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‣ Novos piezoeletretos: desenvolvimento e caracterização; New piezoelectrets: manufacturing process and characterization

Altafim, Ruy Alberto Pisani
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 11/06/2010 Português
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No final da década de 90, foi observado que filmes de espumas poliméricas carregadas eletricamente poderiam apresentar elevados coeficientes piezoelétricos quando eletricamente carregados. Isso devido a macro dipolos elétricos, formados por cargas elétrica opostas aprisionadas nas superfícies internas das cavidades do material. Esses filmes porosos, receberam os nomes de piezoeletretos ou ferroeletretos. Inicialmente produzidos por microextrusão aerada a partir de polipropileno, esses piezoeletretos estimularam as pesquisas devido aos elevados coeficientes piezoelétricos apresentados. No entanto, à baixa estabilidade térmica desse material, limitou sua aplicação em temperaturas mais elevadas. Isso direcionou as pesquisas para técnicas de produção de espumas com outros materiais. Recentemente, uma nova abordagem com dois filme de Teflon FEP moldados e colados por fusão, mostrou-se viável na produção de estruturas com cavidades fechadas. Por meio desse processo termo-moldado, não só estruturas termicamente mais estáveis foram produzidas mas também com elevados coeficientes piezoelétricos. Porém a principal vantagens desse processo foi a possibilidade de produzir estruturas com cavidades bem definidas, o que permite um carregamento mais uniforme e controle na freqüência de ressonância. Contudo...

‣ The morphology, mechanical properties and ageing behavior of porous injection molded starch-based blends for tissue engineering scaffolding

Neves, N. M.; Kouyumdzhiev, A.; Reis, R. L.
Fonte: Elsevier Publicador: Elsevier
Tipo: Artigo de Revista Científica
Publicado em //2005 Português
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One important parameter in the tissue engineering of hard tissues is the scaffold. A scaffold is a support in which cells are seeded and that should create the adequate environment for the cells to attach and proliferate. Furthermore the scaffold should allow the flow of an appropriate culture media, providing nutrients to the cells and simultaneously removing the metabolites resulting from the cells activity. One of the possibilities is to obtain solid foamed structures that will enable the cells to attach, spread into the inner surfaces and start to produce extracellular matrix. Ideally, if the scaffold is produced from a biodegradable material, it should degrade at a pace that is in phase with the formation of the new tissue. In this work it was studied the production of porous structures from biodegradable polymers for use as scaffolds for bone tissue engineering. Two materials were studied, starch compounded with poly(ethylene-vinyl-alcohol) (SEVA-C) and starch with poly(lactic acid) (SPLA). The porous structures were obtained by injection molding with a blowing agent to control the porosity, interconnectivity and degradation rate. In previous attempts, the current starch compounds proved to be very difficult to process by this method. This study includes the characterization of the mechanical properties...

‣ Nuclear IE2 Structures Are Related to Viral DNA Replication Sites during Baculovirus Infection

Mainz, Daniela; Quadt, Ilja; Knebel-Mörsdorf, Dagmar
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /05/2002 Português
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The ie2 gene of Autographa californica multicapsid nuclear polyhedrosis virus is 1 of the 10 baculovirus genes that have been identified as factors involved in viral DNA replication. IE2 is detectable in the nucleus as one of the major early-expressed proteins and exhibits a dynamic localization pattern during the infection cycle (D. Murges, I. Quadt, J. Schröer, and D. Knebel-Mörsdorf, Exp. Cell Res. 264:219-232, 2001). Here, we investigated whether IE2 localized to regions of viral DNA replication. After viral DNA was labeled with bromodeoxyuridine (BrdU), confocal imaging indicated that defined IE2 domains colocalized with viral DNA replication centers as soon as viral DNA replication was detectable. In addition, a subpopulation of IE2 structures colocalized with two further virus-encoded replication factors, late expression factor 3 (LEF-3) and the DNA binding protein (DBP). While DBP and LEF-3 structures always colocalized and enlarged simultaneously with viral DNA replication sites, only those IE2 structures that colocalized with replication sites also colocalized with DBP. Replication and transcription of DNA viruses in association with promyelocytic leukemia protein (PML) oncogenic domains have been observed. By confocal imaging we demonstrated that the human PML colocalized with IE2. Triple staining revealed PML/IE2 domains in the vicinity of viral DNA replication centers...

