• 【Tn10转座中靶位选择的因素: DDE基序在靶DNA捕获中的作用。】 复制标题 收藏 收藏
    DOI:10.1093/emboj/16.10.2646 复制DOI
    作者列表:Junop MS,Haniford DB
    BACKGROUND & AIMS: Tn10, like several other transposons, exhibits a marked preference for integration into particular target sequences. Such sequences are referred to as integration hotspots and have been used to define a consensus target site in Tn10 transposition. We demonstrate that a Tn10 hotspot called HisG1, which was identified originally in vivo, also functions as an integration hotspot in vitro in a reaction where the HisG1 sequence is present on a short DNA oligomer. We use this in vitro system to define factors which are important for the capture of the HisG1 target site. We demonstrate that although divalent metal ions are not essential for HisG1 target capture, they greatly facilitate capture of a mutated HisG1 site. Analysis of catalytic transposase mutants further demonstrates that the DDE motif plays a critical role in 'divalent metal ion-dependent' target capture. Analysis of two other classes of transposase mutants, Exc+ Int- (which carry out transposon excision but not integration) and ATS (altered target specificity), demonstrates that while a particular ATS transposase binds HisG1 mutants better than wild-type transposase, Exc+ Int- mutants are defective in HisG1 capture, further defining the properties of these classes of mutants. Possible mechanisms for the above observations are considered.

    背景与目标: Tn10与其他几个转座子一样,表现出对整合到特定靶序列的明显偏好。此类序列被称为整合热点,并已用于定义Tn10转座中的共有靶位点。我们证明了最初在体内鉴定的称为HisG1的Tn10热点在HisG1序列存在于短DNA寡聚物上的反应中也起着体外整合热点的作用。我们使用此体外系统来定义对于捕获HisG1靶位点很重要的因素。我们证明,尽管二价金属离子对于HisG1靶捕获不是必需的,但它们极大地促进了突变HisG1位点的捕获。催化转座酶突变体的分析进一步表明,DDE基序在 “二价金属离子依赖性” 靶捕获中起关键作用。对另外两类转座酶突变体Exc Int- (进行转座子切除但不整合) 和ATS (改变靶特异性) 的分析表明,尽管特定的ATS转座酶比野生型转座酶更好地结合HisG1突变体,但Exc Int-突变体在HisG1捕获中存在缺陷,进一步定义这些类别的突变体的属性。考虑了上述观察的可能机制。
  • 【Exogean: 在真核基因组DNA中注释蛋白质编码基因的框架。】 复制标题 收藏 收藏
    DOI:10.1186/gb-2006-7-s1-s7 复制DOI
    作者列表:Djebali S,Delaplace F,Roest Crollius H
    BACKGROUND & AIMS: BACKGROUND:Accurate and automatic gene identification in eukaryotic genomic DNA is more than ever of crucial importance to efficiently exploit the large volume of assembled genome sequences available to the community. Automatic methods have always been considered less reliable than human expertise. This is illustrated in the EGASP project, where reference annotations against which all automatic methods are measured are generated by human annotators and experimentally verified. We hypothesized that replicating the accuracy of human annotators in an automatic method could be achieved by formalizing the rules and decisions that they use, in a mathematical formalism. RESULTS:We have developed Exogean, a flexible framework based on directed acyclic colored multigraphs (DACMs) that can represent biological objects (for example, mRNA, ESTs, protein alignments, exons) and relationships between them. Graphs are analyzed to process the information according to rules that replicate those used by human annotators. Simple individual starting objects given as input to Exogean are thus combined and synthesized into complex objects such as protein coding transcripts. CONCLUSION:We show here, in the context of the EGASP project, that Exogean is currently the method that best reproduces protein coding gene annotations from human experts, in terms of identifying at least one exact coding sequence per gene. We discuss current limitations of the method and several avenues for improvement.
