rather than their identity appears to be critical for efficient DNA intercalation. The results are discussed in regard to current models for the porphyrin-DNA intercalation complex.  相似文献   

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