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1.
The effect of intercalating drugs (the anthracycline group of antibiotics, ethidium bromide, actinomycin D) on stepwise melting of DNA was studied by differential scanning calorimetry (DSC). The DSC DNA melting profile of plasmid pJL3-TB5 DNA (5277 base-pairs in length) consists of seven peaks, and all the intercalators caused shifting of these peaks, particularly those formed at the high temperature ranges, to the higher temperature ranges in a characteristic manner depending upon the binding strength of the drug. The analysis of the anthracycline group of antibiotics, such as aclacinomycin A, daunomycin, adriamycin and pyrarubicin, indicates that the difference in binding is due to the sugar moiety at position O-7 of the chromophore in these antibiotics. Analysis on the basis of the helix-coil transition theory suggests that the anthracycline group of antibiotics interact preferentially with the 5'-CG-3' sequences. The effect of various DNA-binding drugs other than intercalators on stepwise melting of DNA was then studied by DSC. The representative drugs examined were distamycin A, peplomycin, cis-dichlorodiamine-platinum(II) (cis-DDP or cis-Platin) and mitomycin C, which differ in their mode of interaction with DNA; namely, minor groove binding, strand cleavage and intrastrand or interstrand cross-linking. Distamycin A caused shifting of the DSC peaks at the low temperature ranges to a higher temperature range, whereas peplomycin and cis-DDP caused shifting of all the DSC peaks to form a broad peak at a lower temperature range, suggesting that the DSC DNA melting profiles are affected in a characteristic manner depending upon the interaction mode of the drug.  相似文献   

2.
The differential scanning calorimetry (DSC) of plasmid ColE1 DNA was carried out. The DSC curve under the solvent condition of 1.0 X SSC buffer gave eleven clear peaks over the temperature range of 83 to 98 degrees C. The DSC curves obtained here were essentially in good agreement with the optical melting curves of ColE1 DNA reported previously. The theoretical melting profiles of ColE1 DNA calculated from its entire nucleotide sequence showed a good agreement with the DSC curves. The theoretical analysis made by constructing the thermal stability map showed that there was the positional correlation between the boundaries of the cooperatively melting regions and the ends of the protein coding regions of genes of ColE1. It was shown that the helix-coil transition of many of the small genes had a single cooperatively melting region. However, the large genes such as cea and mob3 had two or more cooperatively melting regions. It was suggested that this is closely related to the domain structures of the proteins encoded by such genes.  相似文献   

3.
Differential scanning calorimetry (DSC) was carried out to analyze the transition of helix to coil state of DNA, using ColE1 DNA molecules digested with EcoRI. The DSC curves showed multimodal transition, consisting of nine to 11 peaks over a temperature range, depending on the ionic strength of the DNA solution. These DSC curves were essentially in good agreement with the optical melting curves of ColE1 DNA. The theoretical melting profiles of ColE1 DNA were predicted from calculations based on the helix-coil transition theory and the nucleotide sequence of the DNA. These profiles resembled the DSC curves and made it possible to assign the peaks seen in the DSC curves to the helix-coil transition of particular regions of the nucleotide sequence of ColE1. The helix-coil transition of each of the small genes gave rise to a single peak in the DSC curve, while the helix-coil transition of large genes contributed to two or more peaks in the DSC curve. This multimodal transition within a single coding region might correspond to the melting of individual segments encoding the different domains of the proteins. The helix-coil transition at the specific sites including ori, the origin of replication of ColE1, was also found to occur in a particular temperature range. DSC, a simple method, is thus useful for analyzing the multimodal helix-coil transition of DNA, and for providing information on the genetic organization of DNA.  相似文献   

4.
We demonstrate that differential scanning calorimetry (DSC) can be used to yield high‐resolution melting profiles for DNA plasmids that agree in all major features with the corresponding plasmid melting profiles derived using more traditional optical techniques. We further demonstrate that by combining information derived from both calorimetric and optical melting profiles one can glean insights that are unavailable from either melting curve alone. By using both optical and calorimetric observables, we show how one can resolve, identify, and measure the thermodynamic properties of particular sequences/domains of interest within a plasmid. We also show that complementary DSC and optical melting studies on plasmids with and without specifically designed inserts can provide fundamental advantages over the corresponding melting studies on other model system constructs for thermodynamically characterizing nucleic acid sequences/structures. © 1999 John Wiley & Sons, Inc. Biopoly 50: 303–318, 1999  相似文献   

