A methodology to calibrate structural finite element models for reinforced concrete structures subject to blast loads

Civil buildings are not specifically designed to support blast loads, but it is important to take into account these potential scenarios because of their catastrophic effects, on persons and structures. A practical way to consider explosions on reinforced concrete structures is necessary. With this objective we propose a methodology to evaluate blast loads on large concrete buildings, using LS-DYNA code for calculation, with Lagrangian finite elements and explicit time integration. The methodology has three steps. First, individual structural elements of the building like columns and slabs are studied, using continuum 3D elements models subjected to blast loads. In these models reinforced concrete is represented with high precision, using advanced material models such as CSCM_CONCRETE model, and segregated rebars constrained within the continuum mesh. Regrettably this approach cannot be used for large structures because of its excessive computational cost. Second, models based on structural elements are developed, using shells and beam elements. In these models concrete is represented using CONCRETE_EC2 model and segregated rebars with offset formulation, being calibrated with continuum elements models from step one to obtain the same structural response: displacement, velocity, acceleration, damage and erosion. Third, models basedon structural elements are used to develop large models of complete buildings. They are used to study the global response of buildings subjected to blast loads and progressive collapse. This article carries out different techniques needed to calibrate properly the models based on structural elements, using shells and beam elements, in order to provide results of sufficient accuracy that can be used with moderate computational cost.

Decodificar Sejima‐SANAA: Estrategias formales y estructurales en su trayectoria de proyecto, 1987 a 2010; de la Casa Platform I al Centro Rolex

