Using cloud technologies for engaging people with cultural heritage

Cultural heritage is an important asset of Europe which is largely underexplored. One of the main reasons is that the general public do not really incorporate cultural activities in their life style. Currently, curators and professionals in the heritage sector face the toughest challenges on how to attract, engage and retain visitors of heritage institutions (libraries, museums, archives and historical societies). TAG CLOUD FP7 European project seeks to overcome this situation and promote lifelong engagement with culture by personalising the visitors? cultural experiences through cloud technologies.

Optimal Piecewise Linear Function Approximation for GPU-based Applications

Many computer vision and human-computer interaction applications developed in recent years need evaluating complex and continuous mathematical functions as an essential step toward proper operation. However, rigorous evaluation of this kind of functions often implies a very high computational cost, unacceptable in real-time applications. To alleviate this problem, functions are commonly approximated by simpler piecewise-polynomial representations. Following this idea, we propose a novel, efficient, and practical technique to evaluate complex and continuous functions using a nearly optimal design of two types of piecewise linear approximations in the case of a large budget of evaluation subintervals. To this end, we develop a thorough error analysis that yields asymptotically tight bounds to accurately quantify the approximation performance of both representations. It provides an improvement upon previous error estimates and allows the user to control the trade-off between the approximation error and the number of evaluation subintervals. To guarantee real-time operation, the method is suitable for, but not limited to, an efficient implementation in modern Graphics Processing Units (GPUs), where it outperforms previous alternative approaches by exploiting the fixed-function interpolation routines present in their texture units. The proposed technique is a perfect match for any application requiring the evaluation of continuous functions, we have measured in detail its quality and efficiency on several functions, and, in particular, the Gaussian function because it is extensively used in many areas of computer vision and cybernetics, and it is expensive to evaluate.

User experience with immersive and interactive media = Experiencia de usuario en entornos 3D inmersivos e interactivos

