Selective inhibition of the human tie-1 promoter with triplex-forming oligonucleotides targeted to ets binding sites

The Tie receptors (Tie-1 and Tie-2/Tek) are essential for angiogenesis and vascular remodeling/integrity. Tie receptors are up-regulated in tumor-associated endothelium, and their inhibition disrupts angiogenesis and can prevent tumor growth as a consequence. To investigate the potential of anti-gene approaches to inhibit tie gene expression for anti-angiogenic therapy, we have examined triple-helical (triplex) DNA formation at 2 tandem Ets transcription factor binding motifs (designated E-1 and E-2) in the human tie-1 promoter. Various tie-1 promoter deletion/mutation luciferase reporter constructs were generated and transfected into endothelial cells to examine the relative activities of E-1 and E-2. The binding of antiparallel and parallel (control) purine motif oligonucleotides (21-22 bp) targeted to E-1 and E-2 was assessed by plasmid DNA fragment binding and electrophoretic mobility shift assays. Triplex-forming oligonucleotides were incubated with tie-1 reporter constructs and transfected into endothelial cells to determine their activity. The Ets binding motifs in the E-1 sequence were essential for human tie-1 promoter activity in endothelial cells, whereas the deletion of E-2 had no effect. Antiparallel purine motif oligonucleotides targeted at E-1 or E-2 selectively formed strong triplex DNA (K(d) approximately 10(-7) M) at 37 degrees C. Transfection of tie-1 reporter constructs with triplex DNA at E-1, but not E-2, specifically inhibited tie-1 promoter activity by up to 75% compared with control oligonucleotides in endothelial cells. As similar multiple Ets binding sites are important for the regulation of several endothelial-restricted genes, this approach may have broad therapeutic potential for cancer and other pathologies involving endothelial proliferation/dysfunction.

Isolation of nontuberculous mycobacteria (NTM) from household water and shower aerosols in patients with pulmonary disease caused by NTM

It has been postulated that susceptible individuals may acquire infection with nontuberculous mycobacteria (NTM) from water and aerosol exposure. This study examined household water and shower aerosols of patients with NTM pulmonary disease. The mycobacteria isolated from clinical samples from 20 patients included M. avium (5 patients), M. intracellulare (12 patients), M. abscessus (7 patients), M. gordonae (1 patient), M. lentiflavum (1 patient), M. fortuitum (1 patient), M. peregrinum (1 patient), M. chelonae (1 patient), M. triplex (1 patient), and M. kansasii (1 patient). One-liter water samples and swabs were collected from all taps, and swimming pools or rainwater tanks. Shower aerosols were sampled using Andersen six-stage cascade impactors. For a subgroup of patients, real-time PCR was performed and high-resolution melt profiles were compared to those of ATCC control strains. Pathogenic mycobacteria were isolated from 19 homes. Species identified in the home matched that found in the patient in seven (35%) cases: M. abscessus (3 cases), M. avium (1 case), M. gordonae (1 case), M. lentiflavum (1 case), and M. kansasii (1 case). In an additional patient with M. abscessus infection, this species was isolated from potable water supplying her home. NTM grown from aerosols included M. abscessus (3 homes), M. gordonae (2 homes), M. kansasii (1 home), M. fortuitum complex (4 homes), M. mucogenicum (1 home), and M. wolinskyi (1 home). NTM causing human disease can be isolated from household water and aerosols. The evidence appears strongest for M. avium, M. kansasii, M. lentiflavum, and M. abscessus. Despite a predominance of disease due to M. intracellulare, we found no evidence for acquisition of infection from household water for this species.