5.
Thermal denaturation of natural DNA in the absence and presence of antitumor anthracycline antibiotics has been studied by adiabatic differential scanning calorimetry. The helix-coil transition is operationally irreversible as measured by DSC. Both the melting temperature and the overall molar transition enthalpy of the DNA samples was dependent on the percentage of GC base pairs. Calorimetric traces of anthracycline-DNA complexes have qualitatively similar features and the significance of this characteristic is discussed. The unsaturated drug-DNA complex melts through complex thermal transitions with one broad endotherm in the same temperature region as free DNA and the other at a higher temperature which is rf (mol ligand per mol DNA in base pairs) value dependent. Antibiotic binding at concentrations close to saturating conditions (rf = 0.2) reverts the melting range to a value near to its original one and increases the thermal stability of the duplex structure by around 30 degrees C. In addition, the calorimetric enthalpy is increased by between 64% and 150%, depending on which ligand was used.  相似文献   

6.
Nuclear membranes from many tumors contain an unusual redox chain discovered originally in the Hepatoma 22a nuclear membranes7 which catalyzes superoxide dismutase-sensitive adrenaline oxidation to adrenochrome in the presence of either NADPH or NADH as electron donor, the reaction being inhibited by cyanide and azide. This redox chain can reduce anthracycline antitumor antibiotics adriamycin and carminomycin to their free radical states under anaerobic conditions. Evidence has been obtained for a higher stability of the carminomycin radical as compared to that of adriamycin. Operation of the nuclear membrane-bound redox chain can be a source of oxygen radical-mediated single strand breaks in DNA. The role of the nuclear membrane-associated electron transfer chain in augmenting the anticancer action of the anthracycline antibiotics is discussed.  相似文献   

7.
《Free radical research》2013,47(1-5):47-55
Nuclear membranes from many tumors contain an unusual redox chain discovered originally in the Hepatoma 22a nuclear membranes7 which catalyzes superoxide dismutase-sensitive adrenaline oxidation to adrenochrome in the presence of either NADPH or NADH as electron donor, the reaction being inhibited by cyanide and azide. This redox chain can reduce anthracycline antitumor antibiotics adriamycin and carminomycin to their free radical states under anaerobic conditions. Evidence has been obtained for a higher stability of the carminomycin radical as compared to that of adriamycin. Operation of the nuclear membrane-bound redox chain can be a source of oxygen radical-mediated single strand breaks in DNA. The role of the nuclear membrane-associated electron transfer chain in augmenting the anticancer action of the anthracycline antibiotics is discussed.  相似文献   

8.
Differential melting curves (DMCs) of DNAs pA03 and pBR322 in solutions of different ionic strength (0.02 and 0.2M Na+) were obtained. A previously developed procedure of glyxal fixation of partially denatured DNA molecules at temperatures within the melting range was used to construct electron-microscopic melting maps for pBR322 and pAO3 plasmid DNA and for the replicative form of bacteriophage ?X174 DNA, allowing the melting of these DNA molecules to be followed in solutions of low (0.1 × SSC) and high (1 × SSC) ionic strength. In spite of the fact that the melting was at nonequilibrium at the low ionic strength, the melting maps for the two kinds of solutions practically coincided. Experimental data are compared with theoretical calculations based on the Fixman-Freire algorithm. The conclusion is that the melting pattern of these DNAs is, on the whole, correctly described by the theory, although there are appreciable differences between the theoretical and experimental differential melting curves. We have also determined the relation between the melting temperature of a region and its GC content, with allowances made for the boundary conditions of melting in 0.1 × SSC and 1 × SSC solutions, and have analyzed the theoretical shape of peaks of the DMCs.  相似文献   

