Structural basis of nuclear import of flap endonuclease 1 (FEN1)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Biophysical Characterization of Interactions Involving Importin-α during Nuclear Import

Proteins containing the classical nuclear localization sequences (NLSs) are imported into the nucleus by the importin-α/β heterodimer. Importin-α contains the NLS binding site, whereas importin-β mediates the translocation through the nuclear pore. We characterized the interactions involving importin-α during nuclear import using a combination of biophysical techniques (biosensor, crystallography, sedimentation equilibrium, electrophoresis, and circular dichroism). Importin-α is shown to exist in a monomeric autoinhibited state (association with NLSs undetectable by biosensor). Association with importin-β (stoichiometry, 1:1; K D = 1.1 × 10 -8 M) increases the affinity for NLSs; the importin-α/β complex binds representative monopartite NLS (simian virus 40 large T-antigen) and bipartite NLS (nucleoplasmin) with affinities (K D = 3.5 × 10 -8 M and 4.8 × 10 -8 M, respectively) comparable with those of a truncated importin-α lacking the autoinhibitory domain (T-antigen NLS, K D = 1.7 × 10 -8 M; nucleoplasmin NLS, K D = 1.4 × 10 -8 M). The autoinhibitory domain (as a separate peptide) binds the truncated importin-α, and the crystal structure of the complex resembles the structure of full-length importin-α. Our results support the model of regulation of nuclear import mediated by the intrasteric autoregulatory sequence of importin-α and provide a quantitative description of the binding and regulatory steps during nuclear import.

Industrialization in Sub-Saharan Africa and import substitution policy

This article aims to contribute to the understanding of the process of import substitution in Sub-Saharan Africa. The process of industrialization in Sub-Saharan Africa occurred in two phases: a first step, even very early during the colonial regime began around the 1920s and ended in the late forties; a second phase of industrialization began in the late fifties and gained momentum in the sixties, when import substitution was implemented more widely. Although these countries were the last to embark on the strategy of import substitution, they followed the same steps of Latin American countries, and as the structural domestic and external constraints were too strong, the failure of the policy of import substitution arrived early and the negative impact on these economies had a greater magnitude.

Multinationals and Exports in a Large and Protected Developing Country

This paper examines an industry-level model developed to analyze the impact of affiliates of multinational firms (MNFs) on the host country's revealed comparative advantages (RCAs), which predicts that the referred impact is given by both technology service and industry orientation. Based on Brazilian manufacturing industries during the import-substitution industrialization, panel data estimates show that MNFs negatively affected RCA, which is explained by location advantages in industries presenting comparative disadvantages, as reinforced by a location model. Two other important results are: (i) import protection had a stronger anti-export effect on multinationals than on national firms; (ii) MNFs were concentrated in industries with lower world-export growth.

Crystallization and preliminary X-ray diffraction analysis of importin-alpha acomplexed with NLS peptidomimetics

Importin-alpha is the nuclear import receptor that recognizes cargo proteins with nuclear localization sequences (NLSs). Tile study of NLS peptidomimetics can provide a better understanding of the requirements for the molecular recognition of cargo proteins by importin-alpha, and potentially engender a large number of applications in medicine. Importin-a was crystallized with a set of six NLS peptidomimetics, and X-ray diffraction data were collected in the range 2.1-2.5 angstrom resolution. Preliminary electron density calculations show that the ligands are present in the crystals. (c) 2005 Elsevier B.V All rights reserved.

Role of flanking sequences and phosphorylation in the recognition of the simian-virus-40 large T-antigen nuclear localization sequences by importin-alpha

The nuclear import of simian-virus-40 large T-antigen (tumour antigen) is enhanced via phosphorylation by the protein kinase CK2 at Ser(112) in the vicinity of the NLS (nuclear localization sequence). To determine the structural basis of the effect of the sequences flanking the basic cluster KKKRK, and the effect of phosphorylation on the recognition of the NLS by the nuclear import factor importin-alpha (Impalpha), we co-crystallized non-autoinhibited Impalpha with peptides corresponding to the phosphorylated and non-phosphorylated forms of the NLS, and determined the crystal structures of the complexes. The structures show that the amino acids N-terminally flanking the basic cluster make specific contacts with the receptor that are distinct from the interactions between bipartite NLSs and Impalpha. We confirm the important role of flanking sequences using binding assays. Unexpectedly, the regions of the peptides containing the phosphorylation site do not make specific contacts with the receptor. Binding assays confirm that phosphorylation does not increase the affinity of the T-antigen NLS to Impalpha. We conclude that the sequences flanking the basic clusters in NLSs play a crucial role in nuclear import by modulating the recognition of the NLS by Impalpha, whereas phosphorylation of the T-antigen enhances nuclear import by a mechanism that does not involve a direct interaction of the phosphorylated residue with Impalpha.

Structural basis for the specificity of bipartite nuclear localization sequence binding by importin-alpha

Importin-alpha is the nuclear import receptor that recognizes cargo proteins carrying conventional basic monopartite and bipartite nuclear localization sequences (NLSs) and facilitates their transport into the nucleus. Bipartite NLSs contain two clusters of basic residues, connected by linkers of variable lengths. To determine the structural basis of the recognition of diverse bipartite NLSs by mammalian importin-alpha, we co-crystallized a non-autoinhibited mouse receptor protein with peptides corresponding to the NLSs from human retinoblastoma protein and Xenopus laevis phosphoprotein N1N2, containing diverse sequences and lengths of the linker. We show that the basic clusters interact analogously in both NLSs, but the linker sequences adopt different conformations, whereas both make specific contacts with the receptor. The available data allow us to draw general conclusions about the specificity of NLS binding by importin-alpha and facilitate an improved definition of the consensus sequence of a conventional basic/bipartite NLS (KRX10-12KRRK) that can be used to identify novel nuclear proteins.