Estudios recientes promueven la integración de estímulos multisensoriales en activos multimedia con el fin de mejorar la experiencia de usuario mediante la estimulación de nuevos sentidos, más allá de la tradicional experiencia audiovisual. Del mismo modo, varios trabajos proponen la introducción de componentes de interacción capaces de complementar con nuevas características, funcionalidades y/o información la experiencia multimedia. Efectos sensoriales basados en el uso de nuevas técnicas de audio, olores, viento, vibraciones y control de la iluminación, han demostrado tener un impacto favorable en la sensación de Presencia, en el disfrute de la experiencia multimedia y en la calidad, relevancia y realismo de la misma percibidos por el usuario. Asimismo, los servicios basados en dos pantallas y la manipulación directa de (elementos en) la escena de video tienen el potencial de mejorar la comprensión, la concentración y la implicación proactiva del usuario en la experiencia multimedia. El deporte se encuentra entre los géneros con mayor potencial para integrar y explotar éstas soluciones tecnológicas. Trabajos previos han demostrado asimismo la viabilidad técnica de integrar éstas tecnologías con los estándares actualmente adoptados a lo largo de toda la cadena de transmisión de televisión. De este modo, los sistemas multimedia enriquecidos con efectos sensoriales, los servicios interactivos multiplataforma y un mayor control del usuario sobre la escena de vídeo emergen como nuevas formas de llevar la multimedia immersiva e interactiva al mercado de consumo de forma no disruptiva. Sin embargo, existen numerosas interrogantes relativas a los efectos sensoriales y/o soluciones interactivas más adecuadas para complementar un contenido audiovisual determinado o a la mejor manera de de integrar y combinar dichos componentes para mejorar la experiencia de usuario de un segmento de audiencia objetivo. Además, la evidencia científica sobre el impacto de factores humanos en la experiencia de usuario con estas nuevas formas de immersión e interacción en el contexto multimedia es aún insuficiente y en ocasiones, contradictoria. Así, el papel de éstos factores en el potencial de adopción de éstas tecnologías ha sido amplia-mente ignorado. La presente tesis analiza el impacto del audio binaural, efectos sensoriales (de iluminación y olfativos), interacción con objetos 3D integrados en la escena de vídeo e interacción con contenido adicional utilizando una segunda pantalla en la experiencia de usuario con contenidos de deporte. La posible influencia de dichos componentes en las variables dependientes se explora tanto a nivel global (efecto promedio) como en función de las características de los usuarios (efectos heterogéneos). Para ello, se ha llevado a cabo un experimento con usuarios orientado a explorar la influencia de éstos componentes immersivos e interactivos en dos grandes dimensiones de la experiencia multimedia: calidad y Presencia. La calidad de la experiencia multimedia se analiza en términos de las posibles variaciones asociadas a la calidad global y a la calidad del contenido, la imagen, el audio, los efectos sensoriales, la interacción con objetos 3D y la interacción con la segunda pantalla. El posible impacto en la Presencia considera dos de las dimensiones definidas por el cuestionario ITC-SOPI: Presencia Espacial (Spatial Presence) e Implicación (Engagement). Por último, los individuos son caracterizados teniendo en cuenta los siguientes atributos afectivos, cognitivos y conductuales: preferencias y hábitos en relación con el contenido, grado de conocimiento de las tecnologías integradas en el sistema, tendencia a involucrarse emocionalmente, tendencia a concentrarse en una actividad bloqueando estímulos externos y los cinco grandes rasgos de la personalidad: extroversión, amabilidad, responsabilidad, inestabilidad emocional y apertura a nuevas experiencias. A nivel global, nuestro estudio revela que los participantes prefieren el audio binaural frente al sistema estéreo y que los efectos sensoriales generan un aumento significativo del nivel de Presencia Espacial percibido por los usuarios. Además, las manipulaciones experimentales realizadas permitieron identificar una gran variedad de efectos heterogéneos. Un resultado interesante es que dichos efectos no se encuentran distribuidos de forma equitativa entre las medidas de calidad y Presencia. Nuestros datos revelan un impacto generalizado del audio binaural en la mayoría de las medidas de calidad y Presencia analizadas. En cambio, la influencia de los efectos sensoriales y de la interacción con la segunda pantalla se concentran en las medidas de Presencia y calidad, respectivamente. La magnitud de los efectos heterogéneos identificados está modulada por las siguientes características personales: preferencias en relación con el contenido, frecuencia con la que el usuario suele ver contenido similar, conocimiento de las tecnologías integradas en el demostrador, sexo, tendencia a involucrarse emocionalmente, tendencia a a concentrarse en una actividad bloqueando estímulos externos y niveles de amabilidad, responsabilidad y apertura a nuevas experiencias. Las características personales consideradas en nuestro experimento explicaron la mayor parte de la variación en las variables dependientes, confirmando así el importante (y frecuentemente ignorado) papel de las diferencias individuales en la experiencia multimedia. Entre las características de los usuarios con un impacto más generalizado se encuentran las preferencias en relación con el contenido, el grado de conocimiento de las tecnologías integradas en el sistema