9.
目的:应用超声波分散法制备脂质体阿霉素,并比较脂质体阿霉素与游离性阿霉素抗肿瘤活性。方法:以卵磷脂和胆固醇为原料,将阿霉素包封于脂质体中,采用超声分散法制备脂质体阿霉素,对其在290-700nm范围内进行紫外扫描,用SephedexG-50柱分离脂质体阿霉素并计算其包封率。以昆明种小鼠为载体建立肿瘤模型(S180型肉瘤)和细胞荧光染色法研究脂质体阿霉素的抗肿瘤活性,以ZITA SIZER3000型表面电位与粒度测定仪测定其粒径分布。结果:脂质体阿霉素在480nm处有最大吸收峰值,包封率达91.3%,细胞荧光染色显示,脂质体及游离型阿霉素均对S180细胞有明显的抑制作用。结论:此法制备的脂质体阿霉素包封率高,粒径分布集中,脂质体阿霉素较游离型阿霉素有较强的抗肿瘤活性剂及较低的细胞毒作用,对阿霉素的临床应用有一定的参考价值。  相似文献   

10.
Schisanhenol (Sal) did not diminish the antitumor activity of adriamycin in mice bearing P388 ascites tumor. Sal did not antagonize the suppressive effect of adriamycin on DNA synthesis and cell proliferation in an L1210 ascitic tumor cell culture. Furthermore, Sal at the concentration of 0.1, 0.25, or 1 mM accelerated adriamycin-dependent DNA damage in the presence of Fe3+ in vitro. It appears that Sal was able to protect against adriamycin induced heart mitochondrial toxicity, while it did not antagonize the antitumor activity of adriamycin.  相似文献   

11.
A theoretical method is developed for calculation of melting curves of covalent complexes of DNA with antitumor drugs. The method takes into account all the types of chemical modifications of the double helix caused by platinum compounds and DNA alkylating agents: 1) monofunctional adducts bound to one nucleotide; 2) intrastrand cross-links which appear due to bidentate binding of a drug molecule to two nucleotides that are included into the same DNA strand; 3) interstrand cross-links caused by bidentate binding of a molecule to two nucleotides of different strands. The developed calculation method takes into account the following double helix alterations at sites of chemical modifications: 1) a change in stability of chemically modified base pairs and neighboring ones, that is caused by all the types of chemical modifications; 2) a change in the energy of boundaries between helical and melted regions at sites of chemical modification (local alteration of the factor of cooperativity of DNA melting), that is caused by all the types of chemical modifications, too; 3) a change in the loop entropy factor of melted regions that include interstrand cross-links; 4) the prohibition of divergence of DNA strands in completely melted DNA molecules, which is caused by interstrand cross-links only. General equations are derived, and three calculation methods are proposed to calculate DNA melting curves and the parameters that characterize the helix-coil transition.  相似文献   

12.
Abstract

A theoretical method is developed for calculation of melting curves of covalent complexes of DNA with antitumor drugs. The method takes into account all the types of chemical modifications of the double helix caused by platinum compounds and DNA alkylating agents: 1) monofunctional adducts bound to one nucleotide; 2) intrastrand cross-links which appear due to bidentate binding of a drug molecule to two nucleotides that are included into the same DNA strand; 3) interstrand cross-links caused by bidentate binding of a molecule to two nucleotides of different strands. The developed calculation method takes into account the following double helix alterations at sites of chemical modifications: 1) a change in stability of chemically modified base pairs and neighboring ones, that is caused by all the types of chemical modifications; 2) a change in the energy of boundaries between helical and melted regions at sites of chemical modification (local alteration of the factor of cooperativity of DNA melting), that is caused by all the types of chemical modifications, too; 3) a change in the loop entropy factor of melted regions that include interstrand cross-links; 4) the prohibition of divergence of DNA strands in completely melted DNA molecules, which is caused by interstrand cross-links only. General equations are derived, and three calculation methods are proposed to calculate DNA melting curves and the parameters that characterize the helix-coil transition.  相似文献   

