RNA polymerase II large subunit degradation induced by ecteinascidin 743: Molecular characterization and subsequent rational investigation of antitumor mechanisms in cancers with clinical response

Ecteinascidin 743 (Et-743), which is a novel DNA minor groove alkylator with a unique spectrum of antitumor activity, is currently being evaluated in phase II/III clinical trials. Although the precise molecular mechanisms responsible for the observed antitumor activity are poorly understood, recent data suggests that post-translational modifications of RNA polymerase II Large Subunit (RNAPII LS) may play a central role in the cellular response to this promising anticancer agent. The stalling of an actively transcribing RNAPII LS at Et-743-DNA adducts is the initial cellular signal for transcription-coupled nucleotide excision repair (TC-NER). In this manner, Et-743 poisons TC-NER and produces DNA single strand breaks. Et-743 also inhibits the transcription and RNAPII LS-mediated expression of selected genes. Because the poisoning of TC-NER and transcription inhibition are critical components of the molecular response to Et-743 treatment, we have investigated if changes in RNAPII LS contribute to the disruption of these two cellular pathways. In addition, we have studied changes in RNAPII LS in two tumors for which clinical responses were reported in phase I/II clinical trials: renal cell carcinoma and Ewing's sarcoma. Our results demonstrate that Et-743 induces degradation of the RNAPII LS that is dependent on active transcription, a functional 26S proteasome, and requires functional TC-NER, but not global genome repair. Additionally, we have provided the first experimental data indicating that degradation of RNAPII LS might lead to the inhibition of activated gene transcription. A set of studies performed in isogenic renal carcinoma cells deficient in von Hippel-Lindau protein, which is a ubiquitin-E3-ligase for RNAPII LS, confirmed the central role of RNAPII LS degradation in the sensitivity to Et-743. Finally, we have shown that RNAPII LS is also degraded in Ewing's sarcoma tumors following Et-743 treatment and provide data to suggest that this event plays a role in decreased expression of the Ewing's sarcoma oncoprotein, EWS-Fli1. Altogether, these data implicate degradation of RNAPII LS as a critical event following Et-743 exposure and suggest that the clinical activity observed in renal carcinoma and Ewing's sarcoma may be mediated by disruption of molecular pathways requiring a fully functional RNAPII LS. ^

Cleavage of the antithrombin III binding site in heparin by heparinases and its implication in the generation of low molecular weight heparin

Heparin has been used as a clinical anticoagulant for more than 50 years, making it one of the most effective pharmacological agents known. Much of heparin's activity can be traced to its ability to bind antithrombin III (AT-III). Low molecular weight heparin (LMWH), derived from heparin by its controlled breakdown, maintains much of the antithrombotic activity of heparin without many of the serious side effects. The clinical significance of LMWH has highlighted the need to understand and develop chemical or enzymatic means to generate it. The primary enzymatic tools used for the production of LMWH are the heparinases from Flavobacterium heparinum, specifically heparinases I and II. Using pentasaccharide and hexasaccharide model compounds, we show that heparinases I and II, but not heparinase III, cleave the AT-III binding site, leaving only a partially intact site. Furthermore, we show herein that glucosamine 3-O sulfation at the reducing end of a glycosidic linkage imparts resistance to heparinase I, II, and III cleavage. Finally, we examine the biological and pharmacological consequences of a heparin oligosaccharide that contains only a partial AT-III binding site. We show that such an oligosaccharide lacks some of the functional attributes of heparin- and heparan sulfate-like glycosaminoglycans containing an intact AT-III site.

Sequences upstream of the branch site are required to form helix II between U2 and U6 snRNA in a trans-splicing reaction

Three different base paired stems form between U2 and U6 snRNA over the course of the mRNA splicing reaction (helices I, II and III). One possible function of U2/U6 helix II is to facilitate subsequent U2/U6 helix I and III interactions, which participate directly in catalysis. Using an in vitro trans-splicing assay, we investigated the function of sequences located just upstream from the branch site (BS). We find that these upstream sequences are essential for stable binding of U2 to the branch region, and for U2/U6 helix II formation, but not for initial U2/BS pairing. We also show that non-functional upstream sequences cause U2 snRNA stem–loop IIa to be exposed to dimethylsulfate modification, perhaps reflecting a U2 snRNA conformational change and/or loss of SF3b proteins. Our data suggest that initial binding of U2 snRNP to the BS region must be stabilized by an interaction with upstream sequences before U2/U6 helix II can form or U2 stem–loop IIa can participate in spliceosome assembly.

RNA polymerase II transcription initiation: A structural view

In eukaryotes, RNA polymerase II transcribes messenger RNAs and several small nuclear RNAs. Like RNA polymerases I and III, polymerase II cannot act alone. Instead, general initiation factors [transcription factor (TF) IIB, TFIID, TFIIE, TFIIF, and TFIIH] assemble on promoter DNA with polymerase II, creating a large multiprotein–DNA complex that supports accurate initiation. Another group of accessory factors, transcriptional activators and coactivators, regulate the rate of RNA synthesis from each gene in response to various developmental and environmental signals. Our current knowledge of this complex macromolecular machinery is reviewed in detail, with particular emphasis on insights gained from structural studies of transcription factors.

Elongation factor TFIIS contains three structural domains: solution structure of domain II.

