Efficient Trafficking of TGN38 from the Endosome to the trans-Golgi Network Requires a Free Hydroxyl Group at Position 331 in the Cytosolic Domain

TGN38 is one of the few known resident integral membrane proteins of the trans-Golgi network (TGN). Since it cycles constitutively between the TGN and the plasma membrane, TGN38 is ideally suited as a model protein for the identification of post-Golgi trafficking motifs. Several studies, employing chimeric constructs to detect such motifs within the cytosolic domain of TGN38, have identified the sequence 333YQRL336 as an autonomous signal capable of localizing reporter proteins to the TGN. In addition, one group has found that an upstream serine residue, S331, may also play a role in TGN38 localization. However, the nature and degree of participation of S331 in the localization of TGN38 remain uncertain, and the effect has been studied in chimeric constructs only. Here we investigate the role of S331 in the context of full-length TGN38. Mutations that abolish the hydroxyl moiety at position 331 (A, D, and E) lead to missorting of endocytosed TGN38 to the lysosome. Conversely, mutation of S331 to T has little effect on the endocytic trafficking of TGN38. Together, these findings indicate that the S331 hydroxyl group has a direct or indirect effect on the ability of the cytosolic tail of TGN38 to interact with trafficking and/or sorting machinery at the level of the early endosome. In addition, mutation of S331 to either A or D results in increased levels of TGN38 at the cell surface. The results confirm that S331 plays a critical role in the intracellular trafficking of TGN38 and further reveal that TGN38 undergoes a signal-mediated trafficking step at the level of the endosome.

Thermodynamic basis of the enhanced specificity of structured DNA probes

Molecular beacons are DNA probes that form a stem-and-loop structure and possess an internally quenched fluorophore. When they bind to complementary nucleic acids, they undergo a conformational transition that switches on their fluorescence. These probes recognize their targets with higher specificity than probes that cannot form a hairpin stem, and they easily discriminate targets that differ from one another by only a single nucleotide. Our results show that molecular beacons can exist in three different states: bound to a target, free in the form of a hairpin structure, and free in the form of a random coil. Thermodynamic analysis of the transitions between these states reveals that enhanced specificity is a general feature of conformationally constrained probes.

Interaction of voltage-gated sodium channels with the extracellular matrix molecules tenascin-C and tenascin-R

The type IIA rat brain sodium channel is composed of three subunits: a large pore-forming α subunit and two smaller auxiliary subunits, β1 and β2. The β subunits are single membrane-spanning glycoproteins with one Ig-like motif in their extracellular domains. The Ig motif of the β2 subunit has close structural similarity to one of the six Ig motifs in the extracellular domain of the cell adhesion molecule contactin (also called F3 or F11), which binds to the extracellular matrix molecules tenascin-C and tenascin-R. We investigated the binding of the purified sodium channel and the extracellular domain of the β2 subunit to tenascin-C and tenascin-R in vitro. Incubation of purified sodium channels on microtiter plates coated with tenascin-C revealed saturable and specific binding with an apparent Kd of ≈15 nM. Glutathione S-transferase-tagged fusion proteins containing various segments of tenascin-C and tenascin-R were purified, digested with thrombin to remove the epitope tag, immobilized on microtiter dishes, and tested for their ability to bind purified sodium channel or the epitope-tagged extracellular domain of β2 subunits. Both purified sodium channels and the extracellular domain of the β2 subunit bound specifically to fibronectin type III repeats 1–2, A, B, and 6–8 of tenascin-C and fibronectin type III repeats 1–2 and 6–8 of tenascin-R but not to the epidermal growth factor-like domain or the fibrinogen-like domain of these molecules. The binding of neuronal sodium channels to extracellular matrix molecules such as tenascin-C and tenascin-R may play a crucial role in localizing sodium channels in high density at axon initial segments and nodes of Ranvier or in regulating the activity of immobilized sodium channels in these locations.

