KATS: A Knowledge Acquisition Tool Based on Electronic Document Processing

This paper describes a particular knowledge acquisition tool for the construction and maintenance of the knowledge model of an intelligent system for emergency management in the field of hydrology. This tool has been developed following an innovative approach directed to end-users non familiarized in computer oriented terminology. According to this approach, the tool is conceived as a document processor specialized in a particular domain (hydrology) in such a way that the whole knowledge model is viewed by the user as an electronic document. The paper first describes the characteristics of the knowledge model of the intelligent system and summarizes the problems that we found during the development and maintenance of such type of model. Then, the paper describes the KATS tool, a software application that we have designed to help in this task to be used by users who are not experts in computer programming. Finally, the paper shows a comparison between KATS and other approaches for knowledge acquisition.

Modelo de diseño orientado a marcos para sistemas basados en el conocimiento

El objetivo de esta Tesis es crear un Modelo de Diseño Orientado a Marcos que, intermedio entre el Mundo Externo y el Modelo Interno del Mundo que supone el sistema ímplementado, disminuya la pérdida de conocimiento que se produce al formalizar la realidad en Bases de Conocimientos. El modelo disminuye la pérdida de conocimiento al formalizar Bases de Conocimiento, acercando el formalismo de Marcos al Mundo Externo, porque: 1. Crea una base teórica que uniformiza el concepto de Marco en el plano de la Formalización, estableciendo un conjunto de restricciones sintácticas y semánticas que impedirán, al Ingeniero del Conocimiento (IC) cuando formaliza, definir elementos no permitidos o el uso indebido de ellos. 2. Se incrementa la expresividad del formalismo al asociar a cada una de las propiedades de un marco clase un parámetro adicional que simboliza la representatividad de la propiedad en el concepto. Este parámetro, y las técnicas de inferencia que trabajan con él, permitirán al IC introducir en el Modelo Formalizado conocimiento que antes no introducía al construir la base de conocimientos y que, sin embargo, sí existía en la realidad. 3. Se propone una técnica de equiparación que trabaja con el conocimiento incierto presente en el dominio. Esta técnica de equiparación, utiliza la representatividad de las propiedades en los marcos clase y el grado de certeza de las propiedades de las entidades para calcular el valor de equiparación y, así, determinar en qué medida los marcos clase seleccionados son consistentes con la descripción de la situación actual dada por una entidad. 4. Proporciona nuevas técnicas de inferencia basadas en la transferencia de propiedades y modifica las ya existentes. Las transferencias de propiedades realizadas sobre relaciones "ad hoc" definidas por el IC al construir el sistema, es una nueva técnica de inferencia independiente y complementaria a la transferencia de propiedades llamada tradicionalmente Herencia (cesión de propiedades entre padres e hijos). A esta nueva técnica, se le ha llamado Donación, es decir, cesión de propiedades entre marcos sin parentesco. Como aportación práctica, se ha construido un entorno de construcción de Sistemas Basados en el Conocimiento formalizados en Marcos, donde se han introducido todos los nuevos conceptos del Modelo Teórico de la Tesis. Se trata de una cierta anidación. Es decir, son marcos que permiten formalizar cualquier SBC en marcos. El entorno permitirá al IC formalizar bases de conocimientos automáticamente y éste podrá validar el conocimiento del dominio en la fase de formalización en lugar de tener que esperar a que la BC esté implementada. Todo ello lleva a describir el Modelo de Diseño Orientado a Marcos como un puente que aproxima y comunica el Mundo Externo con el Modelo Interno asociado a la realidad e implementado en una computadora, disminuyendo así las diversas pérdidas de conocimiento que si bien no ocurren simultáneamente al construir Sistemas Basados en el Conocimiento, sí coexisten en él.---ABSTRACT---The goal of this thesis is to créate a Frame-Orlented Deslgn Model that, bridging the Outside World and the implemented system's Internal Model of the World, reduces the amount of knowledge lost when reality is formalized in Knowledge Bases (KB). The model diminishes the loss of knowledge when formalizing a KB and brings the Frame-formalized Model closer to the Outside World because: 1. It creates a theory that standardizes the concept of trame at the formalization level to establish a set of syntactic and semantic constraints that will prevent the Knowledge Engineer (KE) from defining forbidden elements or their undue use in the formalization process. 2. The formalism's expressiveness is increased by associating an additional parameter to each of the properties of a class frame to symbolize the representativeness of the concept property. This parameter and the related inference techniques will allow the KE to enter knowledge into the Formalized Model that actually existed but that was not used previously when building the KB. 3. The proposed technique involves matching and works with uncertain knowledge present in the domain. This matching technique takes the representativeness of the properties in the class frame and the degree of certainty of the properties of the entities to calcúlate the matching valué and thus determine to what extent the class frames selected are consistent with the description of the present situation given by an entity. 4. It offers new inference techniques based on property transfer and alters existing ones. Property transfer on ad hoc relations defined by the KE when building a system is a new inference technique independent of and complementary to property transfer traditionally termed Inheritance (transfer of properties between parents and children). This new technique has been callad Donation (transfer of properties between trames without relationships). 5. It improves control of the procedural knowledge defined in the trames by introducing OO concepta. A frame-formalized KBS building environment has been constructed, incorporating all the new concepts of the theoretical model set out in the thesis. There is some embedding, that is, they are trames that provide for any KBS to be formalizad in trames. The environment will enable the KE to formaliza KB automatically, and he will be able to valídate the domain knowledge in the formalization stage instead of havíng to wait until the KB has been implemented. This is a description of the Frame-oriented Design Model, a bridge that brings closer and communicates the Outside World with the Interna! Model associated to reality and implemented on a computar, thus reducing the different losses in knowledge that, though they do not occur simultaneosly when building a Knowledge-based System, coexist within it.

