Telomerase inhibitors based on quadruplex ligands selected by a fluorescence assay

The reactivation of telomerase activity in most cancer cells supports the concept that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to inhibit telomerase activity. We used a fluorescence assay to identify molecules that stabilize G-quadruplexes. Intramolecular folding of an oligonucleotide with four repeats of the human telomeric sequence into a G-quadruplex structure led to fluorescence excitation energy transfer between a donor (fluorescein) and an acceptor (tetramethylrhodamine) covalently attached to the 5′ and 3′ ends of the oligonucleotide, respectively. The melting of the G-quadruplex was monitored in the presence of putative G-quadruplex-binding molecules by measuring the fluorescence emission of the donor. A series of compounds (pentacyclic crescent-shaped dibenzophenanthroline derivatives) was shown to increase the melting temperature of the G-quadruplex by 2–20°C at 1 μM dye concentration. This increase in Tm value was well correlated with an increase in the efficiency of telomerase inhibition in vitro. The best telomerase inhibitor showed an IC50 value of 28 nM in a standard telomerase repeat amplification protocol assay. Fluorescence energy transfer can thus be used to reveal the formation of four-stranded DNA structures, and its stabilization by quadruplex-binding agents, in an effort to discover new potent telomerase inhibitors.

Break-induced replication: A review and an example in budding yeast

Break-induced replication (BIR) is a nonreciprocal recombination-dependent replication process that is an effective mechanism to repair a broken chromosome. We review key roles played by BIR in maintaining genome integrity, including restarting DNA replication at broken replication forks and maintaining telomeres in the absence of telomerase. Previous studies suggested that gene targeting does not occur by simple crossings-over between ends of the linearized transforming fragment and the target chromosome, but involves extensive new DNA synthesis resembling BIR. We examined gene targeting in Saccharomyces cerevisiae where only one end of the transformed DNA has homology to chromosomal sequences. Linearized, centromere-containing plasmid DNA with the 5′ end of the LEU2 gene at one end was transformed into a strain in which the 5′ end of LEU2 was replaced by ADE1, preventing simple homologous gene replacement to become Leu2+. Ade1+ Leu2+ transformants were recovered in which the entire LEU2 gene and as much as 7 kb of additional sequences were found on the plasmid, joined by microhomologies characteristic of nonhomologous end-joining (NHEJ). In other experiments, cells were transformed with DNA fragments lacking an ARS and homologous to only 50 bp of ADE2 added to the ends of a URA3 gene. Autonomously replicating circles were recovered, containing URA3 and as much as 8 kb of ADE2-adjacent sequences, including a nearby ARS, copied from chromosomal DNA. Thus, the end of a linearized DNA fragment can initiate new DNA synthesis by BIR in which the newly synthesized DNA is displaced and subsequently forms circles by NHEJ.

NOMAD: a versatile strategy for in vitro DNA manipulation applied to promoter analysis and vector design.

Molecular analysis of complex modular structures, such as promoter regions or multi-domain proteins, often requires the creation of families of experimental DNA constructs having altered composition, order, or spacing of individual modules. Generally, creation of every individual construct of such a family uses a specific combination of restriction sites. However, convenient sites are not always available and the alternatives, such as chemical resynthesis of the experimental constructs or engineering of different restriction sites onto the ends of DNA fragments, are costly and time consuming. A general cloning strategy (nucleic acid ordered assembly with directionality, NOMAD; WWW resource locator http:@Lmb1.bios.uic.edu/NOMAD/NOMAD.htm l) is proposed that overcomes these limitations. Use of NOMAD ensures that the production of experimental constructs is no longer the rate-limiting step in applications that require combinatorial rearrangement of DNA fragments. NOMAD manipulates DNA fragments in the form of "modules" having a standardized cohesive end structure. Specially designed "assembly vectors" allow for sequential and directional insertion of any number of modules in an arbitrary predetermined order, using the ability of type IIS restriction enzymes to cut DNA outside of their recognition sequences. Studies of regulatory regions in DNA, such as promoters, replication origins, and RNA processing signals, construction of chimeric proteins, and creation of new cloning vehicles, are among the applications that will benefit from using NOMAD.

Telomeric repeats (TTAGGC)n are sufficient for chromosome capping function in Caenorhabditis elegans.