Loss of HMW1 and HMW3 in noncytadhering mutants of Mycoplasma pneumoniae occurs post-translationally

The genomic sequence of Mycoplasma pneumoniae establish this cell-wall-less prokaryote as among the smallest known microorganisms capable of self-replication. However, this genomic simplicity and corresponding biosynthetic austerity are sharply contrasted by the complex terminal structure found in this species. This tip structure (attachment organelle) directs colonization of the human respiratory mucosa, leading to bronchitis and atypical pneumonia. Furthermore, formation of a second tip structure appears to precede cell division, implying temporal regulation. However, the organization, regulation, and assembly of the attachment organelle in M. pneumoniae are poorly understood, and no counterparts have been identified among the walled bacteria. M. pneumoniae possesses a cytoskeleton-like structure required to localize adhesin proteins to the attachment organelle. The cytadherence-associated proteins HMW1, HMW2, and HMW3 are components of the mycoplasma cytoskeleton, with HMW1 localizing strictly along the filamentous extensions from the cell body and HMW3 being a key structural element of the terminal organelle. Disruptions in hmw2 result in the loss of HMW1 and HMW3. However, the hmw1 and hmw3 genes were transcribed and translated at wild-type levels in hmw2 mutants. HMW1 and HMW3 were relatively stable in the wild-type background over 8 h but disappeared in the mutants over this time period. Evaluation of recombinant HMW1 levels in mycoplasma mutants suggested a requirement for the C-terminal domain of HMW1 for turnover. Finally, an apparent defect in the processing of the precursor for the adhesin protein P1 was noted in the HMW− mutants.

The TRANSFAC system on gene expression regulation

The TRANSFAC database on transcription factors and their DNA-binding sites and profiles (http://www.gene-regulation.de/) has been quantitatively extended and supplemented by a number of modules. These modules give information about pathologically relevant mutations in regulatory regions and transcription factor genes (PathoDB), scaffold/matrix attached regions (S/MARt DB), signal transduction (TRANSPATH) and gene expression sources (CYTOMER). Altogether, these distinct database modules constitute the TRANSFAC system. They are accompanied by a number of program routines for identifying potential transcription factor binding sites or for localizing individual components in the regulatory network of a cell.

Simultaneous A8344G heteroplasmy and mitochondrial DNA copy number quantification in Myoclonus Epilepsy and Ragged-Red Fibers (MERRF) syndrome by a multiplex Molecular Beacon based real-time fluorescence PCR

The association of a particular mitochondrial DNA (mtDNA) mutation with different clinical phenotypes is a well-known feature of mitochondrial diseases. A simple genotype–phenotype correlation has not been found between mutation load and disease expression. Tissue and intercellular mosaicism as well as mtDNA copy number are thought to be responsible for the different clinical phenotypes. As disease expression of mitochondrial tRNA mutations is mostly in postmitotic tissues, studies to elucidate disease mechanisms need to be performed on patient material. Heteroplasmy quantitation and copy number estimation using small patient biopsy samples has not been reported before, mainly due to technical restrictions. In order to resolve this problem, we have developed a robust assay that utilizes Molecular Beacons to accurately quantify heteroplasmy levels and determine mtDNA copy number in small samples carrying the A8344G tRNALys mutation. It provides the methodological basis to investigate the role of heteroplasmy and mtDNA copy number in determining the clinical phenotypes.

Neuroimaging studies of word reading

This review discusses how neuroimaging can contribute to our understanding of a fundamental aspect of skilled reading: the ability to pronounce a visually presented word. One contribution of neuroimaging is that it provides a tool for localizing brain regions that are active during word reading. To assess the extent to which similar results are obtained across studies, a quantitative review of nine neuroimaging investigations of word reading was conducted. Across these studies, the results converge to reveal a set of areas active during word reading, including left-lateralized regions in occipital and occipitotemporal cortex, the left frontal operculum, bilateral regions within the cerebellum, primary motor cortex, and the superior and middle temporal cortex, and medial regions in the supplementary motor area and anterior cingulate. Beyond localization, the challenge is to use neuroimaging as a tool for understanding how reading is accomplished. Central to this challenge will be the integration of neuroimaging results with information from other methodologies. To illustrate this point, this review will highlight the importance of spelling-to-sound consistency in the transformation from orthographic (word form) to phonological (word sound) representations, and then explore results from three neuroimaging studies in which the spelling-to-sound consistency of the stimuli was deliberately varied. Emphasis is placed on the pattern of activation observed within the left frontal cortex, because the results provide an example of the issues and benefits involved in relating neuroimaging results to behavioral results in normal and brain damaged subjects, and to theoretical models of reading.

Agrobacterium VirE2 protein mediates nuclear uptake of single-stranded DNA in plant cells.

