DNA loops induced by cooperative binding of transcriptional activator proteins and preinitiation complexes.

DNA conformational changes are essential for the assembly of multiprotein complexes that contact several DNA sequence elements. An approach based on atomic force microscopy was chosen to visualize specific protein-DNA interactions occurring on eukaryotic class II nuclear gene promoters. Here we report that binding of the transcription regulatory protein Jun to linearized plasmid DNA containing the consensus AP-1 binding site upstream of a class II gene promoter leads to bending of the DNA template. This binding of Jun was found to be essential for the formation of preinitiation complexes (PICs). The cooperative binding of Jun and PIC led to looping of DNA at the protein binding sites. These loops were not seen in the absence of either PICs, Jun, or the AP-1 binding site, suggesting a direct interaction between DNA-bound Jun homodimers and proteins bound to the core promoter. This direct visualization of functional transcriptional complexes confirms the theoretical predictions for the mode of gene regulation by trans-activating proteins.

Deletion of high-avidity T cells by thymic epithelium.

Tolerance induction by thymic epithelium induces a state of so-called "split tolerance," characterized in vivo by tolerance and in vitro by reactivity to a given thymically expressed antigen. Using a model major histocompatibility complex class I antigen, H-2Kb (Kb), three mechanisms of thymic epithelium-induced tolerance were tested: induction of tolerance of tissue-specific antigens exclusively, selective inactivation of T helper cell-independent cytotoxic T lymphocytes, and deletion of high-avidity T cells. To this end, thymic anlagen from Kb-transgenic embryonic day 10 mouse embryos, taken before colonization by cells of hemopoietic origin, were grafted to nude mice. Tolerance by thymic epithelium was not tissue-specific, since Kb-bearing skin and spleen grafts were maintained indefinitely. Only strong priming in vivo could partially overcome the tolerant state and induce rejection of some skin grafts overexpressing transgenic Kb. Furthermore, the hypothesis that thymic epithelium selectively inactivates those T cells that reject skin grafts in a T helper-independent fashion could not be supported. Thus, when T-cell help was provided by a second skin graft bearing an additional major histocompatibility complex class II disparity, tolerance to the Kb skin graft was not broken. Finally, direct evidence could be obtained for the avidity model of thymic epithelium-induced negative selection, using Kb-specific T-cell receptor (TCR) transgenic mice. Thymic epithelium-grafted TCR transgenic mice showed a selective deletion of those CD8+ T cells with the highest density of the clonotypic TCR. These cells presumably represent the T cells with the highest avidity for Kb. We conclude that split tolerance induced by thymic epithelium was mediated by the deletion of those CD8+ T lymphocytes that have the highest avidity for antigen.

Genetic analysis of type 1 diabetes using whole genome approaches.

Whole genome linkage analysis of type 1 diabetes using affected sib pair families and semi-automated genotyping and data capture procedures has shown how type 1 diabetes is inherited. A major proportion of clustering of the disease in families can be accounted for by sharing of alleles at susceptibility loci in the major histocompatibility complex on chromosome 6 (IDDM1) and at a minimum of 11 other loci on nine chromosomes. Primary etiological components of IDDM1, the HLA-DQB1 and -DRB1 class II immune response genes, and of IDDM2, the minisatellite repeat sequence in the 5' regulatory region of the insulin gene on chromosome 11p15, have been identified. Identification of the other loci will involve linkage disequilibrium mapping and sequencing of candidate genes in regions of linkage.

Induction of antigen-specific cytolytic T cells in situ in human melanoma by immunization with synthetic peptide-pulsed autologous antigen presenting cells.

Human melanoma cells can process the MAGE-1 gene product and present the processed nonapeptide EADPTGHSY on their major histocompatibility complex class I molecules, HLA-A1, as a determinant for cytolytic T lymphocytes (CTLs). Considering that autologous antigen presenting cells (APCs) pulsed with the synthetic nonapeptide might, therefore, be immunogenic, melanoma patients whose tumor cells express the MAGE-1 gene and who are HLA-A1+ were immunized with a vaccine made of cultured autologous APCs pulsed with the synthetic nonapeptide. Analyses of the nature of the in vivo host immune response to the vaccine revealed that the peptide-pulsed APCs are capable of inducing autologous melanoma-reactive and the nonapeptide-specific CTLs in situ at the immunization site and at distant metastatic disease sites.

Structure and function of a human transcription factor TFIIIB subunit that is evolutionarily conserved and contains both TFIIB- and high-mobility-group protein 2-related domains.