A Semantic Approach to Problem-Based Learning with Conceptual Models

El aprendizaje basado en problemas se lleva aplicando con éxito durante las últimas tres décadas en un amplio rango de entornos de aprendizaje. Este enfoque educacional consiste en proponer problemas a los estudiantes de forma que puedan aprender sobre un dominio particular mediante el desarrollo de soluciones a dichos problemas. Si esto se aplica al modelado de conocimiento, y en particular al basado en Razonamiento Cualitativo, las soluciones a los problemas pasan a ser modelos que representan el compotamiento del sistema dinámico propuesto. Por lo tanto, la tarea del estudiante en este caso es acercar su modelo inicial (su primer intento de representar el sistema) a los modelos objetivo que proporcionan soluciones al problema, a la vez que adquieren conocimiento sobre el dominio durante el proceso. En esta tesis proponemos KaiSem, un método que usa tecnologías y recursos semánticos para guiar a los estudiantes durante el proceso de modelado, ayudándoles a adquirir tanto conocimiento como sea posible sin la directa supervisión de un profesor. Dado que tanto estudiantes como profesores crean sus modelos de forma independiente, estos tendrán diferentes terminologías y estructuras, dando lugar a un conjunto de modelos altamente heterogéneo. Para lidiar con tal heterogeneidad, proporcionamos una técnica de anclaje semántico para determinar, de forma automática, enlaces entre la terminología libre usada por los estudiantes y algunos vocabularios disponibles en la Web de Datos, facilitando con ello la interoperabilidad y posterior alineación de modelos. Por último, proporcionamos una técnica de feedback semántico para comparar los modelos ya alineados y generar feedback basado en las posibles discrepancias entre ellos. Este feedback es comunicado en forma de sugerencias individualizadas que el estudiante puede utilizar para acercar su modelo a los modelos objetivos en cuanto a su terminología y estructura se refiere. ABSTRACT Problem-based learning has been successfully applied over the last three decades to a diverse range of learning environments. This educational approach consists of posing problems to learners, so they can learn about a particular domain by developing solutions to them. When applied to conceptual modeling, and particularly to Qualitative Reasoning, the solutions to problems are models that represent the behavior of a dynamic system. Therefore, the learner's task is to move from their initial model, as their first attempt to represent the system, to the target models that provide solutions to that problem while acquiring domain knowledge in the process. In this thesis we propose KaiSem, a method for using semantic technologies and resources to scaffold the modeling process, helping the learners to acquire as much domain knowledge as possible without direct supervision from the teacher. Since learners and experts create their models independently, these will have different terminologies and structure, giving rise to a pool of models highly heterogeneous. To deal with such heterogeneity, we provide a semantic grounding technique to automatically determine links between the unrestricted terminology used by learners and some online vocabularies of the Web of Data, thus facilitating the interoperability and later alignment of the models. Lastly, we provide a semantic-based feedback technique to compare the aligned models and generate feedback based on the possible discrepancies. This feedback is communicated in the form of individualized suggestions, which can be used by the learner to bring their model closer in terminology and structure to the target models.