Agrobacterium genetically transforms plant cells by transferring a single-stranded DNA (ssDNA) copy of the transferred DNA (T-DNA) element, the T-strand, in a complex with Agrobacterium proteins VirD2, bound to the 5' end, and VirE2. VirE2 binds single-stranded nucleic acid cooperatively, fully coating the T-strand, and the protein localizes to the plant cell nucleus when transiently expressed. The coupling of ssDNA binding and nuclear localizing activities suggests that VirE2 alone could mediate nuclear localization of ssDNA. In this study, fluorescently labeled ssDNA accumulated in the plant cell nucleus specifically when microinjected as a complex with VirE2. Microinjected ssDNA alone remained cytoplasmic. Import of VirE2-ssDNA complex into the nucleus via a protein import pathway was supported by (i) the inhibition of VirE2-ssDNA complex import in the presence of wheat germ agglutinin or a nonhydrolyzable GTP analog, both known inhibitors of protein nuclear import, and (ii) the retardation of import when complexes were prepared from a VirE2 mutant impaired in ssDNA binding and nuclear import.

Contribuições ao processo de inserção das PMES no mercado de capitais brasileiro

O crescimento de uma empresa pode ser apoiado com recursos de terceiros provenientes do mercado de crédito ou do mercado de capitais. Credores ou potenciais investidores disponibilizam recursos a partir de um processo de avaliação de indicadores de performance. Para as PMEs que consideram o crescimento, conhecer como os indicadores relevantes se comportam ao longo de um ciclo de crescimento é uma questão estratégica. Fatores como tamanho, lucratividade, oportunidades de crescimento, composição de ativos das empresas, risco inerente aos resultados, têm sido vinculados a determinantes de uma estrutura de capital. Assim sendo, este trabalho busca verificar quais indicadores podem nortear o desempenho empresarial de PMEs ao longo de um ciclo de crescimento. Como contribuição original, este trabalho apresenta um painel com indicadores em diferentes estágios de crescimento que resultam na geração de valor para os proprietários e potenciais investidores do mercado de capitais. A amostra inicial é composta por 1.610 empresas para o período de 2010 a 2014. Entretanto, considerando a disponibilidade de informações, a amostra final é composta por 28 empresas de porte médio/grande, 387 empresas grandes e 138 empresas listadas na BM&FBOVESPA, totalizando 553 empresas. A metodologia adotada envolve a classificação de porte de empresa do BNDES como critério para definir ciclo de crescimento, e testes de estatística descritiva, análise fatorial, análise de correlação, regressão múltipla linear e montagem de painel. Como resultado, verificou-se que as variáveis tamanho, composição de ativos e lucratividade são fatores que explicam o endividamento de curto prazo para o estágio médias/grandes empresas. Ainda, que esses fatores explicam o endividamento de longo prazo para os estágios grandes e empresas listadas na BM&FBOVESPA. Tais resultados estão de acordo com estudos prévios, mas as variáveis relacionadas a volatilidade e crescimento não foram significantes nos modelos para os estágios de empresas. O painel montado a partir das variáveis de composição de ativos e lucratividade indicou que endividamento de curto prazo, endividamento de longo prazo, receita líquida, retorno sobre o patrimônio líquido e lucros antes de juros, impostos, depreciação e amortização (EBITDA) sinalizam empresas, em cada estágio de ciclo de crescimento do estudo, que buscam o crescimento com rentabilidade acima da média e atendem a critérios valorizados pelo investidor do mercado de capitais. Estes indicadores podem sinalizar o desempenho empresarial de PMEs ao longo de um ciclo de crescimento

Canales, sifones y almenaras: el impacto ambiental de las infraestructuras del Canal de Isabel II (Madrid)

El presente artículo tiene como finalidad, valorar el impacto ambiental de las conducciones del Canal de Isabel II en el contexto del paisaje. Partimos de la idea según la cual, las infraestructuras del Canal de Isabel II, y más que formar parte del paisaje por el que se extienden, son el propio paisaje. Nuestra zona de estudio es el noroeste de la Comunidad de Madrid (síntesis de la interacción de los propios agentes naturales, de la ocupación humana y de los usos del suelo), área a la que nos aproximarnos a través de la investigación de la integración paisajística, entendida ésta como una estrategia de intervención en el territorio, que tiene como objetivo principal orientar las transformaciones del paisaje o corregir las ya realizadas, para conseguir su adaptación al propio paisaje. En definitiva, nos encontramos ante la necesidad de ajustar un objeto o actuación territorial a las características fisonómicas de un paisaje dado, o de algunos de sus componentes, así como a su carácter y a sus contenidos semánticos.