    背景与目标:
  • 【靶向抗龋DNA疫苗的免疫原性和持久性。】 复制标题 收藏 收藏
    DOI:10.1177/154405910608501008 复制DOI
    作者列表:Xu QA,Yu F,Fan MW,Bian Z,Chen Z,Fan B,Jia R,Guo JH
    BACKGROUND & AIMS: :We have previously reported that a targeted anti-caries DNA vaccine, pGJA-P, induced accelerated and increased antibody responses compared with a non-targeted anti-caries DNA vaccine. Recently, pGJA-P/VAX, a new targeted anti-caries DNA vaccine for human trials, was constructed by replacing the pCI vector used in the construction of pGJA-P with pVAX1, the only vector authorized by the US Food and Drug Administration in clinical trials. Here, we report on our exploration of the kinetics of the antibody responses generated following pGJA-P/VAX immunization and the persistence of pGJA-P/VAX at both the inoculation site and the draining lymph nodes. Intranasal vaccination of mice with pGJA-P/VAX induced strong antibody responses that lasted for more than 6 months. Furthermore, pGJA-P/VAX could still be detected at both the inoculation site and the draining cervical lymph nodes 6 months after immunization. Thus, the persistent immune responses are likely due to the DNA depot in the host, which acts as a booster immunization.
    背景与目标: : 我们以前曾报道过,与非靶向抗龋DNA疫苗相比,靶向抗龋DNA疫苗pGJA-P诱导了加速和增加的抗体反应。最近,pGJA-P/VAX是一种用于人体试验的新型靶向抗龋齿DNA疫苗,通过用pVAX1代替用于构建pGJA-P的pCI载体,这是美国食品药品监督管理局在临床试验中唯一授权的载体。在这里,我们报告了我们对pGJA-P/VAX预防接种后产生的抗体反应的动力学以及pGJA-P/VAX在接种部位和引流淋巴结的持久性的探索。用pGJA-P/VAX对小鼠进行鼻内疫苗接种可诱导持续6个月以上的强烈抗体反应。此外预防接种后6个月,在接种部位和引流颈淋巴结中仍可检测到pGJA-P/VAX。因此,持续的免疫反应可能是由于宿主中的DNA储存库而起加强预防接种作用。
  • 【CpG寡脱氧核苷酸作为脊椎动物的DNA佐剂及其在免疫治疗中的应用。】 复制标题 收藏 收藏
    DOI:10.1016/j.intimp.2006.06.001 复制DOI
    作者列表:Chaung HC
    BACKGROUND & AIMS: :The genomes of bacterial and viral DNA contain a much higher frequency of unmethylated CpG dinucleotides than those of vertebrates. This difference in genome structure allows the innate immune system of vertebrates to distinguish bacterial or viral DNA from self-DNA, and consequently to perceive a 'danger signal' when bacterial or viral DNA is encountered. Multiple sources of evidence suggest that CpG motifs, including bacterial DNA and CpG ODNs (synthetic oligodeoxynucleotides containing unmethylated CpG), are capable of evoking a range of immunostimulatory effects in vertebrates and have a tremendous potential to be used as therapeutic agents and adjuvants. CpG motifs with different sequences have been shown to induce various types or levels of immunostimulatory responses whereas the immunostimulatory effects of CpG motifs are species-specific. A better understanding of CpG recognition at the molecular level is fundamental to the identification of those motifs that have desired immunostimulatory responses. It is hoped that this would allow the optimization and application of CpG motifs as therapeutic agents and adjuvants, for numerous diseases in various species.
    背景与目标: : 细菌和病毒DNA的基因组含有比脊椎动物更高的未甲基化CpG二核苷酸的频率。基因组结构的这种差异使脊椎动物的先天免疫系统能够将细菌或病毒DNA与自身DNA区分开,从而在遇到细菌或病毒DNA时感知到 “危险信号”。多种证据表明,CpG基序,包括细菌DNA和CpG odn (含有未甲基化CpG的合成寡脱氧核苷酸),能够在脊椎动物中引起一系列免疫刺激作用,并具有用作治疗剂和佐剂的巨大潜力。已显示具有不同序列的CpG基序可诱导各种类型或水平的免疫刺激反应,而CpG基序的免疫刺激作用是物种特异性的。在分子水平上更好地理解CpG识别对于鉴定具有所需免疫刺激反应的基序至关重要。希望这将允许CpG基序作为治疗剂和佐剂的优化和应用,以治疗各种物种中的许多疾病。
  • 5 Activation volume of DNA duplex formation. 复制标题 收藏 收藏

    【DNA双链体形成的活化体积。】 复制标题 收藏 收藏
    DOI:10.1021/bi963175n 复制DOI
    作者列表:Lin MC,Macgregor RB Jr