‣ Autoradiographic localization of the sites of uptake, cellular transport, and catabolism of low density lipoproteins in the liver of normal and estrogen-treated rats

Chao, Yu-Sheng; Jones, Albert L.; Hradek, Gary T.; Windler, Eberhard E. T.; Havel, Richard J.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1981 Português
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The hepatic uptake and catabolism of low density lipoproteins are stimulated severalfold in rats treated with large amounts of 17α-ethinylestradiol. To determine the sites within the liver at which these processes occur, 125I-labeled human low density lipoproteins were injected intravenously into intact control and estradiol-treated rats or added to perfusates of their isolated livers. The livers were fixed by perfusion and processed for light and electron microscopic autoradiography. Distribution of autoradiographic silver grains was estimated qualitatively in light micrographs and quantitatively in electron micrographs. Many more silver grains were seen in livers from estradiol-treated than from control rats, but the processing of labeled low density lipoprotein was indistinguishable. Three minutes after intravenous injection or perfusion of livers, the grains were concentrated over the microvillous surface of parenchymal cells bordering the space of Disse. Many of these grains were within two half-distances from endocytic pits. Only 5-15% of the grains were seen over endothelial and Kupffer cells. Silver grains were also observed over vesicles beneath the plasma membrane whose size and shape suggested that they were derived from fusion of endocytic vesicles. By 15 min...

‣ Nonmitogenic morphoregulatory action of pp60v-src on multicellular epithelial structures.

Warren, S L; Nelson, W J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1987 Português
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Madin-Darby canine kidney (MDCK) cells form polarized, multicellular epithelial structures in vitro. Low-level expression of pp60v-src in MDCK cells elicits plasticity in these multicellular structures. Plasticity was revealed by the displacement of cells from mechanically stressed regions of the epithelial monolayers; however, the two-dimensional relationship between the cells in the remainder of the monolayer was maintained. Electron microscopy of multicellular structures revealed abnormal separation of the lateral membranes of adjacent cells and selective uncoupling of the junctional complex; the zonula adherens was disrupted, but the zonula occludens and desmosomes were retained. Significantly, this result was not accompanied by transformation of the cells, as judged by the absence of anchorage-independent growth potential. These results demonstrate a nonmitogenic biological activity of pp60v-src which is experimentally dissociable from transformation. This morphoregulatory action on higher-order epithelial structures may reflect a function of related cellular tyrosine kinases.

‣ Prion protein NMR structures of cats, dogs, pigs, and sheep

Lysek, Dominikus A.; Schorn, Christian; Nivon, Lucas G.; Esteve-Moya, Vicent; Christen, Barbara; Calzolai, Luigi; von Schroetter, Christine; Fiorito, Francesco; Herrmann, Torsten; Güntert, Peter; Wüthrich, Kurt
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
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The NMR structures of the recombinant cellular form of the prion proteins (PrPC) of the cat (Felis catus), dog (Canis familiaris), and pig (Sus scrofa), and of two polymorphic forms of the prion protein from sheep (Ovis aries) are presented. In all of these species, PrPC consists of an N-terminal flexibly extended tail with ≈100 amino acid residues and a C-terminal globular domain of ≈100 residues with three α-helices and a short antiparallel β-sheet. Although this global architecture coincides with the previously reported murine, Syrian hamster, bovine, and human PrPC structures, there are local differences between the globular domains of the different species. Because the five newly determined PrPC structures originate from species with widely different transmissible spongiform encephalopathy records, the present data indicate previously uncharacterized possible correlations between local features in PrPC three-dimensional structures and susceptibility of different mammalian species to transmissible spongiform encephalopathies.

‣ Apo and Calcium-Bound Crystal Structures of Alpha-11 Giardin, an Unusual Annexin from Giardia lamblia

Pathuri, Puja; Nguyen, Emily Tam; Svärd, Staffan G.; Luecke, Hartmut
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Alpha-11 giardin is a member of the multi-gene alpha giardin family in the intestinal protozoan, Giardia lamblia. This gene family shares an ancestry with the annexin super family, whose common characteristic is calcium-dependent binding to membranes that contain acidic phospholipids. Several alpha giardins are highly expressed during parasite-induced diarrhea in humans. Despite being a member of a large family of proteins, little is known about the function and cellular localization of alpha-11 giardin, although giardins are often associated with the cytoskeleton. It has been shown that Giardia exhibits high levels of alpha-11 giardin mRNA transcript throughout its life cycle; however, constitutive expression of this protein is lethal to the parasite. Determining the three-dimensional structure of an alpha giardin is essential to identifying functional domains shared in the alpha giardin family. Here we report the crystal structures of the apo and Ca2+-bound forms of alpha-11 giardin, the first alpha giardin to be characterized structurally. Crystals of apo and Ca2+-bound alpha-11 giardin diffracted to 1.1 Å and 2.93 Å, respectively. The crystal structure of selenium-substituted apo alpha-11 giardin reveals a planar array of four tandem repeats of predominantly α-helical domains...