Transcription elongation by RNA polymerase II is regulated by the general elongation factor TFIIS. This factor stimulates RNA polymerase II to transcribe through regions of DNA that promote the formation of stalled ternary complexes. Limited proteolytic digestion showed that yeast TFIIS is composed of three structural domains, termed I, II, and III. The two C-terminal domains (II and III) are required for transcription activity. The structure of domain III has been solved previously by using NMR spectroscopy. Here, we report the NMR-derived structure of domain II: a three-helix bundle built around a hydrophobic core composed largely of three tyrosines protruding from one face of the C-terminal helix. The arrangement of known inactivating mutations of TFIIS suggests that two surfaces of domain II are critical for transcription activity.

Cuerdas y no conmutatividad

Los conceptos geométricos clásicos que usan las ecuaciones de Einstein para describir el espacio, el tiempo y la gravedad no son compatibles con los principios de la mecánica cuántica. A distancias muy cortas, cercanas o por debajo de la longitud de Planck lp 10−36m, se espera que la estructura del espacio-tiempo se haga difusa, con un principio de incertidumbre asociado a las propias coordenadas espacio temporales. Tal principio de incertidumbre podría derivarse de relaciones de conmutación no triviales [xμ, x ] 6= 0 entre operadores asociados a la posición en el espacio-tiempo. Conmutadores de este tipo aparecen de manera natural en teoría de cuerdas, que, por otra parte, contiene y generaliza las ecuaciones de Einstein. Constituye por ello un escenario idóneo para el estudio de la naturaleza no determinista del espacio-tiempo. En efecto, Chu y Ho [4] demostraron que la cuantización canónica de la cuerda abierta en espacio-tiempo de Minkowski con 2-forma B y dilatón constantes conduce a conmutadores no triviales entre los operadores de posición de los extremos de la cuerda. Este hecho sugiere la interpreción de la D-brana sobre la que pueden moverse dichos extremos como un espacio no conmutativo. Seiberg y Witten [5] dieron un paso más y encontraron un límite de baja energía bien definido en el cual la dinámica de los extremos de la cuerda se desacopla de la de los modos internos y se describe como una teoría de Yang-Mills no conmutativa sobre la D-brana. Es díficil trasladar estos resultados a backgrounds más generales para la cuerda. Los modelos de Wess-Zumino-Witten (WZW) [6] constituyen los backgrounds no triviales mejor conocidos, pero no se conoce una caracterización completa de las D-branas en estos modelos. El objetivo de esta tesis es mejorar la comprensión del origen de no conmutatividad a partir de (i) el estudio de la cuerda abierta en backgrounds no triviales y (ii) la caracterización de las D-branas sobre las que pueden moverse sus extremos. El trabajo de esta tesis ha dado lugar a las publicaciones [I], [II] y [III]...

Luxações acromioclaviculares grau III : conceitos

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Pañchatantra I.[-V.] ...

In Sanskrit; t.p. and notes in English.

Novisima recopilación de las Leyes de España : dividida en XII libros : en que se reforma la Recopilación publicada por el Señor Don Felipe II en el año de 1567, reimpresa últimamente en el de 1775, y se incorporan las pragmáticas, cédulas, decretos, órdenes y resoluciones Reales, y otras providencias no recopiladas, y expedidas hasta el de 1804 /

Indices.

Sochinenīi͡a. 1860-1886. Moskva, Tip. M. G. Volchaninova, 1886-1887.

Photocopy (on double leaves)

Gorodskīi︠a︡ poselenīi︠a︡ v Rossīĭskoĭ Imperīi.

Vols. 3-<4, 5, pt. 1> have imprint: S.-Peterburg : V Tip. K. Vulʹfa.

Sochinenīi͡a︡.

Contents.--I. Neslykhannoe divo, ili chestnoĭ sekretarʹ; komedīi͡a︡.--II. Opyt iskusstva, komedīi͡a︡.--III. Tri brata-chudaka.

Una nota a Tucídides I 22, 2

Este trabajo estudia las distintas interpretaciones del inicio del capítulo 22 centrándose en la segunda frase. Se propone una nueva interpretación basada en la construcción sintáctica del participio πυνθανόμενος.

Petrus Hispanus O.P. Auctor Summularum (III) ¿”Petrus Alfonsi” o “Petrus Ferrandi”?

Este artículo pretende ser complemento y continuación de mis anteriores trabajos sobre la figura de Petrus Hispanus O. P., Auctor Summularum. Comienzo presentando algunos nuevos documentos relacionados con las cuestiones ya examinadas en mis artículos de 1997 y 2001. A continuación, me ocupo de las cuestiones aplazadas en el artículo de 2001: los problemas relativos a la figura de “Petrus Ferrandi” y su posible relación con el “auctor Summularum”, así como los argumentos de Tugwell contra la hipótesis de la posible identidad de estas dos figuras, examinados ahora desde la perspectiva del autor de la Legenda prima. Tras analizar testimonios procedentes de muy diversos ámbitos, afirmo, por una parte, que la hipótesis de la identidad entre “Petrus Ferrandi” y “Petrus Hispanus” podría ser correcta y, por otra parte, que no hay argumentos concluyentes que obliguen a afirmar con seguridad que el autor de la Legenda prima es Pedro Ferrando. Aunque los análisis no permiten por el momento determinar si es “Petrus Alfonsi” o “Petrus Ferrandi” el “auctor Summularum”, los testimonios recogidos y las conexiones establecidas contribuirán, sin duda, a orientar futuras investigaciones en torno a la figura de “Petrus Hispanus”.

Mutation in NDUFA13/GRIM19 leads to early onset hypotonia, dyskinesia and sensorial deficiencies, and mitochondrial complex I instability

International audience