13.
The melting curves of 11 vegetable oils have been characterised. Vegetable oil samples that were cooled at a constant rate (5 degrees C/min) from the melt showed between one and seven melting endotherms upon heating at four different heating rates (1, 5, 10 and 20 degrees C/min) in a differential scanning calorimeter (DSC). Triacylglycerol (TAG) profiles and iodine value analyses were used to complement the DSC data. Generally, the melting transition temperature shifted to higher values with increased rates of heating. The breadth of the melting endotherm and the area under the melting peak also increased with increasing heating rate. Although the number of endothermic peaks was dependent on heating rate, the melting curves of the oil samples were not straightforward in that there was no correlation between the number of endothermic peaks and heating rates. Multiple melting behaviour in DSC experiments with different heating rates could be explained by: (1) the melting of TAG populations with different melting points; and (2) TAG crystal reorganisation effects. On the basis of the corollary results obtained, vegetable oils and fats may be distinguished from their offset-temperature (Toff) values in the DSC melting curves. The results showed that Toff values of all oil samples were significantly (p < 0.01) different in the melting curves scanned at four different scanning rates. These calorimetric results indicate that DSC is a valuable technique for studying vegetable oils.  相似文献   

14.
15.
Thermodynamic stability of DNA tandem mismatches   总被引:2,自引:0,他引:2  
The thermodynamics of nine hairpin DNAs were evaluated using UV-monitored melting curves and differential scanning calorimetry (DSC). Each DNA has the same five-base loop and a stem with 8-10 base pairs. Five of the DNAs have a tandem mismatch in the stem, while four have all base pairs. The tandem mismatches examined (ga/ga, aa/gc, ca/gc, ta/ac, and tc/tc) spanned the range of stability observed for this motif in a previous study of 28 tandem mismatches. UV-monitored melting curves were obtained in 1.0 M Na(+), 0.1 M Na(+), and 0.1 M Na(+) with 5 mM Mg(2+). DSC studies were conducted in 0.1 M Na(+). Transition T(m) values were unchanged over a 50-fold range of strand concentration. Model-independent enthalpy changes (DeltaH degrees ) evaluated by DSC were in good agreement (+/-8%) with enthalpy values determined by van't Hoff analyses of the melting curves in 0.1 M Na(+). The average heat capacity change (DeltaC(p)) associated with the hairpin to single strands transitions was estimated from plots of DeltaH degrees and DeltaS degrees with T(m) and ln T(m), respectively, and from profiles of DSC curves. The average DeltaC(p) values (113 +/- 9 and 42 +/- 27 cal x K(-1) x mol(-1) of bp), were in the range of values reported in previous studies. Consideration of DeltaC(p) produced large changes in DeltaH degrees and DeltaS degrees extrapolated from the transition region to 37 degrees C and smaller but significant changes to free energies. The loop free energy of the five tandem mismatches at 37 degrees C varied over a range of approximately 4 kcal x mol(-1) for each solvent.  相似文献   

16.
Interaction of a novel antitumor agent TAS-103 with DNA has been studied by a variety of methods including thermal melting study, UV-Visible spectroscopy, 1H- and 31P-NMR spectroscopy. Thermal melting study indicated that TAS-103 stabilizes the double stranded form of DNA and the relative binding strength of TAS-103 is equal to that of ethidium bromide (EtBr). UV-Visible spectroscopy demonstrated that titration curves are nearly identical with all DNA oligomers producing a hypochromic and hypsochromic effect. A hypsochromic effect of TAS-103 is differ from typical intercalators such as EtBr and Actinomycin D that exhibit a bathochromic effect. 1H- and 31P-NMR spectroscopy revealed that TAS-103 has mainly two binding modes. Major binding mode is outside binding and minor binding mode is intercalation.  相似文献   

17.
The anticancer drugs adriamycin and daunomycin have each been crystallized with the DNA sequence d(CGATCG) and the three-dimensional structures of the complexes solved at 1.7- and 1.5-A resolution, respectively. These antitumor drugs have significantly different clinical properties, yet they differ chemically by only the additional hydroxyl at C14 of adriamycin. In these complexes the chromophore is intercalated at the CpG steps at either end of the DNA helix with the amino sugar extended into the minor groove. Solution of the structure of daunomycin bound to d(CGATCG) has made it possible to compare it with the previously reported structure of daunomycin bound to d(CGTACG). Although the two daunomycin complexes are similar, there is an interesting sequence dependence of the binding of the amino sugar to the A-T base pair outside the intercalation site. The complex of daunomycin with d(CGATCG) has tighter binding than the complex with d(CGTACG), leading us to infer a sequence preference in the binding of this anthracycline drug. The structures of daunomycin and adriamycin with d(CGATCG) are very similar. However, there are additional solvent interactions with the adriamycin C14 hydroxyl linking it to the DNA. Surprisingly, under the influence of the altered solvation, there is considerable difference in the conformation of spermine in these two complexes. The observed changes in the overall structures of the ternary complexes amplify the small chemical differences between these two antibiotics and provide a possible explanation for the significantly different clinical activities of these important drugs.  相似文献   