Narragansett Bay, Rhode Island, 1832 (Raster Image)

This layer is a georeferenced raster image of the historic paper map entitled: Chart of Narraganset Bay : surveyed in 1832 by Capt. Alex. S. Wadsworth ... [et al.] of the U.S. Navy, by order of the Honr. Levi Woodbury, secretary of the Navy. It was published by the U.S. Navy ca. 1832. Scale [1:24,000]. The image inside the map neatline is georeferenced to the surface of the earth and fit to the Rhode Island State Plane Coordinate System (NAD 1983 in Feet) (FIPS 3800). All map collar and inset information is also available as part of the raster image, including any inset maps, profiles, statistical tables, directories, text, illustrations, index maps, legends, or other information associated with the principal map. This map shows coastal features such as lighthouses, buoys, beacons, rocks, channels, points, coves, islands, bottom soil types, wharves, and more. Includes also selected land features such as roads, drainage, land cover, selected buildings, towns, and more. Relief shown by hachures. Depths shown by soundings. Includes notes.This layer is part of a selection of digitally scanned and georeferenced historic maps of New England from the Harvard Map Collection. These maps typically portray both natural and manmade features. The selection represents a range of regions, originators, ground condition dates, scales, and map purposes.

New York Bay and Harbor, and vicinity, 1844 (Image 2 of 6) (Raster Image)

This layer is a georeferenced raster image of the historic paper map entitled: Map of New-York Bay and Harbor and the environs : sheet no. 2, founded upon a trigonometrical survey under the direction of F.R. Hassler, superintendent of the Survey of the Coast of the United States ; triangulation by James Ferguson and Edmund Blunt, assistants ; the hydrography under the direction of Thomas R. Gedney, lieutenant U.S. Navy ; the topography by C. Renard and T.A. Jenkins assists. It was published by Survey of the Coast of the United States in 1844-1845. Scale 1:30,000. This layer is image 2 of 6 total images of the six sheet source map, representing the southeast portion of the map. The image inside the map neatline is georeferenced to the surface of the earth and fit to the Universal Transverse Mercator (UTM) Zone 18N NAD83 projection. All map collar and inset information is also available as part of the raster image, including any inset maps, profiles, statistical tables, directories, text, illustrations, or other information associated with the principal map. This map shows coastal features such as lighthouses, buoys, beacons, rocks, channels, points, coves, islands, bottom soil types, wharves, and more. Includes also selected land features such as roads, drainage, land cover, forts, selected buildings, towns, and more. Relief shown by hachures. Depths are shown by soundings and shading. Includes text, table of currents and stations, notes, sailing directions, 4 coastal panoramas and 2 views of Sandy Hook Light. This layer is part of a selection of digitally scanned and georeferenced historic maps from The Harvard Map Collection as part of the Imaging the Urban Environment project. Maps selected for this project represent major urban areas and cities of the world, at various time periods. These maps typically portray both natural and manmade features at a large scale. The selection represents a range of regions, originators, ground condition dates, scales, and purposes.

New York Bay and Harbor, and vicinity, 1844 (Image 1 of 6) (Raster Image)

This layer is a georeferenced raster image of the historic paper map entitled: Map of New-York Bay and Harbor and the environs : sheet no. 1, founded upon a trigonometrical survey under the direction of F.R. Hassler, superintendent of the Survey of the Coast of the United States ; triangulation by James Ferguson and Edmund Blunt, assistants ; the hydrography under the direction of Thomas R. Gedney, lieutenant U.S. Navy ; the topography by C. Renard and T.A. Jenkins assists. It was published by Survey of the Coast of the United States in 1844-1845. Scale 1:30,000. This layer is image 1 of 6 total images of the six sheet source map, representing the southwest portion of the map. The image inside the map neatline is georeferenced to the surface of the earth and fit to the Universal Transverse Mercator (UTM) Zone 18N NAD83 projection. All map collar and inset information is also available as part of the raster image, including any inset maps, profiles, statistical tables, directories, text, illustrations, or other information associated with the principal map. This map shows coastal features such as lighthouses, buoys, beacons, rocks, channels, points, coves, islands, bottom soil types, wharves, and more. Includes also selected land features such as roads, drainage, land cover, forts, selected buildings, towns, and more. Relief shown by hachures. Depths are shown by soundings and shading. Includes text, table of currents and stations, notes, sailing directions, 4 coastal panoramas and 2 views of Sandy Hook Light. This layer is part of a selection of digitally scanned and georeferenced historic maps from The Harvard Map Collection as part of the Imaging the Urban Environment project. Maps selected for this project represent major urban areas and cities of the world, at various time periods. These maps typically portray both natural and manmade features at a large scale. The selection represents a range of regions, originators, ground condition dates, scales, and purposes.