Telomeres are specialized structures located at the ends of linear eukaryotic chromosomes that ensure their complete replication and protect them from fusion and degradation. We report here the characterization of the telomeres of the nematode Caenorhabditis elegans. We show that the chromosomes terminate in 4-9 kb of tandem repeats of the sequence TTAGGC. Furthermore, we have isolated clones corresponding to 11 of the 12 C. elegans telomeres. Their subtelomeric sequences are all different from each other, demonstrating that the terminal TTAGGC repeats are sufficient for general chromosomal capping functions. Finally, we demonstrate that the me8 meiotic mutant, which is defective in X chromosome crossing over and segregation, bears a terminal deficiency, that was healed by the addition of telomeric repeats, presumably by the activity of a telomerase enzyme. The 11 cloned telomeres represent an important advance for the completion of the physical map and for the determination of the entire sequence of the C. elegans genome.

The multidomain protein Trio binds the LAR transmembrane tyrosine phosphatase, contains a protein kinase domain, and has separate rac-specific and rho-specific guanine nucleotide exchange factor domains.

rho-like GTP binding proteins play an essential role in regulating cell growth and actin polymerization. These molecular switches are positively regulated by guanine nucleotide exchange factors (GEFs) that promote the exchange of GDP for GTP. Using the interaction-trap assay to identify candidate proteins that bind the cytoplasmic region of the LAR transmembrane protein tyrosine phosphatase (PT-Pase), we isolated a cDNA encoding a 2861-amino acid protein termed Trio that contains three enzyme domains: two functional GEF domains and a protein serine/threonine kinase (PSK) domain. One of the Trio GEF domains (Trio GEF-D1) has rac-specific GEF activity, while the other Trio GEF domain (Trio GEF-D2) has rho-specific activity. The C-terminal PSK domain is adjacent to an Ig-like domain and is most similar to calcium/calmodulin-dependent kinases, such as smooth muscle myosin light chain kinase which similarly contains associated Ig-like domains. Near the N terminus, Trio has four spectrin-like repeats that may play a role in intracellular targeting. Northern blot analysis indicates that Trio has a broad tissue distribution. Trio appears to be phosphorylated only on serine residues, suggesting that Trio is not a LAR substrate, but rather that it forms a complex with LAR. As the LAR PTPase localizes to the ends of focal adhesions, we propose that LAR and the Trio GEF/PSK may orchestrate cell-matrix and cytoskeletal rearrangements necessary for cell migration.

Sequence scanning: A method for rapid sequence acquisition from large-fragment DNA clones.

A strategy of "sequence scanning" is proposed for rapid acquisition of sequence from clones such as bacteriophage P1 clones, cosmids, or yeast artificial chromosomes. The approach makes use of a special vector, called LambdaScan, that reliably yields subclones with inserts in the size range 8-12 kb. A number of subclones, typically 96 or 192, are chosen at random, and the ends of the inserts are sequenced using vector-specific primers. Then long-range spectrum PCR is used to order and orient the clones. This combination of shotgun and directed sequencing results in a high-resolution physical map suitable for the identification of coding regions or for comparison of sequence organization among genomes. Computer simulations indicate that, for a target clone of 100 kb, the scanning of 192 subclones with sequencing reads as short as 350 bp results in an approximate ratio of 1:2:1 of regions of double-stranded sequence, single-stranded sequence, and gaps. Longer sequencing reads tip the ratio strongly toward increased double-stranded sequence.

Functional implications for the microtubule-associated protein tau: localization in oligodendrocytes.

We present evidence that the microtubule-associated protein tau is present in oligodendrocytes (OLGs), the central nervous system cells that make myelin. By showing that tau is distributed in a pattern similar to that of myelin basic protein, our results suggest a possible involvement of tau in some aspect of myelination. Tau protein has been identified in OLGs in situ and in vitro. In interfascicular OLGs, tau localization, revealed by monoclonal antibody Tau-5, was confined to the cell somata. However, in cultured ovine OLGs with an exuberant network of processes, tau was detected in cell somata, cellular processes, and membrane expansions at the tips of these processes. Moreover, in such cultures, tau appeared localized adjacent to or coincident with myelin basic protein in membrane expansions along and at the ends of the cellular processes. The presence of tau mRNA was documented using fluorescence in situ hybridization. The distribution of the tau mRNA was similar to that of the tau protein. Western blot analysis of cultured OLGs showed the presence of many tau isoforms. Together, these results demonstrate that tau is a genuine oligodendrocyte protein and pave the way for determining its functional role in these cells.

A structural motif for the voltage-gated potassium channel pore.