Transcription factor TFIIIB plays a central role in transcription initiation by RNA polymerase III on genes encoding tRNA, 5S rRNA, and other small structural RNAs. We report the purification of a human TFIIIB-derived complex containing only the TATA-binding polypeptide (TBP) and a 90-kDa subunit (TFIIIB90) and the isolation of a cDNA clone encoding the 90-kDa subunit. The N-terminal half of TFIIIB90 exhibits sequence similarity to the yeast TFIIIB70 (BRF) and the class II transcription factor TFIIB and interacts weakly with TBP. The C-terminal half of TFIIIB90 contains a high-mobility-group protein 2 (HMG2)-related domain and interacts strongly with TBP. Recombinant TFIIIB90 plus recombinant human TBP substitute for human TFIIIB in a complementation assay for transcription of 5S, tRNA, and VA1 RNA genes, and both the TFIIB-related domain and the HMG2-related domain are required for this activity. TFIIIB90 is also required for transcription of human 7SK and U6 RNA genes by RNA polymerase III, but apparently within a complex distinct from the TBP/TFIIIB90 complex.

Coexpression of two functionally independent p58 inhibitory receptors in human natural killer cell clones results in the inability to kill all normal allogeneic target cells.

In the present study, we define a group of natural killer (NK) clones (group 0) that fails to lyse all of the normal allogeneic target cells analyzed. Their specificity for HLA class I molecules was suggested by their ability to lyse class I-negative target cells and by the fact that they could lyse resistant target cells in the presence of selected anti-class I monoclonal antibodies. The use of appropriate target cells represented by either HLA-homozygous cell lines or cell transfectants revealed that these clones recognized all the HLA-C alleles. By the use of monoclonal antibodies directed to either GL183 or EB6 molecules, we showed that the EB6 molecules were responsible for the recognition of Cw4 and related alleles, while the GL183 molecules recognized Cw3 (and related C alleles). These data suggest that the GL183 and the EB6 molecules can function, in individual NK clones, as independent receptors for two different groups of HLA-C alleles, (which include all known alleles for locus C), thus resulting in their inability to lyse all normal HLA-C+ target cells. Indirect immunofluorescence and fluorescence-activated cell sorting analysis revealed that the presently defined GL183+EB6+ group 0 NK clones brightly express EB6 molecules (EB6bright) while the GL183+EB6+ group 2 clones (unable to recognize Cw4) express an EB6dull phenotype. These data also imply that the density of EB6 receptors may be critical for the generation of an optimal negative signal upon interaction with appropriate HLA-C alleles.

Crystal structure of an H-2Kb-ovalbumin peptide complex reveals the interplay of primary and secondary anchor positions in the major histocompatibility complex binding groove.

Sequence analysis of peptides naturally presented by major histocompatibility complex (MHC) class I molecules has revealed allele-specific motifs in which the peptide length and the residues observed at certain positions are restricted. Nevertheless, peptides containing the standard motif often fail to bind with high affinity or form physiologically stable complexes. Here we present the crystal structure of a well-characterized antigenic peptide from ovalbumin [OVA-8, ovalbumin-(257-264), SIINFEKL] in complex with the murine MHC class I H-2Kb molecule at 2.5-A resolution. Hydrophobic peptide residues Ile-P2 and Phe-P5 are packed closely together into binding pockets B and C, suggesting that the interplay of peptide anchor (P5) and secondary anchor (P2) residues can couple the preferred sequences at these positions. Comparison with the crystal structures of H-2Kb in complex with peptides VSV-8 (RGYVYQGL) and SEV-9 (FAPGNYPAL), where a Tyr residue is used as the C pocket anchor, reveals that the conserved water molecule that binds into the B pocket and mediates hydrogen bonding from the buried anchor hydroxyl group could not be likewise positioned if the P2 side chain were of significant size. Based on this structural evidence, H-2Kb has at least two submotifs: one with Tyr at P5 (or P6 for nonamer peptides) and a small residue at P2 (i.e., Ala or Gly) and another with Phe at P5 and a medium-sized hydrophobic residue at P2 (i.e., Ile). Deciphering of these secondary submotifs from both crystallographic and immunological studies of MHC peptide binding should increase the accuracy of T-cell epitope prediction.