y la tendencia a involucrarse emocionalmente. En particular, los primeros dos factores parecen generar un conflicto de atención hacia el contenido versus las características/elementos técnicos del sistema, respectivamente. Asimismo, la experiencia multimedia de los fans del fútbol parece estar modulada por procesos emociona-les, mientras que para los no-fans predominan los procesos cognitivos, en particular aquellos directamente relacionados con la percepción de calidad. Abstract Recent studies encourage the integration of multi-sensorial stimuli into multimedia assets to enhance the user experience by stimulating other senses beyond sight and hearing. Similarly, the introduction of multi-modal interaction components complementing with new features, functionalities and/or information the multimedia experience is promoted. Sensory effects as odor, wind, vibration and light effects, as well as an enhanced audio quality, have been found to favour media enjoyment and to have a positive influence on the sense of Presence and on the perceived quality, relevance and reality of a multimedia experience. Two-screen services and a direct manipulation of (elements in) the video scene have the potential to enhance user comprehension, engagement and proactive involvement of/in the media experience. Sports is among the genres that could benefit the most from these solutions. Previous works have demonstrated the technical feasibility of implementing and deploying end-to-end solutions integrating these technologies into legacy systems. Thus, sensorially-enhanced media, two-screen services and an increased user control over the displayed scene emerge as means to deliver a new form of immersive and interactive media experiences to the mass market in a non-disruptive manner. However, many questions remain concerning issues as the specific interactive solutions or sensory effects that can better complement a given audiovisual content or the best way in which to integrate and combine them to enhance the user experience of a target audience segment. Furthermore, scientific evidence on the impact of human factors on the user experience with these new forms of immersive and interactive media is still insufficient and sometimes, contradictory. Thus, the role of these factors on the potential adoption of these technologies has been widely ignored. This thesis analyzes the impact of binaural audio, sensory (light and olfactory) effects, interaction with 3D objects integrated into the video scene and interaction with additional content using a second screen on the sports media experience. The potential influence of these components on the dependent variables is explored both at the overall level (average effect) and as a function of users’ characteristics (heterogeneous effects). To these aims, we conducted an experimental study exploring the influence of these immersive and interactive elements on the quality and Presence dimensions of the media experience. Along the quality dimension, we look for possible variations on the quality scores as-signed to the overall media experience and to the media components content, image, audio, sensory effects, interaction with 3D objects and interaction using the tablet device. The potential impact on Presence is analyzed by looking at two of the four dimensions defined by the ITC-SOPI questionnaire, namely Spatial Presence and Engagement. The users’ characteristics considered encompass the following personal affective, cognitive and behavioral attributes: preferences and habits in relation to the content, knowledge of the involved technologies, tendency to get emotionally involved and tendency to get absorbed in an activity and block out external distractors and the big five personality traits extraversion, agreeableness, conscientiousness, neuroticism and openness to experience. At the overall level, we found that participants preferred binaural audio than standard stereo audio and that sensory effects increase significantly the level of Spatial Presence. Several heterogeneous effects were also revealed as a result of our experimental manipulations. Interestingly, these effects were not equally distributed across the quality and Presence measures analyzed. Whereas binaural audio was foud to have an influence on the majority of the quality and Presence measures considered, the effects of sensory effects and of interaction with additional content through the tablet device concentrate mainly on the dimensions of Presence and on quality measures, respectively. The magnitude of these effects was modulated by individual’s characteristics, such as: preferences in relation to the content, frequency of viewing similar content, knowledge of involved technologies, gender, tendency to get emotionally involved, tendency to absorption and levels of agreeableness, conscientiousness and openness to experience. The personal characteristics collected in our experiment explained most of the variation in the dependent variables, confirming the frequently neglected role of individual differences on the media experience. Preferences in relation to the content, knowledge of involved technologies and tendency to get emotionally involved were among the user variables with the most generalized influence. In particular, the former two features seem to present a conflict in the allocation of attentional resources towards the media content versus the technical features of the system, respectively. Additionally, football fans’ experience seems to be modulated by emotional processes whereas for not fans, cognitive processes (and in particular those related to quality judgment) prevail.