    BACKGROUND & AIMS: The denaturation-renaturation thermal hysteresis was used to investigate the kinetics of the helix-coil equilibrium of four 22-base pair homopurine-homopyrimidine duplex oligonucleotides with fractional G x C base pair content (f(G x C)) between 0.14 and 0.5. In 20 mM NaCl and 20 mM Tris-HCl at pH 7.0 and at hydrostatic pressures up to 200 MPa, a two-state bimolecular reaction mechanism adequately described the observed kinetics. At 1 MPa and 47 degrees C, the rate constant for helix formation, k1, increased by a factor of 210, and the reverse rate constant, k(-1), decreased by a factor of 420 upon increasing f(G x C) from 0.14 to 0.5. The activation energies for formation of the duplexes were negative and relatively insensitive to f(G x C). The pressure-induced change in the rate constants is related to the activation volume of the reaction step. Pressure causes k1 to become larger, and the magnitude of the change in k1 with pressure increases the lower the f(G x C) value. Thus, when f(G x C) = 0.14, the activation volume for forward reaction, delta V++(1), equals -20 mL/mol, while when f(G x C) = 0.5, delta V++(1) = -6.7 mL/mol. The rate constant for strand separation, k(-1), decreases at high pressure. The activation volume for this step, delta V++(1), varies from 17 to 1.6 mL/mol when f(G x C) = 0.14 and 0.5, respectively. The delta V for helix formation calculated from the activation parameters changed from -23 mL/mol when f(G x C) = 0.14 to -5.8 mL/mol when f(G x C) = 0.5. From extrapolation, it is estimated that the molar volume change for formation of G x C base pairs in homopurine-homopyrimidine sequences is approximately 0 mL/mol. Parameters calculated from kinetics of other two duplex molecules, when f(G x C) = 0.23 and 0.32, lie between these extremes.

    背景与目标: 变性-复性热滞后用于研究0.14和0.5之间具有分数G x C碱基对含量 (f(G x C)) 的四种22碱基对高嘌呤-高嘧啶双链体寡核苷酸的螺旋-螺旋平衡动力学。在pH 7.0和高达200 MPa的静水压力下,在20 mM NaCl和20 mM Tris-HCl中,两态双分子反应机理充分描述了观察到的动力学。在1 mpa和47 ℃ 下,当f(G x C) 从0.14增加到0.5时,螺旋形成的速率常数k1增加了210倍,反向速率常数k(-1) 降低了420倍。形成双链体的活化能为负,并且对f(G x C) 相对不敏感。压力引起的速率常数变化与反应步骤的活化体积有关。压力导致k1变大,并且k1随压力变化的幅度增加,f(G x C) 值越低。因此,当f(G x C) = 0.14时,正向反应的活化体积 δ V ++(1) 等于-20 mL/mol,而当f(G x C) = 0.5时,δ V ++(1) = -6.7 mL/mol。在高压下,链分离的速率常数k(-1) 降低。当f(G x C) = 0.14和0.5时,该步骤的活化体积 Δ V ++(1) 分别从17至1.6 mL/mol变化。由活化参数计算的螺旋形成的 δ V从f (gxc) = 0.14时的-23 mL/mol变化到f (gxc) = 0.5时的-5.8 mL/mol。根据外推法,估计在均嘌呤-高嘧啶序列中形成G x C碱基对的摩尔体积变化约为0毫升/mol。当f(G x C) = 0.23和0.32时,根据其他两个双链体分子的动力学计算出的参数介于这些极端之间。
  • 【使用聚合酶链反应检测Lindow Man古代遗骸中的大肠杆菌DNA。】 复制标题 收藏 收藏
    DOI:10.1046/j.1472-765x.1997.00066.x 复制DOI
    作者列表:Fricker EJ,Spigelman M,Fricker CR
    BACKGROUND & AIMS: The polymerase chain reaction has been applied to the detection of Escherichia coli DNA in the upper gut contents of Lindow Man, an Iron Age bog body dated to ca 300 BC. With sets of primers from the uidA and lacZ genes, E. coli DNA could be detected reproducibly. Initial attempts at detecting DNA from freshly voided faeces from a healthy volunteer were unsuccessful due to inhibition of the reaction. Development of a method, based on guanidine thiocyanate and silica extraction and purification of the DNA fragments, facilitated the detection of the E. coli DNA in both freshly voided faeces and the upper gut contents of Lindow Man. These findings indicate that it may be possible to study the existence of infectious diseases in ancient civilizations and to learn more about the evolution of microbes.