New York Bay and Harbor, and vicinity, 1844 (Image 4 of 6) (Raster Image)

This layer is a georeferenced raster image of the historic paper map entitled: Map of New-York Bay and Harbor and the environs : sheet no. 4, founded upon a trigonometrical survey under the direction of F.R. Hassler, superintendent of the Survey of the Coast of the United States ; triangulation by James Ferguson and Edmund Blunt, assistants ; the hydrography under the direction of Thomas R. Gedney, lieutenant U.S. Navy ; the topography by C. Renard and T.A. Jenkins assists. It was published by Survey of the Coast of the United States in 1844-1845. Scale 1:30,000. This layer is image 4 of 6 total images of the six sheet source map, representing the southwest portion of the map. The image inside the map neatline is georeferenced to the surface of the earth and fit to the Universal Transverse Mercator (UTM) Zone 18N NAD83 projection. All map collar and inset information is also available as part of the raster image, including any inset maps, profiles, statistical tables, directories, text, illustrations, or other information associated with the principal map. This map shows coastal features such as lighthouses, buoys, beacons, rocks, channels, points, coves, islands, bottom soil types, wharves, and more. Includes also selected land features such as roads, drainage, land cover, forts, selected buildings, towns, and more. Relief shown by hachures. Depths are shown by soundings and shading. Includes text, table of currents and stations, notes, sailing directions, 4 coastal panoramas and 2 views of Sandy Hook Light. This layer is part of a selection of digitally scanned and georeferenced historic maps from The Harvard Map Collection as part of the Imaging the Urban Environment project. Maps selected for this project represent major urban areas and cities of the world, at various time periods. These maps typically portray both natural and manmade features at a large scale. The selection represents a range of regions, originators, ground condition dates, scales, and purposes.

New York Bay and Harbor, and vicinity, 1844 (Image 1 of 6) (Raster Image)

This layer is a georeferenced raster image of the historic paper map entitled: Map of New-York Bay and Harbor and the environs : sheet no. 3, founded upon a trigonometrical survey under the direction of F.R. Hassler, superintendent of the Survey of the Coast of the United States ; triangulation by James Ferguson and Edmund Blunt, assistants ; the hydrography under the direction of Thomas R. Gedney, lieutenant U.S. Navy ; the topography by C. Renard and T.A. Jenkins assists. It was published by Survey of the Coast of the United States in 1844-1845. Scale 1:30,000. This layer is image 3 of 6 total images of the six sheet source map, representing the southwest portion of the map. The image inside the map neatline is georeferenced to the surface of the earth and fit to the Universal Transverse Mercator (UTM) Zone 18N NAD83 projection. All map collar and inset information is also available as part of the raster image, including any inset maps, profiles, statistical tables, directories, text, illustrations, or other information associated with the principal map. This map shows coastal features such as lighthouses, buoys, beacons, rocks, channels, points, coves, islands, bottom soil types, wharves, and more. Includes also selected land features such as roads, drainage, land cover, forts, selected buildings, towns, and more. Relief shown by hachures. Depths are shown by soundings and shading. Includes text, table of currents and stations, notes, sailing directions, 4 coastal panoramas and 2 views of Sandy Hook Light. This layer is part of a selection of digitally scanned and georeferenced historic maps from The Harvard Map Collection as part of the Imaging the Urban Environment project. Maps selected for this project represent major urban areas and cities of the world, at various time periods. These maps typically portray both natural and manmade features at a large scale. The selection represents a range of regions, originators, ground condition dates, scales, and purposes.