Estimación eficiente de atributos demográficos del rostro humano en imágenes

Sin duda, el rostro humano ofrece mucha más información de la que pensamos. La cara transmite sin nuestro consentimiento señales no verbales, a partir de las interacciones faciales, que dejan al descubierto nuestro estado afectivo, actividad cognitiva, personalidad y enfermedades. Estudios recientes [OFT14, TODMS15] demuestran que muchas de nuestras decisiones sociales e interpersonales derivan de un previo análisis facial de la cara que nos permite establecer si esa persona es confiable, trabajadora, inteligente, etc. Esta interpretación, propensa a errores, deriva de la capacidad innata de los seres humanas de encontrar estas señales e interpretarlas. Esta capacidad es motivo de estudio, con un especial interés en desarrollar métodos que tengan la habilidad de calcular de manera automática estas señales o atributos asociados a la cara. Así, el interés por la estimación de atributos faciales ha crecido rápidamente en los últimos años por las diversas aplicaciones en que estos métodos pueden ser utilizados: marketing dirigido, sistemas de seguridad, interacción hombre-máquina, etc. Sin embargo, éstos están lejos de ser perfectos y robustos en cualquier dominio de problemas. La principal dificultad encontrada es causada por la alta variabilidad intra-clase debida a los cambios en la condición de la imagen: cambios de iluminación, oclusiones, expresiones faciales, edad, género, etnia, etc.; encontradas frecuentemente en imágenes adquiridas en entornos no controlados. Este de trabajo de investigación estudia técnicas de análisis de imágenes para estimar atributos faciales como el género, la edad y la postura, empleando métodos lineales y explotando las dependencias estadísticas entre estos atributos. Adicionalmente, nuestra propuesta se centrará en la construcción de estimadores que tengan una fuerte relación entre rendimiento y coste computacional. Con respecto a éste último punto, estudiamos un conjunto de estrategias para la clasificación de género y las comparamos con una propuesta basada en un clasificador Bayesiano y una adecuada extracción de características. Analizamos en profundidad el motivo de porqué las técnicas lineales no han logrado resultados competitivos hasta la fecha y mostramos cómo obtener rendimientos similares a las mejores técnicas no-lineales. Se propone un segundo algoritmo para la estimación de edad, basado en un regresor K-NN y una adecuada selección de características tal como se propuso para la clasificación de género. A partir de los experimentos desarrollados, observamos que el rendimiento de los clasificadores se reduce significativamente si los ´estos han sido entrenados y probados sobre diferentes bases de datos. Hemos encontrado que una de las causas es la existencia de dependencias entre atributos faciales que no han sido consideradas en la construcción de los clasificadores. Nuestro resultados demuestran que la variabilidad intra-clase puede ser reducida cuando se consideran las dependencias estadísticas entre los atributos faciales de el género, la edad y la pose; mejorando el rendimiento de nuestros clasificadores de atributos faciales con un coste computacional pequeño. Abstract Surely the human face provides much more information than we think. The face provides without our consent nonverbal cues from facial interactions that reveal our emotional state, cognitive activity, personality and disease. Recent studies [OFT14, TODMS15] show that many of our social and interpersonal decisions derive from a previous facial analysis that allows us to establish whether that person is trustworthy, hardworking, intelligent, etc. This error-prone interpretation derives from the innate ability of human beings to find and interpret these signals. This capability is being studied, with a special interest in developing methods that have the ability to automatically calculate these signs or attributes associated with the face. Thus, the interest in the estimation of facial attributes has grown rapidly in recent years by the various applications in which these methods can be used: targeted marketing, security systems, human-computer interaction, etc. However, these are far from being perfect and robust in any domain of problems. The main difficulty encountered is caused by the high intra-class variability due to changes in the condition of the image: lighting changes, occlusions, facial expressions, age, gender, ethnicity, etc.; often found in images acquired in uncontrolled environments. This research work studies image analysis techniques to estimate facial attributes such as gender, age and pose, using linear methods, and exploiting the statistical dependencies between these attributes. In addition, our proposal will focus on the construction of classifiers that have a good balance between performance and computational cost. We studied a set of strategies for gender classification and we compare them with a proposal based on a Bayesian classifier and a suitable feature extraction based on Linear Discriminant Analysis. We study in depth why linear techniques have failed to provide competitive results to date and show how to obtain similar performances to the best non-linear techniques. A second algorithm is proposed for estimating age, which is based on a K-NN regressor and proper selection of features such as those proposed for the classification of gender. From our experiments we note that performance estimates are significantly reduced if they have been trained and tested on different databases. We have found that one of the causes is the existence of dependencies between facial features that have not been considered in the construction of classifiers. Our results demonstrate that intra-class variability can be reduced when considering the statistical dependencies between facial attributes gender, age and pose, thus improving the performance of our classifiers with a reduced computational cost.

REFLEXOS INDIVIDUAIS DO USO PESSOAL E EXCESSIVO DE COMUNICADORES INSTANTÂNEOS NO AMBIENTE DE TRABALHO

A Internet está inserida no cotidiano do indivíduo, e torna-se cada vez mais acessível por meio de diferentes tipos de dispositivos. Com isto, diversos estudos foram realizados com o intuito de avaliar os reflexos do seu uso excessivo na vida pessoal, acadêmica e profissional. Esta dissertação buscou identificar se a perda de concentração e o isolamento social são alguns dos reflexos individuais que o uso pessoal e excessivo de aplicativos de comunicação instantânea podem resultar no ambiente de trabalho. Entre as variáveis selecionadas para avaliar os aspectos do uso excessivo de comunicadores instantâneos tem-se a distração digital, o controle reduzido de impulso, o conforto social e a solidão. Através de uma abordagem de investigação quantitativa, utilizaram-se escalas aplicadas a uma amostra de 283 pessoas. Os dados foram analisados por meio de técnicas estatísticas multivariadas como a Análise Fatorial Exploratória e para auferir a relação entre as variáveis, a Regressão Linear Múltipla. Os resultados deste estudo confirmam que o uso excessivo de comunicadores instantâneos está positivamente relacionado com a perda de concentração, e a variável distração digital exerce uma influência maior do que o controle reduzido de impulso. De acordo com os resultados, não se podem afirmar que a solidão e o conforto social exercem relações com aumento do isolamento social, devido à ausência do relacionamento entre os construtos.

