High affinity binding of pyrethroids to the � subunit of brain sodium channels

SUMMARY Na ϩ channels are the primary molecular targets of the pyrethroid insecticides. Na ϩ channels consisting of only a type IIA ␣ subunit expressed in Chinese hamster ovary cells responded to pyrethroid treatment in a normal manner: a sustained Na ϩ current was induced progressively after each depolarizing pulse in a train of stimuli, and this Na ϩ current decayed slowly on repolarization. These modified Na ϩ channels could be reactivated at much more negative membrane potentials (V 0.5 ϭ Ϫ139 mV) than unmodified Na ϩ channels (V 0.5 ϭ Ϫ28 mV). These results indicate that pyrethroids can modify the functional properties of the Na ϩ channel ␣ subunit expressed alone by blocking their inactivation, shifting their voltage dependence of activation, and slowing their deactivation. To demonstrate directly the specific interaction of pyrethroids with the ␣ subunit of voltage-gated Na ϩ channels, a radioactive photosensitive derivative, [ 3 H]RU58487, was used in binding and photolabeling studies. In the presence of a low concentration of the nonionic detergent Triton X-100, specific pyrethroid binding to Na ϩ channels in rat brain membrane preparations could be measured and reached 75% of total binding under optimal conditions. Binding approached equilibrium within 1 hr at 4°, dissociated with a half-time of ϳ10 min, and had K D values of ϳ58 -300 nM for three representative pyrethroids. Specific pyrethroid binding was enhanced by ϳ40% in the presence of 100 nM ␣-scorpion toxin, but no allosteric enhancement was observed in the presence of toxins acting at other Na ϩ channel receptor sites. Extensive membrane washing increased specific binding to 89%. Photolabeling with [ 3 H]RU58487 under these optimal binding conditions revealed a radiolabeled band with an apparent molecular mass of 240 kDa corresponding to the Na ϩ channel ␣ subunit. Anti-peptide antibodies recognizing sequences within the ␣ subunit were able to specifically immunoprecipitate the covalently modified channel. Together, these results demonstrate that the pyrethroids can modify the properties of cells expressing only the ␣ subunit of Na ϩ channels and can bind specifically to a receptor site on the ␣ subunit

Mitoxantrone and Analogues Bind and Stabilize i-Motif Forming DNA Sequences

YesThere are hundreds of ligands which can interact with G-quadruplex DNA, yet very few which target i-motif. To appreciate an understanding between the dynamics between these structures and how they can be affected by intervention with small molecule ligands, more i-motif binding compounds are required. Herein we describe how the drug mitoxantrone can bind, induce folding of and stabilise i-motif forming DNA sequences, even at physiological pH. Additionally, mitoxantrone was found to bind i-motif forming sequences preferentially over double helical DNA. We also describe the stabilisation properties of analogues of mitoxantrone. This offers a new family of ligands with potential for use in experiments into the structure and function of i-motif forming DNA sequences

Human telomeres that contain (CTAGGG)n repeats show replication dependent instability in somatic cells and the male germline

A number of different processes that impact on telomere length dynamics have been identified but factors that affect the turnover of repeats located proximally within the telomeric DNA are poorly defined. We have identified a particular repeat type (CTAGGG) that is associated with an extraordinarily high mutation rate (20% per gamete) in the male germline. The mutation rate is affected by the length and sequence homogeneity of the (CTAGGG)n array. This level of instability was not seen with other sequence-variant repeats, including the TCAGGG repeat type that has the same composition. Telomeres carrying a (CTAGGG)n array are also highly unstable in somatic cells with the mutation process resulting in small gains or losses of repeats that also occasionally result in the deletion of the whole (CTAGGG)n array. These sequences are prone to quadruplex formation in vitro but adopt a different topology from (TTAGGG)n (see accompanying article). Interestingly, short (CTAGGG)2 oligonucleotides induce a DNA damage response (γH2AX foci) as efficiently as (TTAGGG)2 oligos in normal fibroblast cells, suggesting they recruit POT1 from the telomere. Moreover, in vitro assays show that (CTAGGG)n repeats bind POT1 more efficiently than (TTAGGG)n or (TCAGGG)n. We estimate that 7% of human telomeres contain (CTAGGG)n repeats and when present, they create additional problems that probably arise during telomere replication

FASTPCR software for PCR, in silico PCR, and oligonucleotide assembly and analysis

This chapter introduces the software FastPCR as an integrated tools environment for PCR primer and probe design. It also predicts oligonucleotide properties based on experimental studies of PCR efficiency. The software provides comprehensive facilities for designing primers for most PCR applications and their combinations, including standard, multiplex, long-distance, inverse, real-time, group-specific, unique, and overlap extension PCR for multi-fragment assembly in cloning, as well as bisulphite modification assays. It includes a programme to design oligonucleotide sets for long sequence assembly by the ligase chain reaction. The in silico PCR primer or probe search includes comprehensive analyses of individual primers and primer pairs. It calculates the melting temperature for standard and degenerate oligonucleotides including LNA and other modifications, provides analyses for a set of primers with prediction of oligonucleotide properties, dimer and G/C-quadruplex detection, linguistic complexity, and provides a dilution and resuspension calculator. The program includes various bioinformatics tools for analysis of sequences with GC or AT skew, of CG content and purine-pyrimidine skew, and of linguistic sequence complexity. It also permits generation of random DNA sequence and analysis of restriction enzymes of all types. It finds or creates restriction enzyme recognition sites for coding sequences and supports the clustering of sequences. It generates consensus sequences and analyses sequence conservation. It performs efficient and complete detection of various repeat types and displays them. FastPCR allows for sequence file batch processing, which is essential for automation. The FastPCR software is available for download at http://primerdigital.com/fastpcr.html and online version at http://primerdigital.com/tools/pcr.html.Peer reviewe