Seeing it differently: physical education, teacher education and the possibilities of personal construct theory

The celebrated work of Lortie (1975) alerted teacher educators to the extended period of 'apprenticeship' that student teachers have been through before they arrive at teacher education programmes. The subjective implicit theories (Marland, 1992) developed by prospective teachers are shaped by their lifeworld experiences at school and in the case of physical education teachers, their experiences in sport. The biography of physical education teacher education (PETE) students tends to be characterised by ecto-mesomorphic individuals who have been socialised by the rigours of highly competitive sport (Gore, 1990; Macdonald, 1992; Rossi, 1996). We can add to this, the requirements of teacher preparation in physical education which for the most part are dominated by the traditions and rhetoric of the 'natural' bio-physical sciences; largely a legacy of Henry's (1964) work on physical education as an academic discipline, as well as that of Abernathy and Waltz the same year (Abernathy & Waltz, 1964). In the United Kingdom, Curl (1973) further advanced the argument in an attempt to justify human movement as an independent field of study with its own corpus of knowledge. It is little wonder then, that the dominant pedagogical discourse in physical education is, as Tinning (1991) discusses, one of performance pedagogy (see also Hendry, 1986 for an earlier discussion). The knowledge required to support such a discourse could be described as 'official' (Apple, 1993) and it assumes such status by virtue of the power appropriated by and bestowed upon the scientific community in PETE (Macdonald & Tinning, 1995; Sparkes, 1989, 1993). However, there are social reifiers too, and these tend to relate to the social construction of the body (Kirk, 1993; Kirk & Spiller, 1994; Gilroy, 1994) and what Tinning (1985) has termed the Cult of Slenderness. Furthermore the 'slender image' has become a signifier of 'good health'. This is inextricably linked to what might be considered as a health triplex—'exercise = fitness = health' (see Kirk & Colquhoun, 1989; Tinning & Kirk, 1991) which in Australia, underpins curriculum packages such as Daily Physical Education which teachers (often including physical education primary...

Development of an immunomagnetic capture-reverse transcriptase-PCR assay for three pineapple ampeloviruses

A semi-automated, immunomagneticcapture-reverse transcription PCR(IMC-RT-PCR) assay for the detection of three pineapple-infecting ampeloviruses, Pineapple mealybug wilt-associated virus-1, -2 and -3, is described. The assay was equivalent in sensitivity but more rapid than conventional immunocapture RT-PCR. The assay can be used either as a one- or two-step RT-PCR and allows detection of the viruses separately or together in a triplex assay from fresh, frozen or freeze-dried pineapple leaf tissue. This IMC-RT-PCR assay could be used for high throughput screening of pineapple planting propagules and could easily be modified for the detection of other RNA viruses in a range of plant species, provided suitable antibodies are available.