La Tesis decodifica una selección de veinte proyectos representativos de Sejima-SANAA, desde su primer proyecto construido, la Casa Platform I, 1987, hasta el Centro Rolex, 2010, año en que Sejima y Nishizawa –SANAA- reciben el Premio Pritzker. De los veinte proyectos once son de Sejima: Casa Platform I, Casa Platform II, Residencia de Mujeres, Casa N, Pachinco Parlor I, Villa en el Bosque, Comisaría en Chofu, Casa Y, Apartamentos en Gifu, Edificio de equipamientos en la Expo Tokio 96, Pachinko Parlor III; y nueve de SANAA: edificio Multimedia en Oogaki, estudio de viviendas metropolitanas,Park Café en Koga, De Kunstlinie en Almere, Museo de Kanazawa, Pabellón de Toledo, Escuela de Zollverein, Casa Flor y Centro Rolex. La decodificación lee la obra de Sejima-SANAA a la inversa para ‘reconstruir’, en un ejercicio de simulación ficticia, una versión verosímil y coherente de los que podrían haber sido sus procesos proyectuales; podrían, porque los verdaderos son imposibles de dilucidar. Los que se proponen se pretenden exclusivamente verosímiles y plausibles. Con ello se pretende contribuir al entendimiento y comprensión de la arquitectura de Sejima-SANAA y, tangencialmente y en menor medida, a la teoría sobre el ejercicio proyectual arquitectónico. La decodificación se centra en dos aspectos concretos: la forma arquitectónica y el papel proyectual de la estructura portante. Ambas decodificaciones se extienden inevitablemente a otros aspectos relacionados, como, por ejemplo, la naturaleza del espacio arquitectónico. El procedimiento de investigación partió de una descripción objetiva y pormenorizada de los significantes formales y estructurales de cada proyecto desde su propia configuración física y geométrica. Esa descripción ‘objetiva’, llevada al límite, permitió que afloraran estructuras conceptuales y lógicas subyacentes de cada proyecto. Unida a interpretación crítica, –mediante su relación y confrontación con otras arquitecturas y otros modos de hacer conocidos- permitió trazar la reconstitución ficticia que persigue la decodificación. Ese trabajo se materializó en veinte ensayos críticos y se acompañó de un conjunto de otros textos sobre temas sugeridos o reclamados por el proceso de investigación. El conjunto de todos esos textos constituye el material de trabajo de la tesis. A partir de ahí, con una visión de conjunto, la tesis identifica una trayectoria de estrategias formales y una trayectoria de estrategias proyectuales relacionadas con lo portante. Juntas conforman el grueso de la tesis que se expone en los cuatro capítulos centrales. Los precede un capítulo introductorio que expone el recorrido biográfico de K. Sejima y la trayectoria profesional de Sejima-SANAA; y los siguen de unos textos transversales sobre forma, lugar y espacio. La tesis termina con una síntesis de sus conclusiones. Las estrategias formales se exponen en tres capítulos. El primero, ‘Primeras estrategias formales’ agrupa proyectos de la primera etapa de Sejima. El segundo capítulo está dedicado enteramente al proyecto de los apartamentos en Gifu, 1994-98, que según esta tesis, supuso un importante punto de inflexión en la trayectoria de Sejima; tanto el tercer capítulo lleva por nombre ‘Estrategias formales después de Gifu’ y recoge los proyectos que le siguieron. Las ‘Primeras estrategias formales’, varias y balbucientes, se mueven en general en torno a dos modos o procedimientos de composición, bien conocidos: por partes y sistemático. Éste última inicia en la trayectoria de SANAA un aspecto que va a ser relevante de aquí en adelante: entender el proyecto como propuesta genérica en la que, más allá de su realidad específica y tangible, subyace una lógica, en cada proyecto la suya, extrapolable a otros lugares, otras dimensiones, incluso otros programas: cada proyecto podría dar lugar a otros proyectos de la misma familia. La composición sistemática incluye, entre otros, la Casa Platform II, basada en la definición de un elemento constructivo, y la formulación de unas leyes de repetición y de posibles modos de agrupación. Incluye también la Residencia de Mujeres Saishunkan Seiyaku- proyecto que lanzó a Sejima a la fama internacional-, que también sería un sistema, pero distinto: basado en la repetición regular de una serie de elementos a lo largo de una directriz generando un hipotético contenedor infinito del que el proyecto sería tan solo un fragmento. La estrategia formal del edificio de Gifu ahondaría en la voluntad genérica del proyecto, adoptando la lógica de un juego. El proyecto sería una partida del juego, pero no la única posible, podrían jugarse otras. Esta hipótesis del juego está verificada en ‘El Juego de Gifu’ que - tras formular el juego identificando sus elementos (tablero y fichas), reglas y procedimientos- juega una partida: la que habría dado lugar al edificio proyectado por Sejima. Gifu extiende el concepto de ‘repetir’ un elemento constructivo a la de repetir un patrón espacial, lo que conlleva: la desvinculación entre forma y función; y un nuevo concepto de flexibilidad, que deja de referirse al uso flexible del edificio construido para pertenecer al momento proyectual en que se asignan funciones específicas a los patrones espaciales. Esta tesis propone que esa asignación de funciones sería uno de los últimos eslabones del proceso proyectual, algo opuesto a la premisa moderna de “la forma sigue a la función”. Las estrategias formales ‘Después de Gifu’ tienen también lógicas de juego, pero cada estrategia responde a un juego distinto, como dejan entrever sus nombres: ‘Tableros de Juego’, que con distintos grados de madurez estaría presente en varios proyectos; ‘Elementos de Catálogo’ en el Museo de Kanazawa; ‘Forma apriorística’, en la Casa Flor y ‘Repetición de una