Especificación del comportamiento de un avatar en un mundo virtual mediante una ontología

El presente trabajo describe la construcción de una aplicación que controla a un Non Player Character (NPC), en un mundo virtual. La aplicación desarrollada, que tiene como nombre BotManager, realiza dos tareas fundamentales: 1) conectarse al repositorio de conocimiento, que en esta implementación es una ontología expresada en OWL, para obtener las acciones que debe realizar el NPC dentro del mundo virtual; y 2) ordenar al NPC que realice estas acciones en un mundo virtual creado con la plataforma OpenSimulator. BotManager puede tener variadas aplicaciones, por lo tanto puede ser usada como complemento en mundos virtuales aplicados a la educación, simulación, ocio, etc. Ahora bien, la principal razón que motivó el desarrollo del BotManager fue la de crear un sistema de demostración automática de tareas en un mundo virtual destinado a la educación/ entrenamiento. De esta forma, un Sistema Inteligente de Tutoría integrado con un mundo virtual podría demostrar paso a paso a un estudiante cómo realizar una tarea en el mundo virtual. La ontología que lee el BotManager extiende la ontología propuesta en la tesis “Una propuesta de modelado del estudiante basada en ontologías y diagnóstico pedagógico-cognitivo no monótono” de Julia Parraga en el 2011 (Ontología de Julia). La construcción y las pruebas del BotManager se llevaron a cabo en tres etapas: 1) creación de la Ontología de Acciones del NPC que extiende la Ontología de Julia; 2) diseño e implementación de la aplicación en C# que lee la ontología que contiene el plan de acción del NPC, y ordena al NPC realizar las acciones en el mundo virtual; y 3) pruebas de la aplicación con la práctica “preparación de una taza de cafe”, que es parte de un Laboratorio Virtual de Biotecnología. El BotManager se ha diseñado como una aplicación cliente que se conecta a un servidor de Open- Simulator. Por lo tanto, puede ejecutarse en una máquina distinta a la del servidor. Asimismo, en la implementación del BotManager se ha utilizado una librería gratuita denominada LibOpenMetaverse que permite controlar un NPC de forma remota.---ABSTRACT---This paper describes the construction of an application that controls a Non Player Character (NPC), in a virtual world. The application developed, called BotManager, performs two main tasks: 1) the connection to the repository of knowledge, which in this implementation is an ontology expressed in OWL, and retrieving the actions to be performed by the NPC within the virtual world; and 2) commanding the NPC to perform these actions in a virtual world created with the OpenSimulator platform. BotManager can have diverse applications, therefore it can be used as a complement in virtual worlds applied to education, simulation, entertainment, etc. However, the main reason behind the development of BotManager was to create an automatic demonstration of tasks in a virtual world for education / training. Thus, a virtual world integrated with an Intelligent Tutoring Systems could demonstrate step by step to a student how to perform a task in the virtual world. The ontology used by the BotManager extends ontology proposed in the thesis “A proposal for modeling ontologies based student and not monotonous teaching-cognitive diagnosis” by Julia Parraga in 2011 (Julia’s Ontology). Construction and testing of BotManager were conducted in three stages: 1) creation of the NPC Actions Ontology by extending the Julia’s Ontology; 2) design and implementation of the application in C# that reads the ontology containing the plan of action of the NPC, and commands the NPC to perform the read plan in the virtual world; and 3) testing of the application with the practice “preparing a cup of coffee”, which is part of a Virtual Laboratory of Biotechnology. The BotManager has been designed as a client application that connects to an OpenSimulator server. Therefore, it can run on a different machine to the server. To implement the BotManager we have used a free library called libopenmetaverse that allows us to control a NPC remotely.