    背景与目标: 聚合酶链反应已用于检测Lindow Man的上肠内容物中的大肠杆菌DNA,Lindow Man是铁器时代的沼泽体,可追溯至公元前300年。使用uidA和lacZ基因的引物组,可以重复检测大肠杆菌DNA。由于抑制反应,最初尝试从健康志愿者的新鲜排空粪便中检测DNA的尝试均未成功。基于硫氰酸胍和二氧化硅提取和纯化DNA片段的方法的开发,有助于检测新鲜排空的粪便和Lindow Man的上肠内容物中的大肠杆菌DNA。这些发现表明,可能有可能研究古代文明中传染病的存在,并更多地了解微生物的进化。
  • 【小家鼠 β 珠蛋白单倍型Hbbs和Hbbd的DNA片段。】 复制标题 收藏 收藏
    DOI:10.1073/pnas.76.3.1385 复制DOI
    作者列表:Weaver S,Haigwood NL,Hutchison CA 3rd,Edgell MH
    BACKGROUND & AIMS: :Two alternative haplotypes at the complex locus controlling hemoglobin beta chain synthesis in Mus musculus were compared at the DNA level. As expected, Hbbd homozygotes--which as adults synthesize two species of beta chain--have two genes for beta globin. Adult mice homozygous for the Hbbs haplotype make only a single type of beta polypeptide, yet they also have two beta globin genes. Apparently the two Hbbs genes encode identical proteins, or one of the two genes is not detectably expressed. The Hbbs and Hbbd haplotypes are thus more similar at the DNA level than studies of their polypeptide products have indicated.
    背景与目标: : 在DNA水平上比较了控制小家鼠血红蛋白 β 链合成的复杂位点的两种替代单倍型。正如预期的那样,Hbbd纯合子-成年后合成了两个 β 链物种-具有两个 β 球蛋白基因。Hbbs单倍型纯合的成年小鼠仅产生一种 β 多肽,但它们也有两个 β 球蛋白基因。显然,这两个Hbbs基因编码相同的蛋白质,或者这两个基因中的一个无法检测到表达。因此,hbb和Hbbd单倍型在DNA水平上比其多肽产物的研究更相似。
  • 【免疫治疗反应的DNA损伤和修复生物标志物。】 复制标题 收藏 收藏
    DOI:10.1158/2159-8290.CD-17-0226 复制DOI
    作者列表:Mouw KW,Goldberg MS,Konstantinopoulos PA,D'Andrea AD
    BACKGROUND & AIMS: :DNA-damaging agents are widely used in clinical oncology and exploit deficiencies in tumor DNA repair. Given the expanding role of immune checkpoint blockade as a therapeutic strategy, the interaction of tumor DNA damage with the immune system has recently come into focus, and it is now clear that the tumor DNA repair landscape has an important role in driving response to immune checkpoint blockade. Here, we summarize the mechanisms by which DNA damage and genomic instability have been found to shape the antitumor immune response and describe clinical efforts to use DNA repair biomarkers to guide use of immune-directed therapies.Significance: Only a subset of patients respond to immune checkpoint blockade, and reliable predictive biomarkers of response are needed to guide therapy decisions. DNA repair deficiency is common among tumors, and emerging experimental and clinical evidence suggests that features of genomic instability are associated with response to immune-directed therapies. Cancer Discov; 7(7); 675-93. ©2017 AACR.
    背景与目标: : DNA损伤剂广泛用于临床肿瘤学,并利用肿瘤DNA修复中的缺陷。鉴于免疫检查点阻断作为一种治疗策略的作用不断扩大,肿瘤DNA损伤与免疫系统的相互作用最近成为人们关注的焦点,现在很明显,肿瘤DNA修复景观在驱动免疫检查点阻断的反应中具有重要作用。在这里,我们总结了DNA损伤和基因组不稳定性被发现形成抗肿瘤免疫反应的机制,并描述了使用DNA修复生物标志物指导免疫导向疗法的临床努力。意义: 只有一部分患者对免疫检查点阻断有反应,需要可靠的预测反应生物标志物来指导治疗决策。DNA修复缺陷在肿瘤中很常见,新出现的实验和临床证据表明,基因组不稳定性的特征与对免疫导向疗法的反应有关。癌症椎间盘; 7(7); 675-93。©2017 AACR.