situación topológica’, en el Centro Rolex. Todas esas estrategias, o juegos, mantienen aspectos comunes relativos a la forma arquitectónica, precisamente los aspectos Gifu: la repetición aplicada al patrón espacial, y lo que conlleva: desvinculación entre forma y función y la nueva acepción de flexibilidad. ‘Tableros de Juego’ consiste en configurar cada sistema de proyecto (estructura, cerramientos, particiones y mobiliario) eligiendo elementos ofrecidos por una geometría de base, en cada proyecto la suya, en general reticular: intersecciones, líneas, módulos. Cada sistema se configura, en principio, sin relación de subordinación con cualquiera de los demás; cuando esa subordinación es ineludible, el juego determina que el sistema portante no puede materializar el orden geométrico de base, lo que se traduce en que no ejerce el papel dominante. Por lo tanto, ‘Tableros de Juego’ transgrede la lógica de la planta libre moderna: la estructura ni refleja ni revela el orden de base y los sistemas no respetan las relaciones de subordinación jerárquica y encadenada que aquella determinaba. Esta estrategia de ‘Tableros de juego’ deriva en soluciones y proyectos formales muy distintos: los proyectos de Oogaki y Park Café, que presentarían ‘Tableros de Juego’ incipientes; De Kunstlinie en Almere y la Escuela de Zollverein, que presentarían una consolidación de esta estrategia; y el Pabellón de Vidrio de Toledo que resultaría de la subversión de la estrategia. Este último proyecto, además, lleva el concepto de repetición más allá del elemento constructivo y del patrón espacial (que en este caso tiene forma de burbuja) parar acabar afectando a la propia experiencia del espectador, que esté donde esté, siempre tiene la sensación de estar en el mismo sitio. Esta tesis denomina a ese espacio repetitivo como ‘espacio mantra’. La estrategia ‘Elementos de Catálogo’ se ilustra con el Museo de Kanazawa. Su lógica parte de la definición de una serie de elementos, muy pocos, y se basa en el ingente número de posibles combinaciones entre sí. Gifu habría anunciado el catalogo de elementos en la caracterización de sus patrones espaciales. La estrategia ‘Forma Apriorística’ se ilustra con la Casa Flor. La decisión sobre el tipo de forma -en este caso la de una ameba- estaría al principio del proceso proyectual, lo que no quiere decir que sea una forma arbitraria: la forma de la ameba lleva implícita la repetición de un patrón espacial (el seudópodo) y una apoteosis del concepto de repetición que, alcanzando la experiencia espacial, da lugar a un espacio repetitivo o mantra. El ‘Espacio Mantra’ es uno de los leitmotivs, que se emplean como argumento en la última estrategia formal que la Tesis decodifica: el Centro Rolex. Con respecto a la estructura portante, la tesis identifica y traza una trayectoria de cinco estrategias proyectuales: preeminencia, ocultación, disolución, desaparición y desvirtuación. --Ocultación, reduce el papel dominante de la estructura. Al principio es una ocultación literal, casi un tapado de los elementos estructurales, como en Gifu; luego se hace más sofisticada, como la ocultación por camuflaje o la paradójica ocultación por multiplicación de Park Café. --La disolución merma la condición dominante de la estructura que en lugar de configurarse como sistema unitario u homogéneo se fragmenta en varios subsistemas. --La desaparición se refiere a estructuras que desaparecen como sistemas propios y autónomos, a proyectos en los que la función portante es desempeñada por otros sistemas como el de las particiones. La desaparición culmina con la Casa Flor, cuyo perímetro ejerce la función portante y además es transparente, está desmaterializado: la estructura se ha hecho invisible, ha desaparecido. --La desvirtuación se refiere a estructuras que sí se presentan como sistemas propios y autónomos, pero dejan de tener un papel preeminente por cuanto no materializan el orden de base: esta estrategia es correlativa a la estrategia formal ‘Tableros de juego’. Las conclusiones de la tesis están en la propia organización de la tesis: la identificación de las estrategias. Aún así, y como epílogos, se exponen seis. Las dos primeras subrayan el hilo conductor del trabajo realizado, que radica en la cualidad genérica de las estrategias proyectuales en Sejima-SANAA. Las cuatro siguientes dilucidan hasta qué punto hay, en sus proyectos, rasgos o significantes formales y/o estructurales que sean a su vez señales características del panorama arquitectónico contemporáneo; y plantean la pregunta estrella: ¿hay algunos que, apuntando más lejos, supongan aportaciones originales? --Como aportaciones originales la tesis destaca: la identificación entre el ideal genérico y proyecto concreto; y la propuesta de un espacio nuevo, híbrido, una suerte de estadio intermedio entre el espacio subdividido y compartimentado de la tradición y el continuo moderno. --Como síntomas de contemporaneidad se destacan: respecto de la forma, la traslación de la especificidad formal de la parte al conjunto; y respecto de la estructura, la tendencia contemporánea a hacer estructuras cada vez más ligeras y livianas, que tienden a lo evanescente. Ésta última, la tendencia al evanescencia estructural, podría tener la condición de aportación original, no en vano la desaparición de la estructura lleva la evanescencia hacia sus últimas consecuencias, y en el caso de estructuras con presencia física, hace que dejen de ser el sistema ordenador orquestador del proceso proyectual. ABSTRACT The Thesis decodes a selection of twenty representative Sejima-SANAA projects, from the first one built, the Platform I House in 1987, to the Rolex Center in 2010, year in which Sejima and Nishizawa –SANAA- received the Pritzker Prize. Eleven projects