Le trouble spécifique du langage oral chez les enfants bilingues

Le trouble spécifique du langage oral chez les enfants bilingues De par la mobilité grandissante des populations, la proportion d’enfants grandissant dans un environnement plurilingue augmente en France. En région parisienne par exemple, la proportion d’enfants bilingues est estimée à 25%. Or le développement du langage chez l’enfant bilingue reste encore mal connu, en particulier dans son interaction avec un trouble du langage. Notre recherche s’intéresse aux enfants bilingues successifs, c’est-à-dire apprenant leur seconde langue (L2) vers l’âge de 3-4 ans à l’entrée à l’école maternelle. Dans les premières phases d’acquisition de leur L2, les enfants bilingues présentent des difficultés langagières qui se résorbent avec une exposition suffisante et adéquate à cette langue. Cependant, pour environ 5% de ces enfants, les difficultés dans la L2 vont persister malgré une bonne exposition car ils sont porteurs d’un trouble spécifique du langage (TSL) qui, par définition, touche également l'apprentissage de leur langue maternelle (L1). Le premier axe de notre étude vise à décrire le profil langagier des enfants bilingues dans leur L2: Quelles difficultés d’acquisition du français rencontrent-ils ? Quand leurs difficultés se résorbent-elles ? Comment stimuler au mieux leur langage ? Doit-on stimuler leur L2 au détriment de leur L1? Un deuxième axe de notre étude s’intéresse au langage des enfants bilingues successifs porteurs d’un TSL : Comment se manifeste un TSL dans la L2 d’un enfant bilingue? Un TSL est-il plus sévère chez l’enfant bilingue que chez l’enfant monolingue ? Quels bénéfices l’enfant TSL peut-il tirer de son bilinguisme? Doit-on faire le choix d’une seule langue pour aider l’enfant à développer au mieux son langage? A l’heure actuelle, les réponses à ces questions ne sont pas univoques, et très peu d’études à ce jour ont traité de ces questions auprès d’une population d’enfants grandissant dans un contexte de bilinguisme en France. Nous tenterons d’apporter des éléments de réponses à ces questions en présentant les résultats préliminaires de notre recherche menée à l’université d’Amsterdam et l’université de Paris Descartes en collaboration avec le centre référent des troubles du langage et d'apprentissages de l’hôpital de Garches. Nous avons comparé les capacités lexicales et syntaxiques de 10 enfants monolingues avec TSL (âge moyen : 7 ans 3) et 15 enfants monolingues contrôle sans TSL (âge moyen : 7 ans 2) avec les capacités lexicales et syntaxiques en français de 8 enfants bilingues avec TSL (âge moyen : 7 ans 3) et 15 enfants bilingues contrôles sans TSL (âge moyen : 8 ans) apprenant le français comme L2. Cette étude comparative permet une meilleure compréhension de l’effet du bilinguisme et du TSL sur les capacités langagières. Au moyen d’un questionnaire proposé aux parents d’enfants bilingues, nous avons également recueilli des informations sur la situation de bilinguisme de ces enfants, leur degré d’exposition à leur L1 et L2, la place respective de ces deux langues dans la vie quotidienne, ainsi que la façon dont les parents jugent des compétences langagières de leurs enfants dans leurs deux langues. Ces informations permettent de mieux appréhender la manière dont le bilinguisme est différemment investi par les familles d’enfants avec ou sans TSL et de mieux comprendre quelles peuvent être les inquiétudes des parents d’enfants bilingues auxquelles en tant qu’enseignants ou cliniciens il est important de pouvoir répondre par une guidance appropriée

NMR And Biophysical Studies Of G-Quadruplex DNA Within The KRAS Proto-Oncogene Promoter Region

The role of G-quadruplexes (G4) which can coexist with canonical duplex DNA in the human genome is still largely unknown. Although G4s are found throughout the entire genome, much of the attention have been invested in promotor regions of disease-related genes. The human KRAS proto-oncogene contains a nuclease hypersensitive element (4) located upstream of the major transcription start site. This region regulates transcription of KRAS and it was proposed as a new target for drug development. The knowledge of the detailed structure of this target is crucial for the development of new effective drugs. In this study, we report a high-resolution NMR structure of the G-rich element within the KRAS NHE, and their interaction with small ligands. The G-rich elements forms a parallel structure with three G-quartets connected by a four-nucleotide loop, two one-nucleotide double-chain-reversal loops and a thymine bulge. The loops of different lengths and the presence of a bulge between G-quartets are structural elements that can potentially be targeted by small ligands that would further stabilize the structure. In addition, we explore the polymorphism of G-quadruplexes structures within the promotor region. Consequently, our work suggests an alternative route for the development of anticancer agents that could regulate KRAS expression