  • 【“生物碱harmalol与DNA的结合: 光物理和量热方法” 的勘误 [J. Photochem. Photobiol. B: Biol. 130 (2014) 272-280]。】 复制标题 收藏 收藏
    DOI:10.1016/j.jphotobiol.2015.03.003 复制DOI
    作者列表:Sarkar S,Bhadra K
    BACKGROUND & AIMS: -2
    背景与目标: -2
  • 【用重组亚单位或DNA疫苗递送的猪肺炎支原体抗原P37,P42,P46和P95免疫小鼠。】 复制标题 收藏 收藏
    DOI:10.1016/j.vaccine.2012.10.088 复制DOI
    作者列表:Galli V,Simionatto S,Marchioro SB,Fisch A,Gomes CK,Conceição FR,Dellagostin OA
    BACKGROUND & AIMS: :Porcine enzootic pneumonia (PEP), which is caused by the fastidious bacterium Mycoplasma hyopneumoniae, is one of the most economically important diseases in the pig industry worldwide. Commercial bacterins provide only partial protection; therefore, the development of more efficient vaccines against PEP is necessary. In this study, the cellular and humoral immune responses elicited by DNA and recombinant subunit vaccines based on the P37, P42, P46 and P95 antigens of M. hyopneumoniae were evaluated after the intramuscular inoculation of BALB/c mice. The expression of the cytokines INFγ, TNFα and IL1 was evaluated by real-time RT-PCR in splenocytes from vaccinated mice. All antigens delivered as subunit vaccines, especially P42 and P95, and the pcDNA3/P46 DNA vaccine were able to elicit strong immune responses. These vaccines induced cellular immune responses and the production of antibodies able to react with native M. hyopneumoniae proteins. Because both cellular and humoral immune responses were induced, P42 and P95 are promising candidates for a recombinant subunit vaccine and P46 is a promising candidate for a DNA vaccine against PEP.
    背景与目标: : 猪传染性肺炎 (PEP) 是由挑剔的细菌支原体肺炎e引起的,是全球养猪业最重要的经济疾病之一。商业细菌仅提供部分保护; 因此,必须开发针对PEP的更有效疫苗。在这项研究中,在肌内接种BALB/c小鼠后,评估了基于猪肺炎支原体P37,P42,P46和P95抗原的DNA和重组亚基疫苗引起的细胞和体液免疫反应。通过实时rt-pcr评估了免疫小鼠脾细胞中细胞因子inf γ,tnf α 和IL1的表达。作为亚单位疫苗递送的所有抗原,尤其是P42和P95,以及pcDNA3/P46 DNA疫苗能够引发强烈的免疫反应。这些疫苗可诱导细胞免疫反应,并产生能够与天然猪肺炎支原体蛋白反应的抗体。由于诱导了细胞和体液免疫反应,因此P42和P95是重组亚单位疫苗的有希望的候选者,而P46是针对PEP的DNA疫苗的有希望的候选者。
  • 【组蛋白泛素化在DNA损伤反应中的作用。】 复制标题 收藏 收藏
    DOI:10.1016/j.dnarep.2017.06.011 复制DOI
    作者列表:Uckelmann M,Sixma TK
    BACKGROUND & AIMS: :DNA double strand breaks need to be repaired in an organized fashion to preserve genomic integrity. In the organization of faithful repair, histone ubiquitination plays a crucial role. Recent findings suggest an integrated model for DNA repair regulation through site-specific histone ubiquitination and crosstalk to other posttranslational modifications. Here we discuss how site-specific histone ubiquitination is achieved on a molecular level and how different multi-protein complexes work together to integrate different histone ubiquitination states. We propose a model where site-specific H2A ubiquitination organizes the spatio-temporal recruitment of DNA repair factors which will ultimately contribute to DNA repair pathway choice between homologous recombination and non-homologous end joining.