are from Sejima: Platform I, Platform II, Saishunkan Seiyaku Women´s Dormitory, N- House, Pachinco Parlor I, Villa in the Forest, Policy Box at Chofu Station, Y-House, Gifu Kitigata Apartment, World City Expo ´96 Facilities Building, Pachinko Parlor III; and nine from SANAA: Multimedia Workshop in Ogaki, Metropolitan Housing Studies, Park Café in Koga, De Kunstlinie in Almere, Kanazawa Museum, Glass Pavilion at the Toledo Museum of Art, Zollverein School, Flower House and the Rolex Center. This decoding reads the Sejima-SANAA’s projects inversely aiming ‘to reconstruct', in a fictitious simulation exercise, a likely and coherent version of what her/their projectual processes ‘could’ have been; ‘could’, because the true ones are impossible to explain. The ones proposed here pretend only to be likely and reasonable. By so doing the Thesis tries to contribute to the understanding and comprehension of Sejima-SANAA architecture and, tangentially and to a lesser extent, to the theory of architectural projects exercise. Decoding centers in two specific aspects: architectural form, and projectual role of the load bearing structure. Both decodes inevitably extend to other related aspects such as, for example, the nature of space. The research procedure begun by carrying out an objective and detailed description of the formal and structural signifiers of each project; looking at them from their physical and geometric configuration. Taken to the limit, the ‘objective’ descriptions allowed the conceptual structures and underlying logics of each project to arise. Together with critical interpretations, which related and confronted them with other architectures and well-known projectual working ways, it became possible to outline and trace the intended fictitious reconstruction decodes. The descriptive analytical work materialized in twenty critical essays, and was accompanied by a set of other essays on subjects suggested or demanded by the research process. Together, all those texts were the material basis on which thesis work was built. Looking at the whole and taking it from there, the thesis identifies two related projectual trajectories: a trajectory of formal strategies and a trajectory of strategies having to do with structural systems and components. Both, together, constitute the bulk of the thesis, as presented in the four central chapters. Preceding them there is an introductory chapter outlining the biographical path of Kazuyo Sejima and the professional trajectory of Sejima-SANAA. And following them there is another one containing transversal texts on form, place and space. The thesis ends with a synthesis on conclusions. The formal strategies are displayed in three chapters. The first one, `Early formal strategies' groups the first phase projects by Sejima. The second one, ‘Formal strategies of Gifu’s paradigm’, is entirely dedicated to the Gifu apartments project, 1994-98, which according to this thesis meant an important inflexion point in Sejima’s trajectory; so much so that the third chapter is named `Formal strategies after Gifu' and gathers the selected projects that followed it. The ‘Early formal strategies', diverse and tentative, move in general around two well-known projectual composition methods ‘composition by parts’, and ‘systematic composition’. This last one –systematic composition- begins and leads in SANAA’s trajectory an aspect which will remain relevant from here on: the understanding of the project as if it were an specific instance of a generic proposal in which -below and beyond the project tangible reality- there lays a logic that could be applicable at other places, for other dimensions, even with other programs; from each project, other projects of the same family could rise. The set of projects using this systematic composition method include, among others, the ‘Platform II House, based on the definition of a constructive element and of rules having to do with its replicas and their possible groupings. It also includes the Saishunkan Seiyaku Women Residence -project that launched Sejima to international fame- that could also be seen as a system, but of a different kind: a system based on the regular repetition of a series of elements along a directive line, thus generating a hypothetical infinite container of which the project would be only a fragment. The formal strategy of the Gifu apartments building would push further towards the generic project concept, adopting the logic of a game. The project would be a bout, a round, one play…, but not the only possible one; others could be played. The thesis confirms this game hypothesis -after having formulated `The Game of Gifu' and identified its elements (board, chips, rules and procedures)- playing the one play from which the building as projected by Sejima would have raised. Gifu extends the concept of ‘repeating a constructive element’ to that of ‘repeating a space pattern element’, and to what it implies: the decoupling of form and function, leading to a new concept of flexibility that no longer refers to the flexible use of the constructed building but to the projectual moment at which the specific functions are assigned to the space patterns. This thesis proposes that this allocation of functions would be one of the last steps in projectual process, quite opposite from the modern premise: “form follows function”. The Formal strategies after Gifu do also have a game logic; but, as their names reveal, each strategy responds to a different game: ‘Game Boards’, present with different maturity levels in several projects; ‘Elements from a Catalogue’, in the Kanazawa Museum; ‘Aprioristic Form’, in the Flower House; and ‘Repetition of a topologic situation', in the Rolex Center. All of these