Effect on DNA relaxation of the single Thr718Ala mutation in human topoisomerase I: a functional and molecular dynamics study

The functional and dynamical properties of the human topoisomerase I Thr718Ala mutant have been compared to that of the wild-type enzyme using functional assays and molecular dynamics (MD) simulations. At physiological ionic strength, the cleavage and religation rates, evaluated on oligonucleotides containing the preferred topoisomerase I DNA sequence, are almost identical for the wild-type and the mutated enzymes, as is the cleavage/religation equilibrium. On the other hand, the Thr718Ala mutant shows a decreased efficiency in a DNA plasmid relaxation assay. The MD simulation, carried out on the enzyme complexed with its preferred DNA substrate, indicates that the mutant has a different dynamic behavior compared to the wild-type enzyme. Interestingly, no changes are observed in the proximity of the mutation site, whilst a different flexibility is detected in regions contacting the DNA scissile strand, such as the linker and the V-shaped α helices. Taken together, the functional and simulation results indicate a direct communication between the mutation site and regions located relatively far away, such as the linker domain, that with their altered flexibility confer a reduced DNA relaxation efficiency. These results provide evidence that the comprehension of the topoisomerase I dynamical properties are an important element in the understanding of its complex catalytic cycle

Transparent conductive oxide-based architectures for the electrical modulation of the optical response: A spectroscopic ellipsometry study

Transparent conductive oxides are a class of materials that combine high optical transparency with high electrical conductivity. This property makes them uniquely appealing as transparent conductive electrodes in solar cells and interesting for optoelectronic and infrared-plasmonic applications. One of the new challenges that researchers and engineers are facing is merging optical and electrical control in a single device for developing next-generation photovoltaic, optoelectronic devices and energy-efficient solid-state lighting. In this work, the authors investigated the possible variations in the dielectric properties of aluminum-doped ZnO (AZO) upon gating by means of spectroscopic ellipsometry (SE). The authors investigated the electrical-bias-dependent optical response of thin AZO films fabricated by magnetron sputtering within a parallel-plane capacitor configuration. The authors address the possibility to control their optical and electric performances by applying bias, monitoring the effect of charge injection/depletion in the AZO layer by means of in operando SE versus applied gate voltage

Molecular cloning and characterization of a phytochelatin synthase gene, PvPCS1, from Pteris vittata L.

Pteris vittata L. is a staggeringly efficient arsenic hyperaccumulator that has been shown to be capable of accumulating up to 23,000 microg arsenic g(-1), and thus represents a species that may fully exploit the adaptive potential of plants to toxic metals. However, the molecular mechanisms of adaptation to toxic metal tolerance and hyperaccumulation remain unknown, and P. vittata genes related to metal detoxification have not yet been identified. Here, we report the isolation of a full-length cDNA sequence encoding a phytochelatin synthase (PCS) from P. vittata. The cDNA, designated PvPCS1, predicts a protein of 512 amino acids with a molecular weight of 56.9 kDa. Homology analysis of the PvPCS1 nucleotide sequence revealed that it has low identity with most known plant PCS genes except AyPCS1, and the homology is largely confined to two highly conserved regions near the 5'-end, where the similarity is as high as 85-95%. The amino acid sequence of PvPCS1 contains two Cys-Cys motifs and 12 single Cys, only 4 of which (Cys-56, Cys-90/91, and Cys-109) in the N-terminal half of the protein are conserved in other known PCS polypeptides. When expressed in Saccharomyces cerevisae, PvPCS1 mediated increased Cd tolerance. Cloning of the PCS gene from an arsenic hyperaccumulator may provide information that will help further our understanding of the genetic basis underlying toxic metal tolerance and hyperaccumulation

Increased camptothecin toxicity induced in mammalian cells expressing Saccharomyces cerevisiae DNA topoisomerase I