Rhizobium leguminosarum HupE is a highly-specific diffusion facilitator for nickel uptake

Bacteria require nickel transporters for the synthesis of Ni-containing metalloenzymes in natural, low nickel habitats. In this work we carry out functional and topological characterization of Rhizobium leguminosarum HupE, a nickel permease required for the provision of this element for [NiFe] hydrogenase synthesis. Expression studies in the Escherichia coli nikABCDE mutant strain HYD723 revealed that HupE is a medium-affinity permease (apparent Km 227 ! 21 nM; Vmax 49 ! 21 pmol Ni2+ min"1 mg"1 bacterial dry weight) that functions as an energy-independent diffusion facilitator for the uptake of Ni(II) ions. This Ni2+ transport is not inhibited by similar cations such as Mn2+, Zn2+, or Co2+, but is blocked by Cu2+. Analysis of site-directed HupE mutants allowed the identification of several residues (H36, D42, H43, F69, E90, H130, and E133) that are essential for HupE-mediated Ni uptake in E. coli cells. By using translational fusions to reporter genes we demonstrated the presence of five transmembrane domains with a periplasmic N-terminal domain and a C-terminal domain buried in the lipid bilayer. The periplasmic N-terminal domain contributes to stability and functionality of the protein

La representación de la arquitectura moderna : la villa Stein-de Monzie en "L'Oeuvre Complète" de Le Corbusier y Pierre Jeanneret : influencia de la visión cinematográfica