strategies, or games, maintain common aspects having to do with architectural form; aspects that were already present, precisely, in Gifu: repetition of space pattern units, uncoupling of form and function, and a new meaning of flexibility. -`Game Boards’ consists on setting up a base geometry -each project his, generally reticular- and give form to each project system (structure, closings, partitions and furniture) by choosing elements -intersections, lines, modules- it offers. Each project system is formed, in principle, with no subordinated relation with any of the others; when subordination is unavoidable, the game rules determine that the load bearing structural system may not be the one to materialize the base geometric order, which means that it does not exert the dominant role. Therefore, ‘Game Boards' transgresses the Modern logic, because the structure neither reflects nor reveals the base order, and because the systems do not respect any of the hierarchic and chained subordination relations that the ‘free plan’ called for. ‘Game Boards' leads to quite different solutions and formal projects: the Oogaki and Park Coffee projects show incipient Game Boards; The Almere Kunstlinie and the Zollverein School present consolidations of this strategy; and the Toledo’s Glass Pavilion results from subverting the strategy. In addition, the Toledo project takes the repetition concept beyond that of using a constructive element and a space pattern element (in this case with a bubble form) to end up affecting the personal experience of the spectator, who, wherever he is, feels to always be in the same place. This thesis denominates that repetitive space as ‘Mantra space '. -‘Elements from a Catalogue’ is shown with the Kanazawa Museum. Its logic starts from the definition of a series of elements, very few, and it is based on the huge number of possible combinations among them. The ‘Elements from a Catalogue’ approach was announced in the Gifu project when characterizing its space pattern elements. -Aprioristic Form' is illustrated by the Flower House. The decision on the type of form -in this case the form of an amoeba- would be the beginning of the projectual process, but it does not mean it is arbitrary form: the amoeba form implies repeating a space pattern (pseudopodia) and an apotheosis of the repetition concept: embracing the space experience, it gives rise to a repetitive or mantra space. ‘Mantra Space’ is one of leitmotivs used as an argument in the last formal strategy Thesis decodes: the Rolex Center. With respect to the ‘Projectual strategies of the load bearing structure’, the thesis finds and traces a trajectory of five projectual strategies: ‘preeminence, concealment, dissolution, disappearance and desvirtuación’. --Preeminence is present in Sejima’s first works in which she resorts to structures which have a dominant preeminent role in the project in so far as they impersonate the greater scale and/or materialize the base geometric order. In later works that preeminence will be inverted, the projects aiming towards its opposite: lighter, slighter, smaller structures. -Concealment reduces the dominant role of the structure. At the outset concealment is literal, almost hiding the structural elements, as in Gifu; soon it will become more sophisticated, such as the concealment by camouflage or the paradoxical concealment by multiplication in the Koga Park Café. -Dissolution diminishes the dominant condition of the structure: instead of its’ being configured as unitary or homogenous system is fragmented in several subsystems. -Disappearance talks about structures that fade away as self referred and independent systems; projects in which the load bearing function is carried out by other systems such as the set of partitions. Disappearance reaches its zenith at the Flower House, whose perimeter functions structurally being, in addition, transparent, immaterial: its structure has become invisible, has disappeared. -Desvirtuación talks about structures that do appear like independent self-systems, but which that do not longer have a preeminent paper, inasmuch as they do not materialize the base order. This strategy correlates with the ‘Game Boards’ formal strategy. The thesis conclusions are show by the organization of the thesis itself: its identification of the different strategies. Even so, as epilogues, the thesis exposes six ‘Conclusions’. The first two emphasize the leading thread of the work done, rooted in the generic quality of the Sejima-SANAA projectual strategies. The following four expound to what extent their projects show features, or formal and/or structural signifiers, which also are or can be read as characteristic signals of the contemporary architectonic panorama, and raise the key question: aiming farther, may some of them be taken as original contributions? -As original contributions the conclusions highlight: the identification between the generic ideal and the concrete project; and the proposal of a new, hybrid space, kind of an intermediate stage between the traditional subdivided compartmented space and the continuous modern. -As symptoms of contemporaneousness: in relation to the form it highlights the transferring of the formal specificity from the part to the whole; and in relation to the structure, it underscore the contemporary tendency towards lighter and growingly slimmer structures, tending to the evanescent. This last one, the tendency towards structural evanescence, could have condition of being an original contribution, not in vain it carries the structural disappearance towards its last consequences; and in the case of structures with physical presence, it makes them to cease being the ordering system orchestrating the projectual process.