Augustine and the functions of concupiscence

This study analyses Augustine s concept of concupiscentia, or evil desire (together with two cognate terms, libido and cupiditas) in the context of his entire oeuvre. By the aid of systematic analysis, the concept and its development is explored in four distinct ways. It is claimed that Augustine used the concept of concupiscentia for several theological purposes, and the task of the study is to represent these distinct functions, and their connections to Augustine s general theological and philosophical convictions. The study opens with a survey on terminology. A general overview of the occurrences of the negatively connoted words for desire in Latin literature precedes a corresponding examination of Augustine s own works. In this introductory chapter it is shown that, despite certain preferences in the uses of the words, a sufficient degree of synonymy reigns so as to allow an analysis of the concept without tightly discriminating between the terms. The theological functions of concupiscentia with its distinct contexts are analysed in separate chapters. The function of concupiscentia as a divine punishment is explored first (Ch 3). It is seen how Augustine links together concupiscentia and ideas about divine justice, and finally suggests that in the inordinate, psychologically experienced sexual desire, the original theological disobedience of Adam and Eve can be perceived. Augustine was criticized for this solution already in his own times, and the analysis of the function of concupiscentia as a divine punishment ends in a discussion on the critical response of punitive concupiscentia by Julian of Aeclanum. Augustine also attached to concupiscentia another central theological function by viewing evil desire as an inward originating cause for all external evil actions. In the study, this function is analysed by surveying two formally distinct images of evil desire, i.e. as the root (radix) of all evil, and as a threefold (triplex) matrix of evil actions (Ch 4). Both of these images were based on a single verse of the Bible (1 Jn 2, 16 and 1 Tim 6, 10). This function of concupiscentia was formed both parallel to, and in answer to, Manichaean insights into concupiscentia. Being familiar with the traditional philosophical discussions on the nature and therapy of emotions, Augustine situated concupiscentia also into this context. It is acknowledged that these philosophical traditions had an obvious impact into his way of explaining psychological processes in connection with concupiscentia. Not only did Augustine implicitly receive and exploit these traditions, but he also explicitly moulded and criticized them in connection with concupiscentia. Eventually, Augustine conceives the philosophical traditions of emotions as partly useful but also partly inadequate to deal with concupiscentia (Ch 5). The role of concupiscentia in connection to divine grace and Christian renewal is analysed in the final chapter of the study. Augustine s gradual development in internalizing the effects of concupiscentia also into the life of a baptized Christian are elucidated, as are the strong limitations and mitigations Augustine makes to the concept when attaching it into the life under grace (sub gratia). A crucial part in the development of this function is played by Augustine s changing interpretation of Rom 7, and the way concupiscentia appears in Augustine s readings of this text is therefore also analysed. As a result of the analysis of these four distinct functions and contexts of concupiscentia, it is concluded that Augustine s concept of concupiscentia is fairly tightly and coherently connected to his views of central theological importance. Especially the functions of concupiscentia as a punishment and the function of concupiscentia in Christian renewal were both tightly interwoven into Augustine s view of God s being and God s grace. The study shows the importance of reading Augustine s discussions on evil desire with a constant awareness of their role in their larger context, that is, of their function in each situation. The study warns against too simplistic and unifying readings of Augustine s concupiscentia, emphasizing the need to acknowledge both the necessitating, sinful aspects of concupiscentia, and the domesticated features of concupiscentia during Christian renewal.

Structural Polymorphism in d(T).d(A)*d(T) Triple Helices

DNA triple helices containing two thymine strands and one adenine strand have been studied, using model building followed by energy minimisation, for different orientations of the third strand resulting from variation in the hydrogen bonding between the Watson-Crick duplex and the third strand and the glycosidic torsion angle in the third strand. Our results show that the structure with a parallel orientation of the third strand, in which the third strand base forms Hoogsteen hydrogen bonds with the adenine base in the Watson-Crick duplex, is energetically the most favourable. An antiparallel orientation of the third strand is also possible, in which the third strand base hydrogen bonds to both the bases in the Watson-Crick duplex. This structure is energetically comparable to the parallel structure. For the parallel triplex a 200ps molecular dynamics simulation starting from two different starting structures indicates that at 300K significant structural heterogeneity exists in this tripler structure. The results are compared with existing structural data on this class of triplexes derived from theoretical and NMR techniques.

Design and structural analysis of hairpin-TFO for transcriptional activation of genes in S-cerevisiae

Triplex forming oligonucleotides (TFOs) have the potential to modulate gene expression. While most of the experiments are directed towards triplex mediated inhibition of gene expression the strategy potentially could be used for gene specific activation. In an attempt to design a strategy for gene specific activation in vivo applicable to a large number of genes we have designed a TFO based activator-target system which may be utilized in Saccharomyces cerevisiae or any other system where Gal4 protein is ectopically expressed. The total genome sequence of Saccharomyces cerevisiae and expression profiles were used to select the target genes with upstream poly (pu/py) sequences. We have utilized the paradigm of Gal4 protein and its binding site. We describe here the selection of target genes and design of hairpin-TFO including the targeting sequences containing polypurine stretch found in the upstream promoter regions of weakly expressed genes. We demonstrate, the formation of hairpin-TFO, its binding to Gal4 protein, its ability to form triplex with the target duplex in vitro, the effect of polyethylenimine on complex formation and discuss the implication on in vivo transcription activation.