‣ Direct mechanical measurement of geodesic structures in rat mesenchymal stem cells

Maguire, P.; Kilpatrick, J. I.; Kelly, G.; Prendergast, P. J.; Campbell, V. A.; O’Connell, B. C.; Jarvis, S. P.
Fonte: HFSP Publishing Publicador: HFSP Publishing
Tipo: Artigo de Revista Científica
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During numerous biological processes, cell adhesion, cell migration and cell spreading are vital. These basic biological functions are regulated by the interaction of cells with their extracellular environment. To examine the morphology and mechanical changes occurring in mesenchymal stem cells cultured on a mechanically rigid substrate, atomic force microscopy and fluorescence microscopy were employed. Investigations of the cells revealed both linear and geodesic F-actin configurations. No particular cell characteristics or intra-cellular location were implicated in the appearance of the geodesic structures. However, the length of time the cells were cultured on the substrate correlated with the percentage appearance of the geodesic structures. Calculating energy dissipation from cell images acquired by dynamic mode atomic force microscopy, it was observed that the vertices of the geodesic structures had significantly higher energy dissipation compared to the linear F-actin and the glass. This supports work by Lazarides [J. Cell Biol. 68, 202–219 (1976)], who postulated that the vertices of these geodesic structures should have a greater flexibility. Our results also support predictions based on the microfilament tensegrity model. By understanding the basic principles of cell ultrastructure and cell mechanics in relation to different extracellular environments...

‣ Steric confinement of proteins on lipid membranes can drive curvature and tubulation

Stachowiak, Jeanne C.; Hayden, Carl C.; Sasaki, Darryl Y.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
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Deformation of lipid membranes into curved structures such as buds and tubules is essential to many cellular structures including endocytic pits and filopodia. Binding of specific proteins to lipid membranes has been shown to promote membrane bending during endocytosis and transport vesicle formation. Additionally, specific lipid species are found to colocalize with many curved membrane structures, inspiring ongoing exploration of a variety of roles for lipid domains in membrane bending. However, the specific mechanisms by which lipids and proteins collaborate to induce curvature remain unknown. Here we demonstrate a new mechanism for induction and amplification of lipid membrane curvature that relies on steric confinement of protein binding on membrane surfaces. Using giant lipid vesicles that contain domains with high affinity for his-tagged proteins, we show that protein crowding on lipid domain surfaces creates a protein layer that buckles outward, spontaneously bending the domain into stable buds and tubules. In contrast to previously described bending mechanisms relying on local steric interactions between proteins and lipids (i.e. helix insertion into membranes), this mechanism produces tubules whose dimensions are defined by global parameters: domain size and membrane tension. Our results suggest the intriguing possibility that confining structures...

‣ Atomic force microscopy imaging and 3-D reconstructions of serial thin sections of a single cell and its interior structures

Chen, Yong; Cai, Jiye; Zhao, Tao; Wang, Chenxi; Dong, Shuo; Luo, Shuqian; Chen, Zheng W.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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The thin sectioning has been widely applied in electron microscopy (EM), and successfully used for an in situ observation of inner ultrastructure of cells. This powerful technique has recently been extended to the research field of atomic force microscopy (AFM). However, there have been no reports describing AFM imaging of serial thin sections and three-dimensional (3-D) reconstruction of cells and their inner structures. In the present study, we used AFM to scan serial thin sections approximately 60nm thick of a mouse embryonic stem (ES) cell, and to observe the in situ inner ultrastructure including cell membrane, cytoplasm, mitochondria, nucleus membrane, and linear chromatin. The high-magnification AFM imaging of single mitochondria clearly demonstrated the outer membrane, inner boundary membrane and cristal membrane of mitochondria in the cellular compartment. Importantly, AFM imaging on six serial thin sections of a single mouse ES cell showed that mitochondria underwent sequential changes in the number, morphology and distribution. These nanoscale images allowed us to perform 3-D surface reconstruction of interested interior structures in cells. Based on the serial in situ images, 3-D models of morphological characteristics...