The structure and organization within the membrane of the helices composing the pore-forming domain of Bacillus thuringiensis δ-endotoxin are consistent with an “umbrella-like” structure of the pore

The aim of this study was to elucidate the mechanism of membrane insertion and the structural organization of pores formed by Bacillus thuringiensis δ-endotoxin. We determined the relative affinities for membranes of peptides corresponding to the seven helices that compose the toxin pore-forming domain, their modes of membrane interaction, their structures within membranes, and their orientations relative to the membrane normal. In addition, we used resonance energy transfer measurements of all possible combinatorial pairs of membrane-bound helices to map the network of interactions between helices in their membrane-bound state. The interaction of the helices with the bilayer membrane was also probed by a Monte Carlo simulation protocol to determine lowest-energy orientations. Our results are consistent with a situation in which helices α4 and α5 insert into the membrane as a helical hairpin in an antiparallel manner, while the other helices lie on the membrane surface like the ribs of an umbrella (the “umbrella model”). Our results also support the suggestion that α7 may serve as a binding sensor to initiate the structural rearrangement of the pore-forming domain.

The polo-box-dependent induction of ectopic septal structures by a mammalian polo kinase, Plk, in Saccharomyces cerevisiae

Members of the polo subfamily of protein kinases play pivotal roles in cell-cycle control and proliferation. In addition to a high degree of sequence similarity in the kinase domain, polo kinases contain a strikingly conserved motif termed “polo-box” in the noncatalytic C-terminal domain. We have previously shown that the mammalian polo-like kinase Plk is a functional homolog of Saccharomyces cerevisiae Cdc5. Here, we show that, in a polo-box- and kinase activity-dependent manner, ectopic expression of Plk in budding yeast can induce a class of cells with abnormally elongated buds. In addition to localization at spindle poles and cytokinetic neck filaments, Plk induces and localizes to ectopic septin ring structures within the elongated buds. In contrast, mutations in the polo-box abolish both localization to, and induction of, septal structures. Consistent with the polo-box-dependent subcellular localization, the C-terminal domain of Plk, but not its polo-box mutant, is sufficient for subcellular localization. Our data suggest that Plk may contribute a signal to initiate or promote cytokinetic event(s) and that an intact polo-box is required for regulation of these cellular processes.

Endobrevin, a Novel Synaptobrevin/VAMP-Like Protein Preferentially Associated with the Early Endosome

Synaptobrevins/vesicle-associated membrane proteins (VAMPs) together with syntaxins and a synaptosome-associated protein of 25 kDa (SNAP-25) are the main components of a protein complex involved in the docking and/or fusion of synaptic vesicles with the presynaptic membrane. We report here the molecular, biochemical, and cell biological characterization of a novel member of the synaptobrevin/VAMP family. The amino acid sequence of endobrevin has 32, 33, and 31% identity to those of synaptobrevin/VAMP-1, synaptobrevin/VAMP-2, and cellubrevin, respectively. Membrane fractionation studies demonstrate that endobrevin is enriched in membrane fractions that are also enriched in the asialoglycoprotein receptor. Indirect immunofluorescence microscopy establishes that endobrevin is primarily associated with the perinuclear vesicular structures of the early endocytic compartment. The preferential association of endobrevin with the early endosome was further established by electron microscopy (EM) immunogold labeling. In vitro binding assays show that endobrevin interacts with immobilized recombinant α-SNAP fused to glutathione S-transferase (GST). Our results highlight the general importance of members of the synaptobrevin/VAMP protein family in membrane traffic and provide new avenues for future functional and mechanistic studies of this protein as well as the endocytotic pathway.

A Role for the GSG Domain in Localizing Sam68 to Novel Nuclear Structures in Cancer Cell Lines

The GSG (GRP33, Sam68, GLD-1) domain is a protein module found in an expanding family of RNA-binding proteins. The numerous missense mutations identified genetically in the GSG domain support its physiological role. Although the exact function of the GSG domain is not known, it has been shown to be required for RNA binding and oligomerization. Here it is shown that the Sam68 GSG domain plays a role in protein localization. We show that Sam68 concentrates into novel nuclear structures that are predominantly found in transformed cells. These Sam68 nuclear bodies (SNBs) are distinct from coiled bodies, gems, and promyelocytic nuclear bodies. Electron microscopic studies show that SNBs are distinct structures that are enriched in phosphorus and nitrogen, indicating the presence of nucleic acids. A GFP-Sam68 fusion protein had a similar localization as endogenous Sam68 in HeLa cells, diffusely nuclear with two to five SNBs. Two other GSG proteins, the Sam68-like mammalian proteins SLM-1 and SLM-2, colocalized with endogenous Sam68 in SNBs. Different GSG domain missense mutations were investigated for Sam68 protein localization. Six separate classes of cellular patterns were obtained, including exclusive SNB localization and association with microtubules. These findings demonstrate that the GSG domain is involved in protein localization and define a new compartment for Sam68, SLM-1, and SLM-2 in cancer cell lines.