Probing the Specificity of Binding to the Major Nuclear Localization Sequence-binding Site of Importin-alpha Using Oriented Peptide Library Screening

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Structural Basis of Importin-alpha-Mediated Nuclear Transport for Ku70 and Ku80

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Pesquisa educacional com base nas artes: pensando a educação dos professores como experiência estética

Neste artigo, o autor descreve dois estudos, realizados com professores, nos quais objetos de arte são usados como dispositivos deflagradores de reflexão compartilhada. Teoricamente fundamentado em uma nova modalidade de investigação qualitativa no campo da Educação - a Pesquisa Educacional com Base nas Artes (PEBA) -, ele discute particularidades do funcionamento e do papel dessa modalidade de pesquisa no desenvolvimento profissional docente, as diferentes naturezas dos dois objetos de arte utilizados (a fotografia e o espetáculo teatral) e aponta para duas principais vertentes dessa modalidade de pesquisa - a vertente de produção de significados, pela qual o educador de professores e os participantes da pesquisa compartilham e constroem significados ao entrarem em contato com um objeto de arte previamente pronto e confeccionado por um artista profissional; e a vertente representacional, pela qual os professores e educadores participantes constroem, individualmente ou de forma compartilhada, um determinado objeto de arte que reflita e expresse suas representações do mundo da docência. Por fim, o artigo sugere que a PEBA, além de estabelecer contextos reflexivos nos quais alunos e professores têm oportunidades de desvelar a experiência estética, instaura relações alternativas dos participantes com o conhecimento e com a prática pedagógica, evidenciando sua importância social e suas forças revitalizadoras.

Relações entre pesquisa em ensino de Ciências e formação de professores: algumas representações

Este estudo foi realizado com o objetivo de investigar representações de pesquisadores da área de ensino de Física sobre a possível interferência da pesquisa da área de ensino de Ciências na maneira como se formam professores no Brasil. Para tal, foram analisadas as respostas a uma questão formulada em entrevistas a treze pesquisadores do ensino da Física, indicados por seus pares por meio de correio eletrônico encaminhado a pesquisadores do ensino de Ciências, incluindo as subáreas de Biologia, Física, Geociências e Química. A análise de discurso desenvolvida na França por Michel Pêcheux, a partir de subsídios obtidos principalmente em publicações de Eni Orlandi no Brasil, foi o apoio teórico que sustentou o estudo. As representações que os discursos selecionados permitem inferir evidenciam pequena influência. Entretanto, foram enunciados fatores de grande abrangência, incluindo desde aqueles internos à própria área, quanto fatores associados a políticas públicas. Também se observou uma diversidade de posições entre os pesquisadores entrevistados. No conjunto, os fatores abordados pelos entrevistados constituem um amplo quadro configurativo de representações que pode ser elemento de reflexão para outros pesquisadores. Acreditamos que essas representações têm potencial de contribuição para efetivas interferências na forma de realização de políticas públicas, sem a necessidade do uso de prescrições e cada vez menos com recomendações vazias.

Tensor3D: A computer graphics program to simulate 3D real-time deformation and visualization of geometric bodies

Tensor3D is a geometric modeling program with the capacity to simulate and visualize in real-time the deformation, specified through a tensor matrix and applied to triangulated models representing geological bodies. 3D visualization allows the study of deformational processes that are traditionally conducted in 2D, such as simple and pure shears. Besides geometric objects that are immediately available in the program window, the program can read other models from disk, thus being able to import objects created with different open-source or proprietary programs. A strain ellipsoid and a bounding box are simultaneously shown and instantly deformed with the main object. The principal axes of strain are visualized as well to provide graphical information about the orientation of the tensor's normal components. The deformed models can also be saved, retrieved later and deformed again, in order to study different steps of progressive strain, or to make this data available to other programs. The shape of stress ellipsoids and the corresponding Mohr circles defined by any stress tensor can also be represented. The application was written using the Visualization ToolKit, a powerful scientific visualization library in the public domain. This development choice, allied to the use of the Tcl/Tk programming language, which is independent on the host computational platform, makes the program a useful tool for the study of geometric deformations directly in three dimensions in teaching as well as research activities. (C) 2007 Elsevier Ltd. All rights reserved.

Preparation of N2, N2,7-trimethylguanosine affinity columns

2,2,7-trimethylguanosine (TMG) binding proteins from human cells were purified through TMG-affinity columns. TMG synthesis was improved and the TMG obtained was shown to be similar to the TMG in the 5' cap of the UsnRNAs. The eluates obtained with TMG-affinity chromatographies were very different from those isolated with m7G-affinity columns, thus suggesting that specific TMG- binding proteins were obtained. The fraction may be enriched with factors associated with import and/or hypermethylation of UsnRNPs.

Chitosan nanoparticles for non-viral gene therapy

The cationic polysaccharide chitosan has been widely used for non-viral transfection in vitro and in vivo and has many advantages over other polycations. Chitosan is biocompatible and biodegradable and protects DNA against DNase degradation. However following administration the ChitosanDNA polyplexes must overcome a series of barriers before DNA is delivered to the cell nucleus. This paper describes the most important parameters involved in the chitosan-DNA interaction and their effects of on the condensation, shape, size and protection of DNA. Strategies developed for chitosanDNA polyplexes to avoid non-specific interaction with blood components and to overcome intracellular obstacles as the crossing of die cell membrane, endosomal escape and nuclear import are presented. © 2006 American Chemical Society.