‣ History of biological metal utilization inferred through phylogenomic analysis of protein structures

Dupont, Christopher L.; Butcher, Andrew; Valas, Ruben E.; Bourne, Philip E.; Caetano-Anollés, Gustavo
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
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The fundamental chemistry of trace elements dictates the molecular speciation and reactivity both within cells and the environment at large. Using protein structure and comparative genomics, we elucidate several major influences this chemistry has had upon biology. All of life exhibits the same proteome size-dependent scaling for the number of metal-binding proteins within a proteome. This fundamental evolutionary constant shows that the selection of one element occurs at the exclusion of another, with the eschewal of Fe for Zn and Ca being a defining feature of eukaryotic proteomes. Early life lacked both the structures required to control intracellular metal concentrations and the metal-binding proteins that catalyze electron transport and redox transformations. The development of protein structures for metal homeostasis coincided with the emergence of metal-specific structures, which predominantly bound metals abundant in the Archean ocean. Potentially, this promoted the diversification of emerging lineages of Archaea and Bacteria through the establishment of biogeochemical cycles. In contrast, structures binding Cu and Zn evolved much later, providing further evidence that environmental availability influenced the selection of the elements. The late evolving Zn-binding proteins are fundamental to eukaryotic cellular biology...

‣ Rationalisation of the Differences between APOBEC3G Structures from Crystallography and NMR Studies by Molecular Dynamics Simulations

Autore, Flavia; Bergeron, Julien R. C.; Malim, Michael H.; Fraternali, Franca; Huthoff, Hendrik
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 12/07/2010 Português
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The human APOBEC3G (A3G) protein is a cellular polynucleotide cytidine deaminase that acts as a host restriction factor of retroviruses, including HIV-1 and various transposable elements. Recently, three NMR and two crystal structures of the catalytic deaminase domain of A3G have been reported, but these are in disagreement over the conformation of a terminal β-strand, β2, as well as the identification of a putative DNA binding site. We here report molecular dynamics simulations with all of the solved A3G catalytic domain structures, taking into account solubility enhancing mutations that were introduced during derivation of three out of the five structures. In the course of these simulations, we observed a general trend towards increased definition of the β2 strand for those structures that have a distorted starting conformation of β2. Solvent density maps around the protein as calculated from MD simulations indicated that this distortion is dependent on preferential hydration of residues within the β2 strand. We also demonstrate that the identification of a pre-defined DNA binding site is prevented by the inherent flexibility of loops that determine access to the deaminase catalytic core. We discuss the implications of our analyses for the as yet unresolved structure of the full-length A3G protein and its biological functions with regard to hypermutation of DNA.

‣ Cell Surface Attachment Structures Contribute to Biofilm Formation and Xylem Colonization by Erwinia amylovora▿

Koczan, Jessica M.; Lenneman, Bryan R.; McGrath, Molly J.; Sundin, George W.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /10/2011 Português
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Biofilm formation plays a critical role in the pathogenesis of Erwinia amylovora and the systemic invasion of plant hosts. The functional role of the exopolysaccharides amylovoran and levan in pathogenesis and biofilm formation has been evaluated. However, the role of biofilm formation, independent of exopolysaccharide production, in pathogenesis and movement within plants has not been studied previously. Evaluation of the role of attachment in E. amylovora biofilm formation and virulence was examined through the analysis of deletion mutants lacking genes encoding structures postulated to function in attachment to surfaces or in cellular aggregation. The genes and gene clusters studied were selected based on in silico analyses. Microscopic analyses and quantitative assays demonstrated that attachment structures such as fimbriae and pili are involved in the attachment of E. amylovora to surfaces and are necessary for the production of mature biofilms. A time course assay indicated that type I fimbriae function earlier in attachment, while type IV pilus structures appear to function later in attachment. Our results indicate that multiple attachment structures are needed for mature biofilm formation and full virulence and that biofilm formation facilitates entry and is necessary for the buildup of large populations of E. amylovora cells in xylem tissue.