The Dynamin-like Protein DLP1 Is Essential for Normal Distribution and Morphology of the Endoplasmic Reticulum and Mitochondria in Mammalian Cells

The dynamin family of large GTPases has been implicated in vesicle formation from both the plasma membrane and various intracellular membrane compartments. The dynamin-like protein DLP1, recently identified in mammalian tissues, has been shown to be more closely related to the yeast dynamin proteins Vps1p and Dnm1p (42%) than to the mammalian dynamins (37%). Furthermore, DLP1 has been shown to associate with punctate vesicles that are in intimate contact with microtubules and the endoplasmic reticulum (ER) in mammalian cells. To define the function of DLP1, we have transiently expressed both wild-type and two mutant DLP1 proteins, tagged with green fluorescent protein, in cultured mammalian cells. Point mutations in the GTP-binding domain of DLP1 (K38A and D231N) dramatically changed its intracellular distribution from punctate vesicular structures to either an aggregated or a diffuse pattern. Strikingly, cells expressing DLP1 mutants or microinjected with DLP1 antibodies showed a marked reduction in ER fluorescence and a significant aggregation and tubulation of mitochondria by immunofluorescence microscopy. Consistent with these observations, electron microscopy of DLP1 mutant cells revealed a striking and quantitative change in the distribution and morphology of mitochondria and the ER. These data support very recent studies by other authors implicating DLP1 in the maintenance of mitochondrial morphology in both yeast and mammalian cells. Furthermore, this study provides the first evidence that a dynamin family member participates in the maintenance and distribution of the ER. How DLP1 might participate in the biogenesis of two presumably distinct organelle systems is discussed.

Fibroblast growth factors 1 and 2 are distinct in oligomerization in the presence of heparin-like glycosaminoglycans

Fibroblast growth factor (FGF) 1 and FGF-2 are prototypic members of the FGF family, which to date comprises at least 18 members. Surprisingly, even though FGF-1 and FGF-2 share more than 80% sequence similarity and an identical structural fold, these two growth factors are biologically very different. FGF-1 and FGF-2 differ in their ability to bind isoforms of the FGF receptor family as well as the heparin-like glycosaminoglycan (HLGAG) component of proteoglycans on the cell surface to initiate signaling in different cell types. Herein, we provide evidence for one mechanism by which these two proteins could differ biologically. Previously, it has been noted that FGF-1 and FGF-2 can oligomerize in the presence of HLGAGs. Therefore, we investigated whether FGF-1 and FGF-2 oligomerize by the same mechanism or by a different one. Through a combination of matrix-assisted laser desorption ionization mass spectrometry and chemical crosslinking, we show here that, under identical conditions, FGF-1 and FGF-2 differ in the degree and kind of oligomerization. Furthermore, an extensive analysis of FGF-1 and FGF-2 uncomplexed and HLGAG complexed crystal structures enables us to readily explain why FGF-2 forms sequential oligomers whereas FGF-1 forms only dimers. FGF-2, which possesses an interface capable of protein association, forms a translationally related oligomer, whereas FGF-1, which does not have this interface, forms only a symmetrically related dimer. Taken together, these data show that FGF-1 and FGF-2, despite their sequence homology, differ in their mechanism of oligomerization.

Native protein sequences are close to optimal for their structures

How large is the volume of sequence space that is compatible with a given protein structure? Starting from random sequences, low free energy sequences were generated for 108 protein backbone structures by using a Monte Carlo optimization procedure and a free energy function based primarily on Lennard–Jones packing interactions and the Lazaridis–Karplus implicit solvation model. Remarkably, in the designed sequences 51% of the core residues and 27% of all residues were identical to the amino acids in the corresponding positions in the native sequences. The lowest free energy sequences obtained for ensembles of native-like backbone structures were also similar to the native sequence. Furthermore, both the individual residue frequencies and the covariances between pairs of positions observed in the very large SH3 domain family were recapitulated in core sequences designed for SH3 domain structures. Taken together, these results suggest that the volume of sequence space optimal for a protein structure is surprisingly restricted to a region around the native sequence.

Paired Ig-like receptor homologs in birds and mammals share a common ancestor with mammalian Fc receptors

Paired Ig-like receptors (PIR) that can reciprocally modulate cellular activation have been described in mammals. In the present study, we searched expressed sequence tag databases for PIR relatives to identify chicken expressed sequence tags predictive of ≈25% amino acid identity to mouse PIR. Rapid amplification of cDNA ends (RACE)-PCR extension of expressed sequence-tag sequences using chicken splenic cDNA as a template yielded two distinct cDNAs, the sequence analysis of which predicted protein products with related extracellular Ig-like domains. Chicken Ig-like receptor (CHIR)-A was characterized by its transmembrane segment with a positively charged histidine residue and short cytoplasmic tail, thereby identifying CHIR-A as a candidate-activating receptor. Conversely, CHIR-B was characterized by its nonpolar transmembrane segment and cytoplasmic tail with two immunoreceptor tyrosine-based inhibitory motifs, indicating that it may serve as an inhibitory receptor. The use of CHIR amino acid sequences in a search for other PIR relatives led to the recognition of mammalian Fc receptors as distantly related genes. Comparative analyses based on amino acid sequences and three-dimensional protein structures provided molecular evidence for common ancestry of the PIR and Fc receptor gene families.