    背景与目标: : DNA双链断裂需要有组织地修复,以保持基因组完整性。在组织忠实修复中,组蛋白泛素化起着至关重要的作用。最近的发现表明,通过位点特异性组蛋白泛素化和与其他翻译后修饰的串扰,建立了DNA修复调控的综合模型。在这里,我们讨论了如何在分子水平上实现位点特异性组蛋白泛素化,以及不同的多蛋白复合物如何协同工作以整合不同的组蛋白泛素化状态。我们提出了一个模型,其中位点特异性H2A泛素化组织DNA修复因子的时空募集,这最终将有助于同源重组和非同源末端连接之间的DNA修复途径选择。
  • 【对G-四链体DNA表现出高保真度的卤化五甲胺花青染料。】 复制标题 收藏 收藏
    DOI:10.1016/j.bmc.2012.10.008 复制DOI
    作者列表:Nanjunda R,Owens EA,Mickelson L,Alyabyev S,Kilpatrick N,Wang S,Henary M,Wilson WD
    BACKGROUND & AIMS: :Design and optimization of quadruplex-specific small molecules is developing into an attractive strategy for anti-cancer therapeutics with some promising candidates in clinical trials. A number of therapeutically favorable features of cyanine molecules can be effectively exploited to develop them as promising quadruplex-targeting agents. Herein, the design, synthesis and evaluation of a series of dimethylindolenine cyanine dyes with varying halogen substitutions are reported. Their interactions with telomeric and c-myc quadruplexes as well as a reference duplex sequence have been evaluated using thermal melting, biosensor-surface plasmon resonance, circular dichroism, isothermal titration calorimetry and mass spectrometry. Thermal melting analysis indicates that these ligands exhibit significant quadruplex stabilization and a very low duplex binding, with the dimethyl incorporation of paramount importance for decreased duplex affinity. Circular dichroism studies showed that the interaction of cyanines with quadruplex structures are primarily through stacking at one or both ends of the terminal tetrads with the two (trimethylammonium)propyl groups interacting in the accessible quadruplex grooves. Surface plasmon resonance and mass spectral studies shows the formation of an initial strong 1:1 complex followed by a significantly weaker secondary binding. Isothermal calorimetry studies show that the interaction of cyanines is predominantly entropy driven. In line with the design principles, this work provides new insights for further developing potent, highly selective cyanines as promising quadruplex-specific agents.
    背景与目标: : 四链特异性小分子的设计和优化正在发展成为一种有吸引力的抗癌治疗策略,在临床试验中有一些有前途的候选药物。可以有效地利用花青分子的许多治疗上有利的特征来开发它们作为有希望的四链靶向剂。本文报道了一系列具有不同卤素取代的二甲基吲哚烯花青染料的设计,合成和评估。已使用热熔,生物传感器-表面等离子体共振,圆二色性,等温滴定量热法和质谱法评估了它们与端粒和c-myc四链体以及参考双链体序列的相互作用。热熔分析表明,这些配体表现出显着的四链稳定性和非常低的双链体结合,其中二甲基掺入对于降低双链体亲和力至关重要。圆二色性研究表明,花青与四重结构的相互作用主要是通过在末端四重的一端或两端堆叠,其中两个 (三甲基铵) 丙基基团在可及的四重沟槽中相互作用。表面等离子体共振和质谱研究表明,形成了初始的强1:1络合物,随后显着较弱的二级结合。等温量热法研究表明,花青的相互作用主要是熵驱动的。符合设计原则,这项工作为进一步开发有效的,高选择性的花青作为有希望的四链特异性试剂提供了新的见解。
  • 【声刺激促进豚鼠耳蜗中的DNA片段化。】 复制标题 收藏 收藏
    DOI:10.1272/jnms.79.349 复制DOI
    作者列表:Kamio T,Watanabe K,Okubo K
    BACKGROUND & AIMS: :Apoptosis can be described as programmed cell death. Apoptosis regulates cell turnover and is involved in various pathological conditions. The characteristic features of apoptosis are shrinkage of the cell body, chromatin condensation, and nucleic acid fragmentation. During apoptosis, double-stranded DNA is broken down into single-stranded DNA (ssDNA) by proteases. Acoustic trauma is commonly encountered in otorhinolaryngology clinics. Intense noise can cause inner ear damage, such as hearing disturbance, tinnitus, ear fullness, and decreased speech discrimination. In this study, we used immunohistochemical and electrophysiological methods to examine the fragmentation of DNA in the cochleas of guinea pigs that had been exposed to intense noise. Twenty-four guinea pigs weighing 250 to 350 g were used. The animals were divided into 4 groups: (I) a control group (n=6), (II) a group that was exposed to noise for 2 hours (n=6), (III) a group that was exposed to noise for 5 hours (n=6), and (IV) a group that was exposed to noise for 20 hours. The stimulus was a pure tone delivered at a frequency of 2 kHz. The sound pressure level was 120 dBSPL. No threshold shifts were apparent in group I. Group II showed a significant elevation of the hearing threshold (ANOVA, p<0.05(*)). The ABR threshold level was also significantly elevated immediately after the acoustic stimulation in groups III and IV (ANOVA, p<0.01(**)). In groups I, II, and IV, the lateral wall of the ear did not show immunoreactivity to ssDNA but did in group III. No immunoreactivity was apparent in the organ of Corti in group I or II. However, the supporting cells and outer hair cells in groups III and IV showed reactions for ssDNA. The fine structure of the organ of Corti had been destroyed in group IV. The lateral wall showed immunoreactivity for ssDNA only in group III, whereas the organ of Corti showed reactions for ssDNA in groups III and IV. Our study suggests that apoptotic changes occur in patients that suffer acoustic trauma. Once the apoptotic pathway has started, it is irreversible. Thus, early diagnosis and treatment are necessary. Earplugs should also be worn at rock concerts.