Detecção e caracterização moleculares dos paramixovírus aviários tipo 1 em materiais provenientes de aves silvestres utilizando testes para a detecção dos vírus da família viral Paramyxoviridae

As aves silvestres são importantes reservatórios de vírus que podem acometer as aves domésticas. O monitoramento da circulação viral em aves silvestres é de extrema importância para garantir a sanidade dos plantéis avícolas. O presente estudo teve como objetivo 1) comparar dois testes moleculares de RT-PCR para a detecção dos vírus da família Paramyxoviridae em aves silvestres e sinantrópicas; 2) caracterizar os vírus detectados nestas amostras. Dois testes de RT-PCR e testes específicos de RT-PCR em tempo real (RRT-PCR) para o vírus da doença de Newcastle (NDV) e o metapneumovírus aviário (aMPV) foram utilizados para comparar o limite de detecção entre as amostras. As amostras de aves silvestres foram testadas por dois testes de RT-PCR. Um pequeno fragmento da região do sítio de clivagem do gene F das amostras positivas foi sequenciado. Os testes de RT-PCR foram validados com sucesso, mas apresentaram diferenças entre os limites de detecção quando comparados aos testes específicos de RRT-PCR utilizando diferentes vírus. No total, 100 amostras de aves (suabes) foram testados pelo teste RT-PCR que apresentou um limite de detecção similar entre os diferentes agentes virais. O teste selecionado foi capaz de detectar duas amostras de aves silvestres que foram também detectadas pelo testes específico para NDV e relacionadas às amostras de NDV vacinais do genótipo II da classe II referentes aos vírus de NDV lentogênico (113RQGR ↓ L117). Nosso estudo demonstra a deficiência na biosseguridade adotada pelos sistemas avícolas por permitir a saída dos vírus vacinais para as aves silvestres

Calidad de la dieta española según el índice de alimentación saludable

Objetivo: determinar la calidad de la dieta española mediante el Índice de Alimentación-Saludable (IASE) y su relación con variables geográficas y socioeconómicas. Metodología: Estudio descriptivo transversal a partir de Encuesta-Nacional-Salud-2006 (ENS-2006) Se estudiaron 29.478 personas (Mujeres = 15.019; Hombres = 14.459) que respondieron el Cuestionario de Frecuencia de Consumo (CFC). El IASE se compone de 10 variables (Cereales-derivados, Verduras-hortalizas, Frutas, Leche-derivados, Carnes, Legumbres, Embutidos-fiambres, Dulces, Refrescos-azúcar y Variedad-dieta), construidas a partir del CFC y las recomendaciones de las Guías-Alimentarias (Sociedad-Española-Nutrición-Comunitaria-2004). Categorías IASE (puntuación-máxima 100): Alimentación-saludable: > 80 puntos; Necesita-cambios: > 5.080; Poco-saludable: 50. Se realizó un análisis descriptivo, de diferencias de medias (pruebas Kruskal–Wallis y Mann–Whitney), y prueba Chi-Cuadrado, para estudiar la independencia de las variables edad, sexo, clase-social y nivel de estudios con las categorías de IASE. Resultados: El 72% del total de la muestra necesita cambios en su alimentación. La puntuación media para mujeres es 73,7 ± 10,5 y para hombres 69,9 ± 11,3 (p < 0,001). En la categoría saludable obtienen mayor porcentaje (38,8%) el grupo de edad > 65 años y las mujeres (28,3%) frente a los hombres (18,4%). Así mismo, las clases-sociales más altas (clase-I: 24,4%, clase-II: 25,0%, clase-III: 25,8%) presentan mayor índice de alimentación-saludable, (p < 0,001). Las Comunidades-Autónomas: Comunitat Valenciana (5,4%), Illes Balears (4,6%) y Andalucía (4,3%) son las que presentan mayor índice en la categoría poco-saludable. Conclusiones: El IASE es un método rápido y económico de estimación de la calidad de la dieta de la población, porque utiliza datos secundarios procedente de la ENS y de las guías-alimentarias; siendo útil en la planificación de políticas nutricionales en España.

Red supergiants around the obscured open cluster Stephenson 2

Context. Several clusters of red supergiants have been discovered in a small region of the Milky Way close to the base of the Scutum-Crux Arm and the tip of the Long Bar. Population synthesis models indicate that they must be very massive to harbour so many supergiants. Amongst these clusters, Stephenson 2, with a core grouping of 26 red supergiants, is a strong candidate to be the most massive young cluster in the Galaxy. Aims. Stephenson 2 is located close to a region where a strong over-density of red supergiants had been found. We explore the actual cluster size and its possible connection to this over-density. Methods. Taking advantage of Virtual Observatory tools, we have performed a cross-match between the DENIS, USNO-B1 and 2MASS catalogues to identify candidate obscured luminous red stars around Stephenson 2, and in a control nearby region. More than 600 infrared bright stars fulfill our colour criteria, with the vast majority having a counterpart in the I band and >400 being sufficiently bright in I to allow observation with a 4-m class telescope. We observed a subsample of ~250 stars, using the multi-object, wide-field, fibre spectrograph AF2 on the WHT telescope in La Palma, obtaining intermediate-resolution spectroscopy in the 7500–9000 Å range. We derived spectral types and luminosity classes for all these objects and measured their radial velocities. Results. Our targets turned out to be G and K supergiants, late (≥ M4) M giants, and M-type bright giants (luminosity class II) and supergiants. We found ~35 red supergiants with radial velocities similar to Stephenson 2 members, spread over the two areas surveyed. In addition, we found ~40 red supergiants with radial velocities incompatible in principle with a physical association. Conclusions. Our results show that Stephenson 2 is not an isolated cluster, but part of a huge structure likely containing hundreds of red supergiants, with radial velocities compatible with the terminal velocity at this Galactic longitude (and a distance ~6 kpc). In addition, we found evidence of several populations of massive stars at different distances along this line of sight.