The C-terminal SET domains of ALL-1 and TRITHORAX interact with the INI1 and SNR1 proteins, components of the SWI/SNF complex

The ALL-1 gene was discovered by virtue of its involvement in human acute leukemia. Its Drosophila homolog trithorax (trx) is a member of the trx-Polycomb gene family, which maintains correct spatial expression of the Antennapedia and bithorax complexes during embryogenesis. The C-terminal SET domain of ALL-1 and TRITHORAX (TRX) is a 150-aa motif, highly conserved during evolution. We performed yeast two hybrid screening of Drosophila cDNA library and detected interaction between a TRX polypeptide spanning SET and the SNR1 protein. SNR1 is a product of snr1, which is classified as a trx group gene. We found parallel interaction in yeast between the SET domain of ALL-1 and the human homolog of SNR1, INI1 (hSNF5). These results were confirmed by in vitro binding studies and by demonstrating coimmunoprecipitation of the proteins from cultured cells and/or transgenic flies. Epitope-tagged SNR1 was detected at discrete sites on larval salivary gland polytene chromosomes, and these sites colocalized with around one-half of TRX binding sites. Because SNR1 and INI1 are constituents of the SWI/SNF complex, which acts to remodel chromatin and consequently to activate transcription, the interactions we observed suggest a mechanism by which the SWI/SNF complex is recruited to ALL-1/trx targets through physical interactions between the C-terminal domains of ALL-1 and TRX and INI1/SNR1.

Molecular cloning and functional analysis of SUT-1, a sulfate transporter from human high endothelial venules

High endothelial venules (HEV) are specialized postcapillary venules found in lymphoid organs and chronically inflamed tissues that support high levels of lymphocyte extravasation from the blood. One of the major characteristics of HEV endothelial cells (HEVEC) is their capacity to incorporate large amounts of sulfate into sialomucin-type counter-receptors for the lymphocyte homing receptor L-selectin. Here, we show that HEVEC express two functional classes of sulfate transporters defined by their differential sensitivity to the anion-exchanger inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), and we report the molecular characterization of a DIDS-resistant sulfate transporter from human HEVEC, designated SUT-1. SUT-1 belongs to the family of Na+-coupled anion transporters and exhibits 40–50% amino acid identity with the rat renal Na+/sulfate cotransporter, NaSi-1, as well as with the human and rat Na+/dicarboxylate cotransporters, NaDC-1/SDCT1 and NaDC-3/SDCT2. Functional expression studies in cRNA-injected Xenopus laevis oocytes showed that SUT-1 mediates high levels of Na+-dependent sulfate transport, which is resistant to DIDS inhibition. The SUT-1 gene mapped to human chromosome 7q33. Northern blotting analysis revealed that SUT-1 exhibits a highly restricted tissue distribution, with abundant expression in placenta. Reverse transcription–PCR analysis indicated that SUT-1 and the diastrophic dysplasia sulfate transporter (DTD), one of the two known human DIDS-sensitive sulfate transporters, are coexpressed in HEVEC. SUT-1 and DTD could correspond, respectively, to the DIDS-resistant and DIDS-sensitive components of sulfate uptake in HEVEC. Together, these results demonstrate that SUT-1 is a distinct human Na+-coupled sulfate transporter, likely to play a major role in sulfate incorporation in HEV.