Mutation studies have identified a region of the S5-S6 loop of voltage-gated K+ channels (P region) responsible for teraethylammonium (TEA) block and permeation/selectivity properties. We previously modeled a similar region of the Na+ channel as four beta-hairpins with the C strands from each of the domains forming the external vestibule and with charged residues at the beta-turns forming the selectivity filter. However, the K+ channel P region amino acid composition is much more hydrophobic in this area. Here we propose a structural motif for the K+ channel pore based on the following postulates (Kv2.1 numbering). (i) The external TEA binding site is formed by four Tyr-380 residues; P loop residues participating in the internal TEA binding site are four Met-371 and Thr-372 residues. (ii) P regions form extended hairpins with beta-turns in sequence ITMT. (iii) only C ends of hairpins form the inner walls of the pore. (iv) They are extended nonregular strands with backbone carbonyl oxygens of segment VGYGD facing the pore with the conformation BRLRL. (v) Juxtaposition of P loops of the four subunits forms the pore. Fitting the external and internal TEA sites to TEA molecules predicts an hourglass-like pore with the narrowest point (GYG) as wide as 5.5 A, suggesting that selectivity may be achieved by interactions of carbonyls with partially hydrated K+. Other potential cation binding sites also exist in the pore.

Platelet-derived growth factor BB induces functional vascular anastomoses in vivo.

Neovascularization that generates collateral blood flow can limit the extent of tissue damage after acute ischemia caused by occlusion of the primary blood supply. The neovascular response stimulated by the BB homodimeric form of recombinant platelet-derived growth factor (PDGF-BB) was evaluated for its capacity to protect tissue from necrosis in a rat skin flap model of acutely induced ischemia. Complete survival of the tissue ensued, when the original nutritive blood supply was occluded, as early as 5 days after local PDGF-BB application, and the presence of a patent vasculature was evident compared to control flaps. To further evaluate the vascular regenerative response, PDGF-BB was injected into the muscle/connective tissue bed between the separated ends of a divided femoral artery in rats. A patent new vessel that functionally reconnected the ends of the divided artery within the original 3- to 4-mm gap was regenerated 3 weeks later in all PDGF-BB-treated limbs. In contrast, none of the paired control limbs, which received vehicle with an inactive variant of PDGF-BB, had vessel regrowth (P < 0.001). The absence of a sustained inflammatory response and granulation tissue suggests locally delivered PDGF-BB may directly stimulate the angiogenic phenotype in endothelial cells. These findings indicate that PDGF-BB can generate functional new blood vessels and nonsurgically anastomose severed vessels in vivo. This study supports the possibility of a therapeutic modality for the salvage of ischemic tissue through exogenous cytokine-induced vascular reconnection.

Por outras espacialidades: uma cartografia da pedagogização no Parque Ibirapuera, SP

O presente trabalho tem como propósito principal perspectivar analiticamente como os processos de pedagogização forjam e governam determinados espaços e espacialidades. Para tanto, partimos do pressuposto de que as práticas sociais são moduladas por jogos de força que as criam e recriam espacialmente, sobretudo por meio de mecanismos pedagogizantes que ultrapassam o âmbito educacional formal, alastrando-se cada vez mais no cenário urbano contemporâneo. Assim, propomo-nos a analisar a relação entre educação e produção de espaços/espacialidades nas práticas instituídas no Parque Ibirapuera-SP. A investigação tem como marco teórico o pensamento de Michel Foucault, além de mobilizar os trabalhos de Doreen Massey, Edward Soja, Henri Lefebvre, David Harvey e Rodrigo Valverde. A cartografia realizada pleiteia-se, portanto, uma derivação da perspectiva arqueogenealógica foucaultiana. De acordo com tal referencial, assinalaram-se práticas que permitiram dimensionar as transformações pedagógicas e espaciais do Parque desde sua criação, apontando para os modos como atualmente se perfaz o governamento das espacialidades e, em igual medida, as contracondutas aí tornadas possíveis. Além do levantamento bibliográfico, operamos segundo duas frentes de trabalho complementares: em primeiro lugar, a análise de documentos oficiais acerca do Parque, dos Processos da Comissão do IV Centenário da Cidade de São Paulo e de discursos jornalísticos veiculados a seu respeito pelo jornal O Estado de S. Paulo em diferentes décadas; em segundo, a observação e o registro de práticas contemporâneas ali em curso. Os resultados evidenciam uma racionalidade vincada em práticas de pedagogização, e estas, aliadas a tecnologias específicas de governamento dos espaços. Um cenário que apresenta lógicas de governo heterogêneas, que coexistem através do tempo, ajustando-se, desajustando-se ou alterando-se. Não obstante, um campo vivo de forças, de sujeitos e práticas de subjetivação. A história arqueogenealógica do Parque, assim como dos diferentes mecanismos pedagogizantes ali em voga, possibilitou deslindar o trânsito dos espaços e o governo das espacialidades pela população que dele faz uso, além das diferentes contestações dos arranjos até então configurados, demarcando traços fugidios de uma heterotopia urbana.