Recent Developments in the Chemistry and Biology of G-Quadruplexes with Reference to the DNA Groove Binders

DNA is the chemotherapeutic target for treating diseases of genetic origin. Besides well-known double-helical structures (A, B, Z, parallel stranded-DNA etc.), DNA is capable of forming several multi-stranded structures (triplex, tetraplex, i-motif etc.) which have unique biological significance. The G-rich 3'-ends of chromosomes, called telomeres, are synthesized by telomerase, a ribonucleoprotein, and over-expression of telomerase is associated with cancer. The activity of telomerase is suppressed if the G-rich region is folded into the four stranded structures, called G-quadruplexes (G4-DNAs) using small synthetic ligands. Thus design and synthesis of new G4-DNA ligands is an attractive strategy to combat cancer. G4-DNA forming sequences are also prevalent in other genomic regions of biological significance including promoter regions of several oncogenes. Effective gene regulation may be achieved by inducing a G4-DNA structure within the G-rich promoter sequences. To date, several G4-DNA stabilizing ligands are known. DNA groove binders interact with the duplex B-DNA through the grooves (major and minor groove) in a sequence-specific manner. Some of the groove binders are known to stabilize the G4-DNA. However, this is a relatively under explored field of research. In this review, we focus on the recent advances in the understanding of the G4-DNA structures, particularly made from the human telomeric DNA stretches. We summarize the results of various investigations of the interaction of various organic ligands with the G4-DNA while highlighting the importance of groove binder-G4-DNA interactions.

溴代胞嘧啶对三螺旋DNA 稳定性的影响

研究了三螺旋DNA 中胞嘧啶被5 - 溴胞嘧啶取代后的稳定性问题。通过建立CGC , CGBrC , BrCGC 和BrCGBrC 4 种模型, 并采用Insight Ⅱ软件包中的Discover 程序进行计算模拟, 发现用5 - 溴胞嘧啶取代三 螺旋DNA 分子中的胞嘧啶后, 整个分子的稳定性有所降低。同时, 当用5 - 溴胞嘧啶取代三螺旋DNA 不 同链上的胞嘧啶时, 被取代链的碱基堆积能显著增高, 导致分子链内稳定性降低, 但被取代链和相邻链间 相互作用能有所降低, 使得链间作用趋向稳定。

Analysis of the energies of the triple base triplets

Sixty-four sets of three-dimensional models of DNA triplex base triplets (TBT) were built up based on codons by homologous modeling method and their energies were minimized. According to sequence of TBT and orientation of the third ODN strand third, the energies of monomers and water-K+-TBT ternary complexes of TBT were analyzed. The results showed: (i) The energies of the symmetric parallel monomers are generally lower than those of the symmetric anti-parallel monomers of TBT, but the energies of the symmetric parallel ternary complexes are higher than those of the symmetric anti-parallel ternary complexes of TBT. (ii) No matter TBTs are monomers or ternary complexes, the energies of asymmetric parallel TBTs are generally lower than those of the asymmetric anti-parallel ones. (iii) Although the energies of the parallel TBTs are correlated with those of the anti-parallel ones, the energy differences are significant between them. The results here suggest the sequences of TBTs and the orientations of the third ODN strands are two of the key factors that can influence the formation and stability of TBT. (C) 2002 Elsevier Science B.V. All rights reserved.

The comparative analysis of the energies of conjugated triple base triplets and their applications

We built 64 sets of 3D models of DNA triplex base triplets (TBT) and minimized their energies. The TBTs were divided into 32 pairs of conjugated ones on the basis of their sequence characteristic, and the energies of each pair of them were compared and analyzed, the results showed: (i) The duplex DNA of which any strand contains at least a couple of A or T, has a preference for selecting the oligodeoxyribonucleic acid (ODN) strand containing abundant T to form TBT. (ii) The duplex DNA of which any strand contains at least a couple of G or C has a preference for selecting ODN containing abundant G to form symmetric antiparallel TBT, but selecting ODN containing abundant C to form asymmetric parallel TBT. (iii) The duplex DNA of which any strand contains only one of A, T, G or C has a preference for selecting ODN containing abundant pyrimidines (T or C) to form antiparallel TBT. Additionally, two examples of TBTs applications, in designing ODN to form triplex with duplex were presented. The energy calculation result revealed that 15-TCG is the best ligand of the HIV PPT duplex. The comparative analysis of energies of the conjugated TBTs provides directive significance for designing ODN strand that is easy to form triplex in theory. (C) 2002 Elsevier Science B.V. All rights reserved.