ETS target genes: Identification of Egr1 as a target by RNA differential display and whole genome PCR techniques

ETS transcription factors play important roles in hematopoiesis, angiogenesis, and organogenesis during murine development. The ETS genes also have a role in neoplasia, for example in Ewing’s sarcomas and retrovirally induced cancers. The ETS genes encode transcription factors that bind to specific DNA sequences and activate transcription of various cellular and viral genes. To isolate novel ETS target genes, we used two approaches. In the first approach, we isolated genes by the RNA differential display technique. Previously, we have shown that the overexpression of ETS1 and ETS2 genes effects transformation of NIH 3T3 cells and specific transformants produce high levels of the ETS proteins. To isolate ETS1 and ETS2 responsive genes in these transformed cells, we prepared RNA from ETS1, ETS2 transformants, and normal NIH 3T3 cell lines and converted it into cDNA. This cDNA was amplified by PCR and displayed on sequencing gels. The differentially displayed bands were subcloned into plasmid vectors. By Northern blot analysis, several clones showed differential patterns of mRNA expression in the NIH 3T3-, ETS1-, and ETS2-expressing cell lines. Sixteen clones were analyzed by DNA sequence analysis, and 13 of them appeared to be unique because their DNA sequences did not match with any of the known genes present in the gene bank. Three known genes were found to be identical to the CArG box binding factor, phospholipase A2-activating protein, and early growth response 1 (Egr1) genes. In the second approach, to isolate ETS target promoters directly, we performed ETS1 binding with MboI-cleaved genomic DNA in the presence of a specific mAb followed by whole genome PCR. The immune complex-bound ETS binding sites containing DNA fragments were amplified and subcloned into pBluescript and subjected to DNA sequence and computer analysis. We found that, of a large number of clones isolated, 43 represented unique sequences not previously identified. Three clones turned out to contain regulatory sequences derived from human serglycin, preproapolipoprotein C II, and Egr1 genes. The ETS binding sites derived from these three regulatory sequences showed specific binding with recombinant ETS proteins. Of interest, Egr1 was identified by both of these techniques, suggesting strongly that it is indeed an ETS target gene.

Cloning of a novel T-cell protein FYB that binds FYN and SH2-domain-containing leukocyte protein 76 and modulates interleukin 2 production

T cell receptor ζ (TcRζ)/CD3 ligation initiates a signaling cascade that involves src kinases p56lck and ζ-associated protein 70, leading to the phosphorylation of substrates such as TcRζ, Vav, SH2-domain-containing leukocyte protein 76 (SLP-76), cbl, and p120/130. FYN binding protein (FYB or p120/130) associates with p59fyn, the TcRζ/CD3 complex, and becomes tyrosine-phosphorylated in response to receptor ligation. In this study, we report the cDNA cloning of human and murine FYB and show that it is restricted in expression to T cells and myeloid cells and possesses an overall unique hydrophilic sequence with several tyrosine-based motifs, proline-based type I and type II SH3 domain binding motifs, several putative lysine/glutamic acid-rich nuclear localization motifs, and a SH3-like domain. In addition to binding the src kinase p59fyn, FYB binds specifically to the hematopoietic signaling protein SLP-76, an interaction mediated by the SLP-76 SH2 domain. In keeping with this, expression of FYB augmented interleukin 2 secretion from a T cell hybridoma, DC27.10, in response to TcRζ/CD3 ligation. FYB is therefore a novel hematopoietic protein that acts as a component of the FYN and SLP-76 signaling cascades in T cells.

Apoprotein B100 has a prolonged interaction with the translocon during which its lipidation and translocation change from dependence on the microsomal triglyceride transfer protein to independence

When lipid synthesis is limited in HepG2 cells, apoprotein B100 (apoB100) is not secreted but rapidly degraded by the ubiquitin-proteasome pathway. To investigate apoB100 biosynthesis and secretion further, the physical and functional states of apoB100 destined for either degradation or lipoprotein assembly were studied under conditions in which lipid synthesis, proteasomal activity, and microsomal triglyceride transfer protein (MTP) lipid-transfer activity were varied. Cells were pretreated with a proteasomal inhibitor (which remained with the cells throughout the experiment) and radiolabeled for 15 min. During the chase period, labeled apoB100 remained associated with the microsomes. Furthermore, by crosslinking sec61β to apoB100, we showed that apoB100 remained close to the translocon at the same time apoB100–ubiquitin conjugates could be detected. When lipid synthesis and lipoprotein assembly/secretion were stimulated by adding oleic acid (OA) to the chase medium, apoB100 was deubiquitinated, and its interaction with sec61β was disrupted, signifying completion of translocation concomitant with the formation of lipoprotein particles. MTP participates in apoB100 translocation and lipoprotein assembly. In the presence of OA, when MTP lipid-transfer activity was inhibited at the end of pulse labeling, apoB100 secretion was abolished. In contrast, when the labeled apoB100 was allowed to accumulate in the cell for 60 min before adding OA and the inhibitor, apoB100 lipidation and secretion were no longer impaired. Overall, the data imply that during most of its association with the endoplasmic reticulum, apoB100 is close to or within the translocon and is accessible to both the ubiquitin-proteasome and lipoprotein-assembly pathways. Furthermore, MTP lipid-transfer activity seems to be necessary only for early translocation and lipidation events.