Struggling, Coping, And Thriving: Sense-Making In Stepfamily Couples' Narratives About Coparenting

The term coparenting implies a bioparental dyad that often excludes the stepparent's role in sharing parenting across joint-custody households. Focusing solely on this dyad also precludes gaining an understanding of how stepfamily couples manage together the communication and sharing of parental responsibilities with the parent(s) in the shared children's other home. In a departure from this bioparental dyad-focused approach, this study locates the stepfamily couple at the center of an inquiry into managing coparenting across households. This mixed methods design study included in-depth interviews of 32 stepfamily couples whose narratives about coparenting were analyzed using grounded theory methods. Forty-one percent of stepparents engage in direct coparenting communication, sometimes manifested as the coactive approach identified in this study. Stepfamily couples also involve the stepparent indirectly in coparenting communication, through the conferred and consultative approaches. As well, the couples' narratives about coparenting identify them as either united, where they share the experience, or divided, where coparenting is reserved exclusively for the bioparent to manage. The stepfamily couples' narratives about significant coparenting experiences revealed that they experience and make sense of coparenting as 1) struggling, 2) coping, or 3) thriving. No significant relationship was found between marital satisfaction and experiencing coparenting as strugglers, copers or thrivers. Grounded theory analysis of these narratives also reflects the four dichotomous dimensions of 1) regard-disregard, 2) decency-duplicity, 3) facilitation-interference, and 4) accommodation-inflexibility. Significant incidents located along these dimensions contribute to the stepfamily couples' identification as struggling, coping, or thriving in coparenting. Experiences on the extreme ends of the dichotomous dimensions generate positive and negative turning points for the coparenting interactions and relationships. As well, experiences on the negative end of the dimensional poles can present challenges for the stepfamily couples. Finally, a synthesis of the findings related to the dichotomous dimensions generates a theory of shared parenting values expectancy.

Instrumentos y prácticas de enseñanza de las ciencias físicas y químicas en la Universidad de Valencia, durante el siglo XIX

This study is an attempt to reconstruct the evolution of spaces and scientific equipments used for the teaching of physics and chemistry in the University of Valencia in the 19th century. Through an analysis of the instruments' characteristics and their spatial distribution, the teaching programs and textbooks, as well as the profile of the target public, we try in this essay to understand the evolution of the didactic methods employed by the successive teachers and assistants when teaching physics and chemistry in the existing cabinets, amphitheatres and laboratories.

L’episcopio del Tolmo de Minateda (Albacete, Spagna). Architettura e funzione degli ambienti tra la fine del VI e l’inizio dell’VIII secolo

The episcopal complex of Eio, located at El Tolmo de Minateda, was built between the end of the sixth century and the beginning of the seventh century. It may have been created as a result of a political decision taken by the authority of the Visigothic kingdom (based at Toletum) to control the Byzantine territories of south-east Hispania. With a comprehensive study of the construction phases, and of the decoration and location of spaces, we can recreate and interpret the function of each space in the episcopal palatium or episcopium, and detail the chronological development of the building. After the Arab-Berber conquest of Hispania in the early eight century, the whole complex underwent alterations that converted the religious and monumental public area into a private, residential and artisan Islamic quarter.

Aportaciones al estudio de las vías de época islámica en la Meseta Norte

Justificación de la importancia histórica del estudio de las vías islámicas de Al-Andalus. Se hacen diversas aportaciones al mapa de los caminos de época andalusí en la Meseta Norte de la Península Ibérica utilizándose esta metodología estructuralista de estudio de los espacios. Al final se enumeran los itinerarios principales de la región, según son descritos por las fuentes árabes.

Postcolonial healing landscapes and mental health in a remote Indigenous community in subarctic Ontario, Canada

The concept of therapeutic landscape is concerned with a holistic, socio-ecological model of health, but most studies have attempted to explore land-health links from a Western perspective. On an Indigenous reserve in Northern Ontario, part of the Canadian subarctic, we explore the importance of spaces and places in creating postcolonial therapeutic landscapes to treat the wounds inflicted by colonialism. The aim of this research is to gain insight from views and experiences of First Nations residents living on reservations that are undergoing a process of regaining traditional spiritual beliefs and teachings to construct therapeutic spaces to face mental health problems caused by legal opioid analgesic abuse. This qualitative study used semi-structured interviews with Cree and Ojibwe participants to understand how they are reconnecting with earth, spirituality and traditional animist beliefs on their way to recovery. We find that practices such as taking part in ceremonies and ritual spaces, and seeking knowledge and advice from Elders assist with personal healing and enable Indigenous people to be physically and mentally healthy. Our research findings provide important insights into the relationship between space, healing and culture as determinants of health and well-being and document some key factors that contribute to substance abuse recovery.