PNA(T).DNA(AT) triplexes with Hoogsteen base pairing are more favorable

Peptide nucleic acids (PNAs) are nucleic acid analogs with the deoxyribose phosphate backbone replaced by pseudo-peptide polymers to which the nucleobases are linked. The achiral, uncharged and rather flexible properties of the peptide backbone permit peptide nucleic acids more potential than oligonucleotides in application to antisence and antigenic reagents. The process of PNA binding to DNA duplex and forming triplex is the first step of PNA interacting with PNA. But there are no PNA.2DNA triplex crystal data up to date and little has been reported on the structure features and the force of the PNA.2DNA triplex. In this work, PNA(T).DNA(AT) triplexes are successfully built and the structures and forces to stabilize the triplex after optimizations and molecule dynamics are systematically examined, which are expected to aid in the application of PNAs as anticense and antigene agents.

日本血吸虫三链形成寡核苷酸结合位点的全基因组分析及其应用探讨

血吸虫病是一种广泛分布于亚、非、拉美州的寄生虫病，威胁着成千上万人的健康安全。然而，目前血吸虫病防治面临着一系列的挑战。 “三链形成寡核苷酸（TFO）”能位点特异性地结合双链DNA，已被用于抑制基因的转录表达、介导DNA突变和目标基因的敲除、诱导DNA重组及介导双链DNA的位点特异性切割等。因而该技术对于血吸虫的功能基因组学研究和血吸虫病的防治具有重要的应用前景。由于高亲和性、特异的TFO结合位点是该技术发挥作用的先决条件，因而想要实现该应用，就必须首先了解血吸虫基因组中的“三链形成寡核苷酸结合位点（TTS）”的分布、含量情况以及分析它们作为有效作用靶点的可能性。 本研究通过对日本血吸虫全基因组的搜索与分析，发现该基因组中基因的上游序列（起始密码子到上游1500bp间）较之转录区及整个基因组中的平均水平具有更高的TTS含量，且长TTS含量也更高。并鉴定出共有7576个基因可以在scaffolds数据中提取出基因区（上游序列+基因转录区），其中98%都含有至少1个高亲和性的TTS。在这些基因中鉴定出了5177个只在某一基因中独有的TTS，同时反之也鉴定出了2878个含有至少1个这种独有TTS的基因，其中包括25个编码代谢途径关键酶的基因和231个在人类基因组中没有同源基因且暂无功能注释的基因，这些基因均可能成为药物的作用靶标。此外，还鉴定出了5689个人类基因组中所没有而日本血吸虫具有的TTS，其中有1013个出现于日本血吸虫的基因区。这些TTS有可能为各种基于TFO技术的基因打靶，或直接利用TFO技术设计抗血吸虫药物提供特异的靶位点。因此，本研究揭示出了日本血吸虫基因组中存在着丰富的高亲和性和特异的三螺旋形成位点，提示TFO技术可以作为血吸虫功能基因组学研究的潜在重要手段。同时，该技术也可为血吸虫病的防治提供了一条重要的途径。