Retina-specific nuclear receptor: A potential regulator of cellular retinaldehyde-binding protein expressed in retinal pigment epithelium and Müller glial cells

In an effort to identify nuclear receptors important in retinal disease, we screened a retina cDNA library for nuclear receptors. Here we describe the identification of a retina-specific nuclear receptor (RNR) from both human and mouse. Human RNR is a splice variant of the recently published photoreceptor cell-specific nuclear receptor [Kobayashi, M., Takezawa, S., Hara, K., Yu, R. T., Umesono, Y., Agata, K., Taniwaki, M., Yasuda, K. & Umesono, K. (1999) Proc. Natl. Acad. Sci. USA 96, 4814–4819] whereas the mouse RNR is a mouse ortholog. Northern blot and reverse transcription–PCR analyses of human mRNA samples demonstrate that RNR is expressed exclusively in the retina, with transcripts of ≈7.5 kb, ≈3.0 kb, and ≈2.3 kb by Northern blot analysis. In situ hybridization with multiple probes on both primate and mouse eye sections demonstrates that RNR is expressed in the retinal pigment epithelium and in Müller glial cells. By using the Gal4 chimeric receptor/reporter cotransfection system, the ligand binding domain of RNR was found to repress transcriptional activity in the absence of exogenous ligand. Gel mobility shift assays revealed that RNR can interact with the promoter of the cellular retinaldehyde binding protein gene in the presence of retinoic acid receptor (RAR) and/or retinoid X receptor (RXR). These data raise the possibility that RNR acts to regulate the visual cycle through its interaction with cellular retinaldehyde binding protein and therefore may be a target for retinal diseases such as retinitis pigmentosa and age-related macular degeneration.

HIV-1 Tat transactivator recruits p300 and CREB-binding protein histone acetyltransferases to the viral promoter

In cells infected with HIV type 1 (HIV-1), the integrated viral promoter is present in a chromatin-bound conformation and is transcriptionally silent in the absence of stimulation. The HIV-1 Tat protein binds to a stem-loop structure at the 5′ end of viral mRNA and relieves this inhibition by inducing a remodeling of the nucleosome arrangement downstream of the transcription-initiation site. Here we show that Tat performs this activity by recruiting to the viral long terminal repeat (LTR) the transcriptional coactivator p300 and the closely related CREB-binding protein (CBP), having histone acetyltransferase (HAT) activity. Tat associates with HAT activity in human nuclear extracts and binds to p300 and CBP both in vitro and in vivo. Integrity of the basic domain of Tat is essential for this interaction. By a quantitative chromatin immunoprecipitation assay we show that the delivery of recombinant Tat induces the association of p300 and CBP with the chromosomally integrated LTR promoter. Expression of human p300 in both human and rodent cells increases the levels of Tat transactivation of the integrated LTR. These results reinforce the evidence that p300 and CBP have a pivotal function at both cellular and viral promoters and demonstrate that they also can be recruited by an RNA-targeted activator. Additionally, these findings have important implications for the understanding of the mechanisms of HIV-1 latency and reactivation.

The amphiphysin-like protein 1 (ALP1) interacts functionally with the cABL tyrosine kinase and may play a role in cytoskeletal regulation