The yeast Saccharomyces cerevisiae has been useful in establishing the phenotypic effects of specific mutations on the enzymatic activity and camptothecin sensitivity of yeast and human DNA topoisomerase I. To determine whether these phenotypes were faithfully reiterated in higher eukaryotic cells, wild-type and mutant yeast Top1 proteins were epitope-tagged at the amino terminus and transiently overexpressed in mammalian COS cells. Camptothecin preferentially induced apoptosis in cells expressing wild-type eScTop1p yet did not appreciably increase the cytotoxic response of cells expressing a catalytically inactive (eSctop1Y727F) or a catalytically active, camptothecin-resistant eSctop1vac mutant. Using an epitope-specific antibody, immobilized precipitates of eScTop1p were active in DNA relaxation assays, whereas immunoprecipitates of eScTop1Y727Fp were not. Thus, the enzyme retained catalytic activity while tethered to a support. Interestingly, the mutant eSctop1T722A, which mimics camptothecin-induced cytotoxicity in yeast through stabilization of the covalent enzyme-DNA intermediate, induced apoptosis in COS cells in the absence of camptothecin. This correlated with increased DNA cleavage in immunoprecipitates of eScTop1T722Ap, in the absence of the drug. The observation that the phenotypic consequences of expressing wild-type and mutant yeast enzymes were reiterated in mammalian cells suggests that the mechanisms underlying cellular responses to DNA topoisomerase I-mediated DNA damage are conserved between yeast and mammalian cells

Severe bleeding from esophageal varices resistant to endoscopic treatment in a non cirrhotic patient with portal hypertension

A non cirrhotic patient with esophageal varices and portal vein thrombosis had recurrent variceal bleeding unsuccessfully controlled by endoscopy and esophageal transection. Emergency transhepatic portography confirmed the thrombosed right branch of the portal vein, while the left branch appeared angulated, shifted and stenotic. A stent was successfully implanted into the left branch and the collateral vessels along the epatoduodenal ligament disappeared. In patients with esophageal variceal hemorrhage and portal thrombosis if endoscopy fails, emergency esophageal transection or nonselective portocaval shunting are indicated. The rare patients with only partial portal thrombosis can be treated directly with stenting through an angioradiologic approach

Safe use of human anatomical preparations in frontal and interactive teaching

In the institute of Human Anatomy of Pavia, the use of cadaver dissection is not economically feasible. In order to improve students’ preparation related to topography of the central nervous system, we decided to use formalin-fixed brains and cranial sections belonging to the collection of cadaveric specimens. These specimens, preserved in formalin, however cannot be manipulated as such by the students because formalin can cause headaches, burning sensation in the throat, difficult breathing and can trigger or aggravate asthma symptoms [1, 2]. Furthermore, formalin is known to be a human carcinogen [3]. In order to minimize toxic effects, whole brains were extensively washed in running water and then sliced according to different reference planes using a “home-made” device enabling cuts according to parallel planes. Finally, the resulting sections were inserted into transparent plastic envelopes, immerged in a solution composed by 0.5% agar and 1% sodium azide as preservative. Medical students can now use these human brain sections to test their own ability to recognize nervous system structures. This strategy optimize specimen’s choice and focalize student’s attention on peculiar, selected human samples in full compliance with current security laws

The deubiquitinating enzyme Doa4p protects cells from DNA topoisomerase I poisons

DNA topoisomerase I (Top1p) catalyzes changes in DNA topology via the formation of an enzyme-DNA covalent complex that is reversibly stabilized by the antitumor drug, camptothecin (CPT). During S-phase, collisions with replication forks convert these complexes into cytotoxic DNA lesions that trigger cell cycle arrest and cell death. To investigate cellular responses to CPT-induced DNA damage, a yeast genetic screen identified conditional tah mutants with enhanced sensitivity to self-poisoning DNA topoisomerase I mutant (Top1T722Ap), which mimics the action of CPT. Mutant alleles of three genes, DOA4, SLA1 and SLA2, were recovered. A nonsense mutation in DOA4 eliminated the catalytic residues of the Doa4p deubiquitinating enzyme, yet retained the rhodanase domain. At 36 degrees C, this doa4-10 mutant exhibited increased sensitivity to CPT, osmotic stress, and hydroxyurea, and a reversible petite phenotype. However, the accumulation of pre-vacuolar class E vesicles that was observed in doa4Delta cells was not detected in the doa4-10 mutant. Mutations in SLA1 or SLA2, which alter actin cytoskeleton architecture, induced a conditional synthetic lethal phenotype in combination with doa4-10 in the absence of DNA damage. Here actin cytoskeleton defects coincided with the enhanced fragility of large-budded cells. In contrast, the enhanced sensitivity of doa4-10 mutant cells to Top1T722Ap was unrelated to alterations in endocytosis and was selectively suppressed by increased dosage of the ribonucleotide reductase inhibitor Sml1p. Additional studies suggest a role for Doa4p in the Rad9p checkpoint response to Top1p poisons. These findings indicate a functional link between ubiquitin-mediated proteolysis and cellular resistance to CPT-induced DNA damage