‣ Replication Protein A Unfolds G-Quadruplex Structures with a Varying Degree of Efficiency

Qureshi, Mohammad H.; Ray, Sujay; Sewell, Abby L.; Basu, Soumitra; Balci, Hamza
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Replication Protein A (RPA) is known to interact with G-rich sequences that adopt G-quadruplex (GQ) structures. Most studies in the literature have been performed on GQ formed by homogenous sequences, such as the human telomeric repeat, and RPA’s ability to unfold GQ structures of differing stability is not known. We compared the thermal stability of three potential GQ forming DNA sequences (PQS) to their stability against RPA mediated unfolding using single molecule FRET and bulk biophysical and biochemical experiments. One of these sequences is the human telomeric repeat and the other two located in the promoter region of tyrosine hydroxylase gene are highly heterogeneous sequences, which better represent PQS in the genome. The three GQ constructs have thermal stabilities that are significantly different from each other. Our measurements showed that the most thermally stable structure (Tm= 86 °C) was also the most stable against RPA mediated unfolding, although the least thermally stable structure (Tm= 69 °C) had at least an order of magnitude higher stability against RPA mediated unfolding compared to the structure with intermediate thermal stability (Tm= 78 °C). The significance of this observation becomes more evident when considered within the context of cellular environment where protein-DNA interactions can be an important determinant of GQ viability. Considering these...

‣ Biogenesis and Dynamics of the Coronavirus Replicative Structures

Hagemeijer, Marne C.; Rottier, Peter J.M.; de Haan, Cornelis A.M.
Fonte: MDPI Publicador: MDPI
Tipo: Artigo de Revista Científica
Publicado em 21/11/2012 Português
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Coronaviruses are positive-strand RNA viruses that are important infectious agents of both animals and humans. A common feature among positive-strand RNA viruses is their assembly of replication-transcription complexes in association with cytoplasmic membranes. Upon infection, coronaviruses extensively rearrange cellular membranes into organelle-like replicative structures that consist of double-membrane vesicles and convoluted membranes to which the nonstructural proteins involved in RNA synthesis localize. Double-stranded RNA, presumably functioning as replicative intermediate during viral RNA synthesis, has been detected at the double-membrane vesicle interior. Recent studies have provided new insights into the assembly and functioning of the coronavirus replicative structures. This review will summarize the current knowledge on the biogenesis of the replicative structures, the membrane anchoring of the replication-transcription complexes, and the location of viral RNA synthesis, with particular focus on the dynamics of the coronavirus replicative structures and individual replication-associated proteins.

‣ Hsp70 Architecture: The Formation of Novel Polymeric Structures of Hsp70.1 and Hsc70 after Proteotoxic Stress

Steel, Rohan; Cross, Ryan S.; Ellis, Sarah L.; Anderson, Robin L.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 19/12/2012 Português
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Heat induces Hsp70.1 (HSPA1) and Hsc70 (HSPA8) to form complex detergent insoluble cytoplasmic and nuclear structures that are distinct from the cytoskeleton and internal cell membranes. These novel structures have not been observed by earlier immunofluorescence studies as they are obscured by the abundance of soluble Hsp70.1/Hsc70 present in cells. While resistant to detergents, these Hsp70 structures display complex intracellular dynamics and are efficiently disaggregated by ATP, indicating that this pool of Hsp70.1/Hsc70 retains native function and regulation. Hsp70.1 promotes the repair of proteotoxic damage and cell survival after stress. In heated fibroblasts expressing Hsp70.1, Hsp70.1 and Hsc70 complexes are efficiently disaggregated before the cells undergo-heat induced apoptosis. In the absence of Hsp70.1, fibroblasts have increased rates of heat-induced apoptosis and maintain stable insoluble Hsc70 structures. The differences in the intracellular distribution of Hsp70.1 and Hsc70, combined with the ability of Hsp70.1, but not Hsc70, to promote the disaggregation of insoluble Hsp70.1/Hsc70 complexes, indicate that these two closely related proteins perform distinctly different cellular functions in heated cells.

‣ When secondary comes first – the importance of non-canonical DNA structures

Saini, Natalie; Zhang, Yu; Usdin, Karen; Lobachev, Kirill S.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Secondary structure-forming DNA motifs have achieved infamy because of their association with a variety of human diseases and cancer. The 3rd FASEB summer conference on dynamic DNA structures focused on the mechanisms responsible for the instabilities inherent to repetitive DNA and presented many exciting and novel aspects related to the metabolism of secondary structures. In addition, the meeting encompassed talks and posters on the dynamic structures that are generated during DNA metabolism including nicked DNA, Holliday junctions and RNA:DNA hybrids. New approaches for analysis and sequencing technologies put forth secondary structures and other DNA intermediates as vital regulators of a variety of cellular processes that contribute to evolution, polymorphisms and diseases.