cABL is a protooncogene, activated in a subset of human leukemias, whose protein product is a nonreceptor tyrosine kinase of unknown function. cABL has a complex structure that includes several domains and motifs found in proteins implicated in signal transduction pathways. An approach to elucidate cABL function is to identify proteins that interact directly with cABL and that may serve as regulators or effectors of its activity. To this end, a protein-interaction screen of a phage expression library was undertaken to identify proteins that interact with specific domains of cABL. An SH3-domain-containing protein has been identified that interacts with sequences in the cABL carboxyl terminus. The cDNA encoding ALP1 (amphiphysin-like protein 1) was isolated from a 16-day mouse embryo. ALP1 has high homology to BIN1, a recently cloned myc-interacting protein, and also shows significant homology to amphiphysin, a neuronal protein cloned from human and chicken. The amino terminus has homology to two yeast proteins, Rvs167 and Rvs161, which are involved in cell entry into stationary phase and cytoskeletal organization. ALP1 binds cABL in vitro and in vivo. Expression of ALP1 results in morphological transformation of NIH 3T3 fibroblasts in a cABL-dependent manner. The properties of ALP1 suggest that it may be involved in possible cytoskeletal functions of the cABL kinase. Additionally, these results provide further evidence for the importance of the cABL carboxyl terminus and its binding proteins in the regulation of cABL function.

Mice transgenic for human CD4 and CCR5 are susceptible to HIV infection

HIV entry into human cells is mediated by CD4 acting in concert with one of several members of the chemokine receptor superfamily. The resistance to HIV infection observed in individuals with defective CCR5 alleles indicated that this particular chemokine receptor plays a crucial role in the initiation of in vivo HIV infection. Expression of human CD4 transgene does not render mice susceptible to HIV infection because of structural differences between human and mouse CCR5. To ascertain whether expression of human CD4 and CCR5 is sufficient to make murine T lymphocytes susceptible to HIV infection, the lck promoter was used to direct the T cell-specific expression of human CD4 and CCR5 in transgenic mice. Peripheral blood mononuclear cells and splenocytes isolated from these mice expressed human CD4 and CCR5 and were infectible with selected M-tropic HIV isolates. After in vivo inoculation, HIV-infected cells were detected by DNA PCR in the spleen and lymph nodes of these transgenic mice, but HIV could not be cultured from these cells. This indicated that although transgenic expression of human CD4 and CCR5 permitted entry of HIV into the mouse cells, significant HIV infection was prevented by other blocks to HIV replication present in mouse cells. In addition to providing in vivo verification for the important role of CCR5 in T lymphocyte HIV infection, these transgenic mice represent a new in vivo model for understanding HIV pathogenesis by delineating species-specific cellular factors required for productive in vivo HIV infection. These mice should also prove useful for the assessment of potential therapeutic and preventative modalities, particularly vaccines.

Urokinase-Type Plasminogen Activator Receptor Is Internalized by Different Mechanisms in Polarized and Nonpolarized Madin–Darby Canine Kidney Epithelial Cells

Accumulated data indicate that endocytosis of the glycosylphosphatidyl-inositol-anchored protein urokinase plasminogen activator receptor (uPAR) depends on binding of the ligand uPA:plasminogen activator inhibitor-1 (PAI-1) and subsequent interaction with internalization receptors of the low-density lipoprotein receptor family, which are internalized through clathrin-coated pits. This interaction is inhibited by receptor-associated protein (RAP). We show that uPAR with bound uPA:PAI-1 is capable of entering cells in a clathrin-independent process. First, HeLaK44A cells expressing mutant dynamin efficiently internalized uPA:PAI-1 under conditions in which transferrin endocytosis was blocked. Second, in polarized Madin–Darby canine kidney (MDCK) cells, which expressed human uPAR apically, the low basal rate of uPAR ligand endocytosis, which could not be inhibited by RAP, was increased by forskolin or phorbol ester (phorbol 12-myristate 13-acetate), which selectively up-regulate clathrin-independent endocytosis from the apical domain of epithelial cells. Third, in subconfluent nonpolarized MDCK cells, endocytosis of uPA:PAI-1 was only decreased marginally by RAP. At the ultrastructural level uPAR was largely excluded from clathrin-coated pits in these cells and localized in invaginated caveolae only in the presence of cross-linking antibodies. Interestingly, a larger fraction of uPAR in nonpolarized relative to polarized MDCK cells was insoluble in Triton X-100 at 0°C, and by surface labeling with biotin we also show that internalized uPAR was mainly detergent insoluble, suggesting a correlation between association with detergent-resistant membrane microdomains and higher degree of clathrin-independent endocytosis. Furthermore, by cryoimmunogold labeling we show that 5–10% of internalized uPAR in nonpolarized, but not polarized, MDCK cells is targeted to lysosomes by a mechanism that is regulated by ligand occupancy.