Esta tesis doctoral trata de investigar cuánto hubo de presencia del cine en el pensamiento de Le Corbusier y Pierre Jeanneret, a la hora de acometer la publicación y maquetación de las páginas dedicadas a la Villa Stein-de Monzie en Garches, en “Le Corbusier et Pierre Jeanneret. Oeuvre Complète 1910-1929”. Qué mecanismos cinematográficos pusieron en juego, cuando montaban las páginas de L´Oeuvre Complète. Más que encontrar elementos cinematográficos utilizados de manera directa; el objetivo de esta tesis es profundizar en los mecanismos, procedimientos, sistemas de generación de ideas a lo largo del proceso de proyecto; en las maneras de percibir y experimentar los espacios, o de observar las formas. El armazón lo compone por lo tanto el análisis del modo en que Le Corbusier representa la villa en L´Oeuvre Complète, sus intenciones y la función pedagógica de este modo de representar; así como sus diferencias con respecto a lo realmente construido. Una realidad elaborada sobre un tablero; maquetando su representación, eligiendo los fragmentos, y componiendo las láminas (cercana a la de un director de cine). La justificación del objeto a estudiar se plantea en el primer capítulo: L´Oeuvre Complète (1937), la reedición en francés, alemán e inglés de la primera edición en alemán, Ihr Gesamtes Werk (1929).Se establecen las intenciones de Le Corbusier de que se convierta en un modelo de Tratado de Arquitectura Moderna, eminentemente visual. Se estudian los mecanismos formales y geométricos de composición del libro, y el modo en el que L´Oeuvre Complète debe ser leída. El desarrollo de los siguientes capítulos (del 2 al 9) recoge el método principal de investigación de esta tesis, basado en una lectura longitudinal, crítica y sistemática, a partir de la observación atenta de la representación de la villa Stein-de Monzie en Garches, en las páginas nº 140 a 149 de L´Oeuvre Complète. Se efectúa mediante un desarrollo lineal y secuenciado, como si de un guión cinematográfico se tratara. Cada capítulo describe y analiza cada uno de los diferentes fragmentos, permitiendo al mismo tiempo enlazar temas de interés que ayudan a comprender aspectos de la villa de Garches, de su concepción en la intensa labor de proyecto (con numerosas variantes y propuestas), e incluso de su aparición en el cine. Además, la tesis arroja luz sobre unos documentos bastante desconocidos: las láminas de la colección del Museo Cooper-Hewitt de nueva York, para la villa de Garches. El análisis de la presentación de la villa de Garches en L´Oeuvre Complète, constata que para Le Corbusier, el fragmento, per se, ha de ser perfecto, produciendo la máxima emoción. Como un prestidigitador, Le Corbusier los manipula, o le niega información al espectador mediante el uso de la elipsis en el relato. Los textos concatenan las imágenes, soportan el hilo de la narración. Los bocetos quieren siempre seducir al espectador: son dibujos que rezuman vitalidad, con una técnica muy cercana a la ligne claire del cómic. Las plantas son un laboratorio para demostrar su jerarquía y su libertad de composición; eliminando elementos, distorsionando la valoración de líneas y apareciendo algún elemento no ejecutado. Los alzados, esquemáticos y abstractos, demuestran el control de la geometría para garantizar la emoción. Las fotografías son controladas en su fase de captura (elección del punto de vista, cuidada puesta en escena de los objetos, composición con la luz, uso de las sombras para la aparición del fuera de campo); pero también en la fase de postproducción y edición, donde son cortadas, alisadas superficies, borrando o dibujando elementos sobre ellas. El montaje compone asimismo una representación dinámica, fragmentada y múltiple de la villa. Como sucede en el cine, los fragmentos sólo encuentran su razón de ser una vez son re-creados y montados en la cabeza del espectador. La falta de raccord es un mecanismo buscado por Le Corbusier, trasladando a la representación una de las primordiales características de la villa de Garches: su permanente dualidad simultánea. Todos estos mecanismos son desplegados por Le Corbusier, para ofrecer una versión idealizada de la villa, que recoja todas las virtudes de los distintos proyectos e incorpore el factor tiempo. ABSTRACT This doctoral thesis tries to investigate how much the cinema affected Le Corbusier and Pierre Jeanneret´s thoughts, at the moment of undertaking the publication and layout of the pages dedicated to the villa Stein-de Monzie in Garches, in the book “Le Corbusier et Pierre Jeanneret. Oeuvre Complète 1910-1929”. Which cinematographic mechanisms they brought into play, when they were mounting those pages. Instead of finding cinematographic elements, used directly; the aim of this thesis is to go deeply into the mechanisms, methods, systems of generation of ideas along the project process; into the ways of seeing and feeling the spaces, or of watching the forms. The body is composed therefore by the analysis of the way in which Le Corbusier represents the villa in L'Oeuvre Complète, his intentions and the pedagogic function of that way of representation; as well as its differences with the real built villa. One reality elaborated on a board; laying out its representation, choosing the fragments, and composing the sheets (near to the work of a director of cinema). The justification of the object to studying appears in the first chapter: L'Oeuvre Complète (1937), the reissue in French, German and English of the first edition in German, Ihr Gesamtes Werk (1929). This chapter shows the intentions of Le Corbusier of turning the book into a model of modern architecture, highly visual. The formal and geometric mechanisms of composition of the book are studied, and the way in which L'Oeuvre Complète must be read. The development of the following chapters (from 2 to 9) gathers the principal method of investigation of this thesis, based on a longitudinal, critical and systematic reading; from the watching of the representation of the villa Stein-de Monzie in Garches, in the pages nr. 140 to 149 of L'Oeuvre Complète. It is carried out by a linear and sequenced development, as a cinematographic script. Every chapter describes and analyzes each of the different fragments, allowing at the same time to connect interesting issues that help to understand aspects of the villa in Garches, of its conception in the intense project process (with numerous variants and designs), and even of its appearance in films. Also, the thesis throws light on some unknown documents: the sheets of the collection of the Museum Cooper-Hewitt in New York, for the villa in Garches. The analysis of the presentation of the villa in Garches in L'Oeuvre Complète, proves that for Le Corbusier, the fragment, itself, has to be perfect, getting the maximum emotion. As a prestidigitator, Le Corbusier manipulates them, or denies information to the spectator by means of the use of the ellipsis in the story. The texts concatenate the images, support the thread of the story. The sketches always attempt to seduce the spectator: they are drawings that leak vitality, with lines very near to the ligne claire of the cómic. The plans are a laboratory to demonstrate their hierarchy and their freedom of composition; deleting elements, distorting the thickness of lines and showing some not executed elements. The elevations, schematic and abstract, shows the control of the geometry to guarantee the emotion in architecture. The pictures are controlled in their instant of capture (choice of the point of view, elegant mise-en-scène of the objects, composition with light, use of the shadows for the appearance of out of vision); but also in the postproduction and edition time, when surfaces are cut, smoothing, erasing or drawing elements in them. The montage composes in the same way a dynamic, fragmented and multiple representation of the villa. As in the films, the fragments only find their raison d'être once they have been re-created and mounted into the mind of the viewer. The continuity error is a mechanism allowed by Le Corbusier, transfering to the representation in the book one of the basic characteristics of the villa in Garches: its constant simultaneous duality. All these methods are displayed by Le Corbusier, to offer an idealized version of the villa, which gathers all the virtues of the different projects, and incorporates the time factor.