Boston, Massachusetts Region 1:5,000 Color Ortho Imagery (1/2-meter Resolution), 2001

1/2-meter resolution 1:5,000 orthophoto image of the Boston region from April 2001. This datalayer is a subset (covering only the Boston region) of the Massachusetts statewide orthophoto image series available from MassGIS. It consists of 23 orthophoto quads mosaicked together (MassGIS orthophoto quad ID: 229890, 229894, 229898, 229902, 233886, 233890, 233894, 233898, 233902, 233906, 233910, 237890, 237894, 237898, 237902, 237906, 237910, 241890, 241894, 241898, 241902, 245898, 245902). These medium resolution true color images are considered the new "basemap" for the Commonwealth by MassGIS and the Executive Office of Environmental Affairs (EOEA). MassGIS/EOEA and the Massachusetts Highway Department jointly funded the project. The photography for the mainland was captured in April 2001 when deciduous trees were mostly bare and the ground was generally free of snow. The geographic extent of this dataset is the same as that of the MassGIS dataset: Boston, Massachusetts Region LIDAR First Return Elevation Data, 2002 [see cross references].

Doença celíaca num doente com síndrome poliglandular autoimune tipo III : caso clínico

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

Short Peptide Vaccine Induces CD4+ T Helper Cells in Patients with Different Solid Cancers

Previous cancer vaccination trials often aimed to activate CD8(+) cytotoxic T-cell (CTL) responses with short (8-10mer) peptides and targeted CD4(+) helper T cells (TH) with HLA class II-binding longer peptides (12-16 mer) that were derived from tumor antigens. Accordingly, a study of immunomonitoring focused on the detection of CTL responses to the short, and TH responses to the long, peptides. The possible induction of concurrent TH responses to short peptides was widely neglected. In a recent phase I vaccination trial, 53 patients with different solid cancers were vaccinated with EMD640744, a cocktail of five survivin-derived short (9- or 10-mer) peptides in Montanide ISA 51VG. We monitored 49 patients and found strong CD8(+) T-cell responses in 63% of the patients. In addition, we unexpectedly found CD4(+) TH cell responses against at least two of the five short peptides in 61% (23/38) of the patients analyzed. The two peptides were recognized by HLA-DP4- and HLA-DR-restricted TH1 cells. Some short peptide-reactive (sp)CD4 T cells showed high functional avidity. Here, we show that a short peptide vaccine is able to activate a specific CD4(+) T-cell repertoire in many patients, facilitating a strong combined CD4(+)/CD8(+) T-cell response. Cancer Immunol Res; 4(1); 18-25. ©2015 AACR.

American state papers : documents, legislative and executive, of the Congress of the United States ... /

Nos. 1-38 of the Congressional series.

Tuning the metal-to-metal charge transfer energy of cyano-bridged dinuclear complexes

The metal-to-metal charge transfer (MMCT) transitions of a series of Class II mixed valence dinuclear complexes bearing cyano bridging ligands may be varied systematically by variations to either the hexacyanometallate(II) donor or Co-III acceptor moieties. Specifically, the new dinuclear species trans-[(LCoNCFe)-Co-14S(CN)(5)](-) (L-14S = 6-methyl-1,11-diaza-4,8-dithia- cyclotetradecane-6-amine) and trans-[(LCoNCRu)-Co-14(CN)(5)]-(L-14 = 6-methyl-1,4,8,11-tetraazacyclotetradecane-6-amine) have been prepared and their spectroscopic and electrochemical properties are compared with the relative trans-[(LCoNCFe)-Co-14(CN)(5)](-). The crystal structures of Na{trans-[(LCoNCFe)-Co-14S(CN)(5)]}.51/2H(2)O.1/2EtOH, Na{trans-[(LCoNCRu)-Co-14(CN)(5)]}.3H(2)O and Na{trans-[(LCoNCRu)-Co-14(CN)(5)]}.8H(2)O are also reported. The ensuing changes to the MMCT energy have been examined within the framework of Hush theory, and it was found that the free energy change between the redox isomers was the dominant effect in altering the energy of the MMCT transition.