18.
Chemical reduction of the highly active quinone-containing antitumor drugs, adriamycin and daunorubicin formed the same partially reduced free radical previously reported [9] by microsomal activation. In vitro incubation of the chemically activated free radical intermediates with DNA resulted in covalent binding of these drugs to DNA. The adriamycin semiquinone radical has a greater affinity for DNA and covalent complexes up to one adriamycin per 12 nucleotides were obtained. The daunorubicin semiquinone radical, on the other hand, showed a lesser binding affinity and gave rise to complexes in which one drug molecule was covalently bound per 135 nucleotides. The stronger covalent binding of adriamycin to DNA may account for more severe DNA damage induced by this drug.  相似文献   

19.
Lin CH  Chen YC  Pan TM 《PloS one》2011,6(12):e29101
Quantitative real-time PCR (qPCR) is the gold standard for the quantification of specific nucleic acid sequences. However, a serious concern has been revealed in a recent report: supercoiled plasmid standards cause significant over-estimation in qPCR quantification. In this study, we investigated the effect of plasmid DNA conformation on the quantification of DNA and the efficiency of qPCR. Our results suggest that plasmid DNA conformation has significant impact on the accuracy of absolute quantification by qPCR. DNA standard curves shifted significantly among plasmid standards with different DNA conformations. Moreover, the choice of DNA measurement method and plasmid DNA conformation may also contribute to the measurement error of DNA standard curves. Due to the multiple effects of plasmid DNA conformation on the accuracy of qPCR, efforts should be made to assure the highest consistency of plasmid standards for qPCR. Thus, we suggest that the conformation, preparation, quantification, purification, handling, and storage of standard plasmid DNA should be described and defined in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to assure the reproducibility and accuracy of qPCR absolute quantification.  相似文献   

20.
Effects of different end sequences on stability, circular dichroism spectra (CD), and enzyme binding properties were investigated for six 22-base pair, non-self-complementary duplex DNA oligomers. The center sequences of these deoxyoligonucleotides have 8-14 base pairs in common and are flanked on both sides by sequences differing in context and A-T content. Temperature-induced melting transitions monitored by differential scanning calorimetry (DSC) and ultraviolet absorbance were measured for the six duplexes in buffered 115 mM Na(+) solutions. Values of the melting transition enthalpy, DeltaH(cal), and entropy, DeltaS(cal), were obtained directly from DSC experiments. Melting transition parameters, DeltaH(vH) and DeltaS(vH), were also estimated from van't Hoff analysis of optical melting curves collected as a function of DNA concentration, assuming a two-state melting transition. Melting free energies (20 degrees C) of the six DNAs evaluated from DSC experiments ranged from -18.7 to -32.7 kcal/mol. van't Hoff estimates of the free energies ranged from -18.5 to -48.0 kcal/mol. With either method, the trends in free energy as a function of sequence were identical. Equilibrium binding by BamHI restriction endonuclease to the 22-base pair DNAs was also investigated. The central eight base pairs of all six molecules, 5'-A-GGATCC-A-3', contained a BamHI recognition sequence bounded by A-T base pairs. Magnesium free binding assays were performed by titering BamHI against a constant concentration of each of the deoxyoligonucleotide substrates and analyzing reaction products by gel retardation. Binding isotherms of the total amount of bound DNA versus protein concentration were constructed which provided semiquantitative estimates of the equilibrium dissociation constants for dissociation of BamHI from the six DNA oligomers. Dissociation constants ranged from 0.5 x 10(-)(9) to 12.0 x 10(-)(9) M with corresponding binding free energies of -12.5 to -10.6 (+/-0. 1) kcal/mol. An inverse relationship is found when binding and stability are compared.  相似文献   

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