O PERFIL PROFISSIONAL DO SUPERVISOR DE ENSINO

Numa perspectiva sociológica, este trabalho tem o objetivo de identificar o perfil profissional do supervisor de ensino que atua na Secretaria de Estado da Educação de São Paulo, verificando se sua atuação está voltada ao pedagógico ou ao administrativo e qual o compromisso político e as concepções educacionais destes profissionais. Apoiando-se em Tragtenberg, Silva Jr e Huert, entre outros autores que abordam, especificamente, a supervisão de ensino, procurou-se fazer uma analise histórica da legislação, de planilhas e dos dados que foram colhidos através de depoimentos e entrevistas com supervisores de ensino efetivos de três Diretorias de Ensino da Grande São Paulo. O resultado da pesquisa aponta para a predominância de um perfil burocrático e de fiscalização na ação do supervisor de ensino, bem como de executor das políticas públicas e não partícipe da sua elaboração. O supervisor de ensino é um representante de um poder político que favorece uma determinada ordem, mesmo que exista uma tentativa de superação de uma representatividade unilateral dentro da supervisão de ensino.(AU)

O PERFIL PROFISSIONAL DO SUPERVISOR DE ENSINO

Numa perspectiva sociológica, este trabalho tem o objetivo de identificar o perfil profissional do supervisor de ensino que atua na Secretaria de Estado da Educação de São Paulo, verificando se sua atuação está voltada ao pedagógico ou ao administrativo e qual o compromisso político e as concepções educacionais destes profissionais. Apoiando-se em Tragtenberg, Silva Jr e Huert, entre outros autores que abordam, especificamente, a supervisão de ensino, procurou-se fazer uma analise histórica da legislação, de planilhas e dos dados que foram colhidos através de depoimentos e entrevistas com supervisores de ensino efetivos de três Diretorias de Ensino da Grande São Paulo. O resultado da pesquisa aponta para a predominância de um perfil burocrático e de fiscalização na ação do supervisor de ensino, bem como de executor das políticas públicas e não partícipe da sua elaboração. O supervisor de ensino é um representante de um poder político que favorece uma determinada ordem, mesmo que exista uma tentativa de superação de uma representatividade unilateral dentro da supervisão de ensino.(AU)

Modulation of nicotinic acetylcholine receptors by strychnine

Strychnine, a potent and selective antagonist at glycine receptors, was found to inhibit muscle (α1β1γδ, α1β1γ, and α1β1δ) and neuronal (α2β2 and α2β4) nicotinic acetylcholine receptors (AcChoRs) expressed in Xenopus oocytes. Strychnine alone (up to 500 μM) did not elicit membrane currents in oocytes expressing AcChoRs, but, when applied before, concomitantly, or during superfusion of acetylcholine (AcCho), it rapidly and reversibly inhibited the current elicited by AcCho (AcCho-current). Although in the three cases the AcCho-current was reduced to the same level, its recovery was slower when the oocytes were preincubated with strychnine. The amount of AcCho-current inhibition depended on the receptor subtype, and the order of blocking potency by strychnine was α1β1γδ > α2β4 > α2β2. With the three forms of drug application, the Hill coefficient was close to one, suggesting a single site for the receptor interaction with strychnine, and this interaction appears to be noncompetitive. The inhibitory effects on muscle AcChoRs were voltage-independent, and the apparent dissociation constant for AcCho was not appreciably changed by strychnine. In contrast, the inhibitory effects on neuronal AcChoRs were voltage-dependent, with an electrical distance of ≈0.35. We conclude that strychnine regulates reversibly and noncompetitively the embryonic type of muscle AcChoR and some forms of neuronal AcChoRs. In the former case, strychnine presumably inhibits allosterically the receptor by binding at an external domain whereas, in the latter case, it blocks the open receptor-channel complex.