固氮植物菌根的普遍性及其联合共生的研究

豆科和非豆科固氮植物不仅能够固定大气中氮素，增加土壤中的氮素营养，而且由于具有较强的抗逆性，是恢复和重建退化农林生态系统的优良先锋树种。但在逆境条件下对固氮植物的固氮能力最经常的限制因子就是磷素供应，而菌根的形成保证了固氮作用对磷素的需求。目前国内外针对固N植物菌根这样联合共生的研究多停留在现象的描述上，对其机理缺乏深入研究。本文针对典型的固氮树种进行联合共生体人工纯培养研究，试图探讨联合共生体之间相互关系及作用机理，为更好地利用固氮植物资源及菌根技术提供理论依据。通过对共生资源的收集、培养及刺槐联合共生体的纯培养研究，得出如下结论：(1)固N植物同时受菌根侵染不仅具有普遍性，更有其生态学意义。在半干旱地区刺槐林下的VA菌主要有摩西球囊霉(Glomus mosseae)、聚丛球囊霉(G. aggregatum)、细凹无梗囊霉(Acaulospora scrobiculata)三种，外生菌根真菌有尖顶地星(Geastrum triplex)、笼头菌(Clathrus sp.)两种，初步证实了刺槐除具根瘤外以同时受VA菌和外生菌根真菌的侵染；在沙棘林下优势种较明显只有一种，即聚丛球囊霉(G. aggregatum)。(2)对外生菌根菌进行了营养条件的选择实验，供试菌种对碳源的利用较为广泛，葡萄糖、果糖为其最适碳源，平均生长量比对照高出4.4倍；供试菌种对有机氮的利用利用机氮，平均生长量比无机氮源高出1.6倍。(3)在人工纯增减条件下，接种的VA菌、外生菌根菌和根瘤菌均成功侵染了刺槐无菌苗，并形成了内生、外生菌根和根瘤，更充分地证实了刺槐与VA菌、外生菌和固N菌之间的联合共生关系。(4)联合共生对刺槐生长、菌根侵染、根瘤菌的结瘤固N能力均有不同程度的促进作用。VA菌根菌中的苏格兰球囊霉(G. caledonium)和外生菌根菌中的毛边华狙伞(H. mesophaseum)是刺槐比较适合的菌根菌；VA菌和外生菌双接种对宿主植物生长有明显的促进作用，混合接种苏格兰球囊霉(G. caledonium) + 毛边华锈伞(H. mesophaseum) + 劣味乳菇(L. insulsus)及混合接种摩西球囊霉(G. mosseae) + 苏格兰球囊霉(G. caledonium) + 毛边华锈伞(H. mesophaseum)的组合效果最好；接种固N菌对菌根菌的影响不明显，而接种菌根菌对刺槐固N能力有明显的促进作用，尤以两种VA菌混接和两种外生菌混接对刺槐根际结瘤量和固N活性的促进作用最为明显；同时接种VA菌、外生菌和固N菌无论是对刺槐生长、结瘤固N还是菌根侵染都有显著的促进作用。其中最佳接种组合是苏格兰球囊霉(G. caledonium) + 毛边华锈伞(H. mesophaseum) + 劣味乳菇(L. insulsus) + 根瘤菌，宿主植物株高、地径、生物量、侧根数分别比对照增加46%、102%、213%、82%，结瘤量、固N活性分别比单接种固N菌增加500%、451%，菌根侵染率高达100%。(5)接种Frankia菌同时配合适度的P肥施用对沙棘的结瘤固N有明显的促进作用。由于沙棘的固N和供N作用，杨树-沙棘混交林中土壤有效态N、P的含量比杨树纯林分别增加36.7%、17.1%。综上所述，本文在刺槐联合共生体人工纯培养实验中得出具有重要意交的结论：从形态解剖上观测到了VA菌、外生菌根菌的侵染特征；定量研究了不同接种方式对刺槐生长、菌根菌的发育状况及结瘤固N能力的影响，选择出了最佳的接种组合。对宿主植物、菌根菌、固N菌之间相互作用及机理进行了初步的探讨。为固N植物联合共生体接种技术提供了理论依据。

Studies of the intermolecular DNA triplexes of C+center dot GC and T center dot AT triplets by electrospray ionization Fourier-transform ion cyclotron resonance mass spectrometry

Formation and stabilities of four 14-mer intermolecular DNA triplexes, consisting of third strands with repeating sequence CTCT, CCTT, CTT, or TTT, were studied by electrospray ionization Fourier-transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) in the gas phase. The gas-phase stabilities of the triplexes were compared with their CD spectra and melting behaviors in solution, and parallel correlation between two phases were obtained. In the presence of 20 mm NH4+ (pH 5.5), the formation of the TTT triplex was not detected in both solution and the gas phase.