A single mutation in the 729 residue modulates human DNA topoisomerase IB DNA binding and drug resistance

Human DNA topoisomerase I (hTop1p) catalyzes the relaxation of supercoiled DNA and constitutes the cellular target of the antitumor drug camptothecin (CPT). The X-ray crystal structure of the enzyme covalently joined to DNA and bound to the CPT analog Topotecan suggests that there are two classes of mutations that can produce a CPT-resistant enzyme. The first class includes changes in residues that directly interact with the drug, whereas a second class alters interactions with the DNA and thereby destabilizes the drug binding site. The Thr729Ala, that is part of a hydrophobic pocket in the enzyme C-terminal domain, belongs to a third group of mutations that confer CPT resistance, but do not interact directly with the drug or the DNA. To understand the contribution of this residue in drug resistance, we have studied the effect on hTop1p catalysis and CPT sensitivity of four different substitutions in the Thr729 position (Thr729Ala, Thr729Glu, Thr729Lys and Thr729Pro). Tht729Glu and Thr729Lys mutants show severe CPT resistance and furthermore, Thr729Glu shows a remarkable defect in DNA binding. We postulate that the maintenance of the hydrophobic pocket integrity, where Thr729 is positioned, is crucial for drug sensitivity and DNA binding

Planeamiento estratégico de la escuela naval del Perú

La Escuela Naval del Perú es el alma mater de la oficialidad de la Marina de Guerra del Perú, en ella se forman bajo altos estándares académicos, físicos y morales, los futuros oficiales que dotaran las unidades y dependencias. La importancia que esta dependencia tiene es fundamental debido a que en ella se imparten no solo los conocimientos teóricos y prácticos correspondientes a la formación académica del futuro oficial de marino, sino que es en sus ambientes en donde el candidato a oficial absorbe la mística y tradición que caracterizan al marino. El éxito del proceso formativo redundará de manera directa en un mejor desempeño del integrante de la institución y por ende de la institución en sí. Actualmente producto de limitaciones presupuestales y circunstancias exógenas del país, el proceso de captación ha sufrido disminución en calidad y cantidad habiendo sido esta realidad reflejada en la disminución, en número de integrantes de las promociones que egresan así como en nuevos y más problemas disciplinarios. El desarrollo del presente Plan Estratégico busca efectuar un análisis de las actuales fortalezas, oportunidades, debilidades y amenazas, con la finalidad de proponer estrategias para revertir las actuales limitaciones en aspectos de convocatoria y captación, con la finalidad de mejorar el producto entregado por la Escuela Naval, lo que implicara a su vez en la disminución de situaciones disciplinarias no deseadas. Finalmente, el presente trabajo pone a disposición del Alto Mando de la Marina de Guerra del Perú, estrategias orientadas a mejorar las condiciones de captación y de presentación del producto Escuela Naval del Perú buscando que sea más demandado por el público de interés.Peru Navy School is the alma mater of the officers of the Navy of Peru, there is formed under high academic standards, physical and moral, that will provide the future officers for the units and departments. The importance that this unit has is critical because it not only taught the knowledge and skills relevant to the academic training of future marine officer, because it is in their environments where the officer candidate absorbs mystical tradition that characterizes the sailors. The success of the training process directly results in improved performance of the member of the institution and therefore the institution itself. Currently product exogenous budget constraints and circumstances of the country, the recruitment process has been declining in quality and quantity having been this reality reflected in the decrease in number of members who graduate promotions as well as new and more disciplinary problems. The development of this Strategic Plan seeks to make an analysis of the current strengths, weaknesses, opportunities and threats, in order to propose strategies to reverse the current limitations in aspects of marketing and income, in order to improve the product delivered by the Navy School, which in turn involve decreasing unwanted disciplinary situations. Finally, this paper provides the High Command of the Navy of Peru, strategies to improve the uptake and product presentation Peru Naval School looking to be demanded by the public interest.Tesi