‣ Open and shut: Crystal structures of the dodecylmaltoside solubilized mechanosensitive channel of small conductance from Escherichia coli and Helicobacter pylori at 4.4 Å and 4.1 Å resolutions

Lai, Jeffrey Y; Poon, Yan Shuen; Kaiser, Jens T; Rees, Douglas C
Fonte: Wiley Subscription Services, Inc., A Wiley Company Publicador: Wiley Subscription Services, Inc., A Wiley Company
Tipo: Artigo de Revista Científica
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The mechanosensitive channel of small conductance (MscS) contributes to the survival of bacteria during osmotic downshock by transiently opening large diameter pores for the efflux of cellular contents before the membrane ruptures. Two crystal structures of the Escherichia coli MscS are currently available, the wild type protein in a nonconducting state at 3.7 Å resolution (Bass et al., Science 2002; 298:1582–1587) and the Ala106Val variant in an open state at 3.45 Å resolution (Wang et al., Science 2008; 321:1179–1183). Both structures used protein solubilized in the detergent fos-choline-14. We report here crystal structures of MscS from E. coli and Helicobacter pylori solubilized in the detergent β-dodecylmaltoside at resolutions of 4.4 and 4.2 Å, respectively. While the cytoplasmic domains are unchanged in these structures, distinct conformations of the transmembrane domains are observed. Intriguingly, β-dodecylmaltoside solubilized wild type E. coli MscS adopts the open state structure of A106V E. coli MscS, while H. pylori MscS resembles the nonconducting state structure observed for fos-choline-14 solubilized E. coli MscS. These results highlight the sensitivity of membrane protein conformational equilibria to variations in detergent...

‣ Origin and Evolution of Protein Fold Designs Inferred from Phylogenomic Analysis of CATH Domain Structures in Proteomes

Bukhari, Syed Abbas; Caetano-Anollés, Gustavo
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
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The spatial arrangements of secondary structures in proteins, irrespective of their connectivity, depict the overall shape and organization of protein domains. These features have been used in the CATH and SCOP classifications to hierarchically partition fold space and define the architectural make up of proteins. Here we use phylogenomic methods and a census of CATH structures in hundreds of genomes to study the origin and diversification of protein architectures (A) and their associated topologies (T) and superfamilies (H). Phylogenies that describe the evolution of domain structures and proteomes were reconstructed from the structural census and used to generate timelines of domain discovery. Phylogenies of CATH domains at T and H levels of structural abstraction and associated chronologies revealed patterns of reductive evolution, the early rise of Archaea, three epochs in the evolution of the protein world, and patterns of structural sharing between superkingdoms. Phylogenies of proteomes confirmed the early appearance of Archaea. While these findings are in agreement with previous phylogenomic studies based on the SCOP classification, phylogenies unveiled sharing patterns between Archaea and Eukarya that are recent and can explain the canonical bacterial rooting typically recovered from sequence analysis. Phylogenies of CATH domains at A level uncovered general patterns of architectural origin and diversification. The tree of A structures showed that ancient structural designs such as the 3-layer (αβα) sandwich (3.40) or the orthogonal bundle (1.10) are comparatively simpler in their makeup and are involved in basic cellular functions. In contrast...

‣ Quantitative Visualization of DNA G-quadruplex Structures in Human Cells

Biffi, Giulia; Tannahill, David; McCafferty, John; Balasubramanian, Shankar
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Four-stranded G-quadruplex nucleic acid structures have been of great interest as their high thermodynamic stability under near-physiological conditions suggests that they could form in cells. Here, we report the generation and application of an engineered, structure-specific antibody that was employed to visualize quantitatively DNA G-quadruplex structures in human cells. We explicitly show that G-quadruplex formation in DNA is modulated during cell cycle progression and that endogenous G-quadruplex DNA structures can be stabilized by a small molecule ligand. Together these findings provide substantive evidence for the formation of G-quadruplex structures in the genome of mammalian cells and corroborate the application of stabilizing ligands in a cellular context to target G-quadruplexes and intervene with their function.