A dimeric crystal structure for the N-terminal two domains of intercellular adhesion molecule-1

The 3.0-Å structure of a 190-residue fragment of intercellular adhesion molecule-1 (ICAM-1, CD54) reveals two tandem Ig-superfamily (IgSF) domains. Each of two independent molecules dimerizes identically with a symmetry-related molecule over a hydrophobic interface on the BED sheet of domain 1, in agreement with dimerization of ICAM-1 on the cell surface. The residues that bind to the integrin LFA-1 are well oriented for bivalent binding in the dimer, with the critical Glu-34 residues pointing away from each other on the periphery. Residues that bind to rhinovirus are in the flexible BC and FG loops at the tip of domain 1, and these and the upper half of domain 1 are well exposed in the dimer for docking to virus. By contrast, a residue important for binding to Plasmodium falciparum-infected erythrocytes is in the dimer interface. The presence of A′ strands in both domains 1 and 2, conserved hydrogen bonds at domain junctions, and elaborate hydrogen bond networks around the key integrin binding residues in domain 1 make these domains suited to resist tensile forces during adhesive interactions. A subdivision of the intermediate (I) set of IgSF domains is proposed in which domain 1 of ICAM-1 and previously described I set domains belong to the I1 set and domain 2 of ICAM-1, ICAM-2, and vascular cell adhesion molecule-1 belong to the I2 set.

A human homolog of the Saccharomyces cerevisiae REV3 gene, which encodes the catalytic subunit of DNA polymerase ζ

To get a better understanding of mutagenic mechanisms in humans, we have cloned and sequenced the human homolog of the Saccharomyces cerevisiae REV3 gene. The yeast gene encodes the catalytic subunit of DNA polymerase ζ, a nonessential enzyme that is thought to carry out translesion replication and is responsible for virtually all DNA damage-induced mutagenesis and the majority of spontaneous mutagenesis. The human gene encodes an expected protein of 3,130 residues, about twice the size of the yeast protein (1,504 aa). The two proteins are 29% identical in an amino-terminal region of ≈340 residues, 39% identical in a carboxyl-terminal region of ≈850 residues, and 29% identical in a 55-residue region in the middle of the two genes. The sequence of the expected protein strongly predicts that it is the catalytic subunit of a DNA polymerase of the pol ζ type; the carboxyl-terminal domain possesses, in the right order, the six motifs characteristic of eukaryotic DNA polymerases, most closely resembles yeast pol ζ among all polymerases in the GenBank database, and is different from the human α, δ, and ɛ enzymes. Human cells expressing high levels of an hsREV3 antisense RNA fragment grow normally, but show little or no UV-induced mutagenesis and are slightly more sensitive to killing by UV. The human gene therefore appears to carry out a function similar to that of its yeast counterpart.

Identification of the binding site for Gqα on its effector Bruton’s tyrosine kinase

Heterotrimeric G proteins and tyrosine kinases are two major cellular signal transducers. Although G proteins are known to activate tyrosine kinases, the activation mechanism is not clear. Here, we demonstrate that G protein Gqα binds directly to the nonreceptor Bruton’s tyrosine kinase (Btk) to a region composed of a Tec-homology (TH) domain and a sarcoma virus tyrosine kinase (Src)-homology 3 (SH3) domain both in vitro and in vivo. Only active GTP-bound Gqα, not inactive GDP-bound Gqα, can bind to Btk. Mutations of Btk that disrupt its ability to bind Gqα also eliminate Btk stimulation by Gqα, suggesting that this interaction is important for Btk activation. Remarkably, the structure of this TH (including a proline-rich sequence) -SH3 fragment of the Btk family of tyrosine kinases shows an intramolecular interaction. Furthermore, the crystal structure of the Src family of tyrosine kinases reveals that the intramolecular interaction of SH3 and its ligand is the major determining factor keeping the kinase inactive. Thus, we propose an activation model that entails binding of Gqα to the TH-SH3 region, thereby disrupting the TH-SH3 intramolecular interaction and activating Btk.