    背景与目标: : 细胞凋亡可以描述为程序性细胞死亡。凋亡调节细胞更新,并参与各种病理状况。细胞凋亡的特征是细胞体收缩,染色质浓缩和核酸片段化。在细胞凋亡过程中,双链DNA被蛋白酶分解为单链DNA (ssDNA)。耳鼻喉科诊所通常会遇到声学创伤。强烈的噪声会引起内耳的损伤,如听力障碍、耳鸣、耳饱满、言语辨别能力下降等。在这项研究中,我们使用免疫组织化学和电生理学方法来检查暴露于强烈噪音的豚鼠耳蜗中DNA的片段化。使用了24只体重250至350g的豚鼠。将动物分为4组 :( I) 对照组 (n = 6),(II) 暴露于噪声2小时的组 (n = 6),(III) 暴露于噪声5小时的组 (n = 6),(IV) 暴露于噪音20小时的人群。刺激是以2 kHz的频率传递的纯音。声压级为120 dBSPL。组I中没有明显的阈值变化。组II显示听力阈值显着升高 (ANOVA,p<0.05(*))。在第III组和第IV组的声刺激后,ABR阈值水平也显着升高 (ANOVA,p<0.01(**))。在I,II和IV组中,耳朵的侧壁未显示对ssDNA的免疫反应性,但在III组中显示。在第一组或第二组的Corti器官中没有明显的免疫反应性。然而,III和IV组中的支持细胞和外部毛细胞显示出对ssDNA的反应。第四组破坏了Corti器官的精细结构。侧壁仅在III组中显示出对ssDNA的免疫反应性,而Corti器官在III和IV组中显示出对ssDNA的反应。我们的研究表明,遭受声学创伤的患者会发生凋亡变化。一旦凋亡途径开始,它是不可逆的。因此,早期诊断和治疗是必要的。摇滚音乐会上也应该戴耳塞。
  • 【O-甲基鸟嘌呤-DNA甲基转移酶的丢失赋予拓扑异构酶II介导的阿霉素抗性三阴性乳腺癌细胞对卡莫司汀的附带敏感性。】 复制标题 收藏 收藏
    DOI:10.1016/j.bcp.2012.10.020 复制DOI
    作者列表:Raguz S,Adams C,Masrour N,Rasul S,Papoutsoglou P,Hu Y,Cazzanelli G,Zhou Y,Patel N,Coombes C,Yagüe E
    BACKGROUND & AIMS: :Triple-negative breast cancer is characterized by aggressive tumours whose cells lack oestrogen and progesterone receptors and do not over-express HER2. It accounts for approximately 10-15% of breast cancer cases. We sought to generate a cellular model of chemotherapy drug resistance for this type of disease to provide the tools for the development of new therapies. Doxorubicin is a component of some chemotherapy regimes used to treat this form of cancer but resistance preventing disease eradication frequently occurs, mainly due to over-expression of drug transporters such as P-glycoprotein. CALDOX cells were generated by exposure of CAL51 to doxorubicin. Resistance to doxorubicin did not involve drug transporters, as the both parental and resistant cells accumulated doxorubicin to comparable levels. CALDOX cells had slower proliferation rate and an extended G1 cell cycle stage than the parental line, mainly due to an intrinsic activation of CDNK1 (p21), but this cell cycle block was not involved in the mechanism of resistance. CALDOX cells had reduced levels of TOP2A (topoisomerase IIα) and were cross resistant to the topoisomerase II inhibitors etoposide and mitoxantrone. CALDOX cells showed collateral sensitivity to carmustine due to the lack of O⁶-methylguanine-DNA-methyltransferase (MGMT) expression, related to the hypermethylation of its promoter. The collateral sensitivity of CALDOX cells to carmustine provides the rationale to evaluate MGMT promoter methylation status to design better therapeutic strategies for triple negative breast cancer.
    背景与目标: : 三阴性乳腺癌的特征是侵袭性肿瘤,其细胞缺乏雌激素和孕激素受体,并且不会过度表达her2。它约占乳腺癌病例的10-15%。我们试图为这种类型的疾病生成化学疗法耐药性的细胞模型,以为开发新疗法提供工具。阿霉素是用于治疗这种癌症的某些化学疗法方案的组成部分,但经常发生抗药性预防疾病根除,这主要是由于药物转运蛋白 (例如P-糖蛋白) 的过表达。CALDOX细胞是通过将CAL51暴露于阿霉素而产生的。对阿霉素的耐药性不涉及药物转运蛋白,因为亲本和耐药细胞都积累了阿霉素的水平相当。与亲本系相比,钙氧化细胞的增殖速度较慢,G1细胞周期阶段延长,这主要是由于CDNK1 (p21) 的内在激活,但这种细胞周期阻滞不参与抗性机制。钙氧化细胞的TOP2A (拓扑异构酶II α) 水平降低,并且对拓扑异构酶II抑制剂依托泊苷和米托蒽醌具有交叉抗性。由于缺乏o-甲基鸟嘌呤-DNA-甲基转移酶 (MGMT) 表达,CALDOX细胞对卡莫司汀表现出附带敏感性,这与其启动子的高甲基化有关。CALDOX细胞对卡莫司汀的附带敏感性为评估MGMT启动子甲基化状态以设计更好的三阴性乳腺癌治疗策略提供了依据。
  • 【使用尿素滴定曲线确定基于DNA的纳米设备和纳米开关的折叠和结合自由能。】 复制标题 收藏 收藏
    DOI:10.1093/nar/gkx498 复制DOI
    作者列表:Idili A,Ricci F,Vallée-Bélisle A
    BACKGROUND & AIMS: :DNA nanotechnology takes advantage of the predictability of DNA interactions to build complex DNA-based functional nanoscale structures. However, when DNA functional and responsive units that are based on non-canonical DNA interactions are employed it becomes quite challenging to predict, understand and control their thermodynamics. In response to this limitation, here we demonstrate the use of isothermal urea titration experiments to estimate the free energy involved in a set of DNA-based systems ranging from unimolecular DNA-based nanoswitches to more complex DNA folds (e.g. aptamers) and nanodevices. We propose here a set of fitting equations that allow to analyze the urea titration curves of these DNA responsive units based on Watson-Crick and non-canonical interactions (stem-loop, G-quadruplex, triplex structures) and to correctly estimate their relative folding and binding free energy values under different experimental conditions. The results described herein will pave the way toward the use of urea titration experiments in the field of DNA nanotechnology to achieve easier and more reliable thermodynamic characterization of DNA-based functional responsive units. More generally, our results will be of general utility to characterize other complex supramolecular systems based on different biopolymers.
    背景与目标: : DNA纳米技术利用DNA相互作用的可预测性来构建复杂的基于DNA的功能性纳米级结构。但是,当使用基于非规范DNA相互作用的DNA功能和响应单元时,预测,理解和控制其热力学变得非常具有挑战性。针对这一限制,在这里,我们演示了使用等温尿素滴定实验来估计一组基于DNA的系统中涉及的自由能,这些系统从基于单分子DNA的纳米开关到更复杂的DNA折叠 (例如适体) 和纳米设备。我们在这里提出了一组拟合方程,允许基于Watson-Crick和非规范相互作用 (茎环,G-四链,三链结构) 分析这些DNA响应单元的尿素滴定曲线,并正确估计它们在不同实验条件下的相对折叠和结合自由能值。本文所述的结果将为在DNA纳米技术领域中使用尿素滴定实验铺平道路,以实现基于DNA的功能响应单元的更容易且更可靠的热力学表征。更一般地说,我们的结果将普遍用于表征基于不同生物聚合物的其他复杂超分子系统。

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