Protein phosphatase 2C dephosphorylates and inactivates cystic fibrosis transmembrane conductance regulator

cAMP-dependent phosphorylation activates the cystic fibrosis transmembrane conductance regulator (CFTR) in epithelia. However, the protein phosphatase (PP) that dephosphorylates and inactivates CFTR in airway and intestinal epithelia, two major sites of disease, is not certain. We found that in airway and colonic epithelia, neither okadaic acid nor FK506 prevented inactivation of CFTR when cAMP was removed. These results suggested that a phosphatase distinct from PP1, PP2A, and PP2B was responsible. Because PP2C is insensitive to these inhibitors, we tested the hypothesis that it regulates CFTR. We found that PP2Cα is expressed in airway and T84 intestinal epithelia. To test its activity on CFTR, we generated recombinant human PP2Cα and found that it dephosphorylated CFTR and an R domain peptide in vitro. Moreover, in cell-free patches of membrane, addition of PP2Cα inactivated CFTR Cl− channels; reactivation required readdition of kinase. Finally, coexpression of PP2Cα with CFTR in epithelia reduced the Cl− current and increased the rate of channel inactivation. These results suggest that PP2C may be the okadaic acid-insensitive phosphatase that regulates CFTR in human airway and T84 colonic epithelia. It has been suggested that phosphatase inhibitors could be of therapeutic value in cystic fibrosis; our data suggest that PP2C may be an important phosphatase to target.

Three distinct domains of SSI-1/SOCS-1/JAB protein are required for its suppression of interleukin 6 signaling

Cytokine-inducible protein SSI-1 [signal transducers and activators of transcription (STAT)-induced STAT inhibitor 1, also referred to as SOCS-1 (suppressor of cytokine signaling 1) or JAB (Janus kinase-binding protein)] negatively regulates cytokine receptor signaling by inhibition of JAK kinases. The SSI family of proteins includes eight members that are structurally characterized by an SH2 domain and a C-terminal conserved region that we have called the SC-motif. In this study, we investigated the roles of these domains in the function of SSI-1. Results of reporter assays demonstrated that the pre-SH2 domain (24 aa in front of the SH2 domain) and the SH2 domain of SSI-1 were required for the suppression by SSI-1 of interleukin 6 signaling. Coexpression studies of COS7 cells revealed that these domains also were required for inhibition of three JAKs (JAK1, JAK2, and TYK2). Furthermore, deletion of the SH2 domain, but not the pre-SH2 domain, resulted in loss of association of SSI-1 with TYK2. Thus, SSI-1 associates with JAK family kinase via its SH2 domain, and the pre-SH2 domain is required for the function of SSI-1. Deletion of the SC-motif markedly reduced expression of SSI-1 protein in M1 cells, and this reduction was reversed by treatment with proteasome inhibitors, suggesting that this motif is required to protect the SSI-1 molecule from proteolytic degradation. Based on these findings, we concluded that three distinct domains of SSI-1 (the pre-SH2 domain, the SH2 domain, and the SC-motif) cooperate in the suppression of interleukin 6 signaling.

Multiple domains of repressor activator protein 1 contribute to facilitated binding of glycolysis regulatory protein 1

The function of repressor activator protein 1 (Rap1p) at glycolytic enzyme gene upstream activating sequence (UAS) elements in Saccharomyces cerevisiae is to facilitate binding of glycolysis regulatory protein 1 (Gcr1p) at adjacent sites. Rap1p has a modular domain structure. In its amino terminus there is an asymmetric DNA-bending domain, which is distinct from its DNA-binding domain, which resides in the middle of the protein. In the carboxyl terminus of Rap1p lie its silencing and putative activation domains. We carried out a molecular dissection of Rap1p to identify domains contributing to its ability to facilitate binding of Gcr1p. We prepared full-length and three truncated versions of Rap1p and tested their ability to facilitate binding of Gcr1p by gel shift assay. The ability to detect ternary complexes containing Rap1p⋅DNA⋅Gcr1p depended on the presence of binding sites for both proteins in the probe DNA. The DNA-binding domain of Rap1p, although competent to bind DNA, was unable to facilitate binding of Gcr1p. Full-length Rap1p and the amino- and carboxyl-truncated versions of Rap1p were each able to facilitate binding of Gcr1p at an appropriately spaced binding site. Under these conditions, Gcr1p displayed an approximately 4-fold greater affinity for Rap1p-bound DNA than for otherwise identical free DNA. When spacing between Rap1p- and Gcr1p-binding sites was altered by insertion of five nucleotides, the ability to form ternary Rap1p⋅DNA⋅Gcr1p complexes was inhibited by all but the DNA-binding domain of Rap1p itself; however, the ability of each individual protein to bind the DNA probe was unaffected.