Insulin-like growth factor 1 regulates developing brain glucose metabolism

The brain has enormous anabolic needs during early postnatal development. This study presents multiple lines of evidence showing that endogenous brain insulin-like growth factor 1 (Igf1) serves an essential, insulin-like role in promoting neuronal glucose utilization and growth during this period. Brain 2-deoxy-d- [1-14C]glucose uptake parallels Igf1 expression in wild-type mice and is profoundly reduced in Igf1−/− mice, particularly in those structures where Igf1 is normally most highly expressed. 2-Deoxy-d- [1-14C]glucose is significantly reduced in synaptosomes prepared from Igf1−/− brains, and the deficit is corrected by inclusion of Igf1 in the incubation medium. The serine/threonine kinase Akt/PKB is a major target of insulin-signaling in the regulation of glucose transport via the facilitative glucose transporter (GLUT4) and glycogen synthesis in peripheral tissues. Phosphorylation of Akt and GLUT4 expression are reduced in Igf1−/− neurons. Phosphorylation of glycogen synthase kinase 3β and glycogen accumulation also are reduced in Igf1−/− neurons. These data support the hypothesis that endogenous brain Igf1 serves an anabolic, insulin-like role in developing brain metabolism.

Two different neurodegenerative diseases caused by proteins with similar structures

The downstream prion-like protein (doppel, or Dpl) is a paralog of the cellular prion protein, PrPC. The two proteins have ≈25% sequence identity, but seem to have distinct physiologic roles. Unlike PrPC, Dpl does not support prion replication; instead, overexpression of Dpl in the brain seems to cause a completely different neurodegenerative disease. We report the solution structure of a fragment of recombinant mouse Dpl (residues 26–157) containing a globular domain with three helices and a small amount of β-structure. Overall, the topology of Dpl is very similar to that of PrPC. Significant differences include a marked kink in one of the helices in Dpl, and a different orientation of the two short β-strands. Although the two proteins most likely arose through duplication of a single ancestral gene, the relationship is now so distant that only the structures retain similarity; the functions have diversified along with the sequence.

Structures of two molluscan hemocyanin genes: Significance for gene evolution

We present here the description of genes coding for molluscan hemocyanins. Two distantly related mollusks, Haliotis tuberculata and Octopus dofleini, were studied. The typical architecture of a molluscan hemocyanin subunit, which is a string of seven or eight globular functional units (FUs, designated a to h, about 50 kDa each), is reflected by the gene organization: a series of eight structurally related coding regions in Haliotis, corresponding to FU-a to FU-h, with seven highly variable linker introns of 174 to 3,198 bp length (all in phase 1). In Octopus seven coding regions (FU-a to FU-g) are found, separated by phase 1 introns varying in length from 100 bp to 910 bp. Both genes exhibit typical signal (export) sequences, and in both cases these are interrupted by an additional intron. Each gene also contains an intron between signal peptide and FU-a and in the 3′ untranslated region. Of special relevance for evolutionary considerations are introns interrupting those regions that encode a discrete functional unit. We found that five of the eight FUs in Haliotis each are encoded by a single exon, whereas FU-f, FU-g, and FU-a are encoded by two, three and four exons, respectively. Similarly, in Octopus four of the FUs each correspond to an uninterrupted exon, whereas FU-b, FU-e, and FU-f each contain a single intron. Although the positioning of the introns between FUs is highly conserved in the two mollusks, the introns within FUs show no relationship either in location nor phase. It is proposed that the introns between FUs were generated as the eight-unit polypeptide evolved from a monomeric precursor, and that the internal introns have been added later. A hypothesis for evolution of the ring-like quaternary structure of molluscan hemocyanins is presented.

An Arabidopsis GSK3/shaggy-Like Gene That Complements Yeast Salt Stress-Sensitive Mutants Is Induced by NaCl and Abscisic Acid

GSK3/shaggy-like genes encode kinases that are involved in a variety of biological processes. By functional complementation of the yeast calcineurin mutant strain DHT22-1a with a NaCl stress-sensitive phenotype, we isolated the Arabidopsis cDNA AtGSK1, which encodes a GSK3/shaggy-like protein kinase. AtGSK1 rescued the yeast calcineurin mutant cells from the effects of high NaCl. Also, the AtGSK1 gene turned on the transcription of the NaCl stress-inducible PMR2A gene in the calcineurin mutant cells under NaCl stress. To further define the role of AtGSK1 in the yeast cells we introduced a deletion mutation at the MCK1 gene, a yeast homolog of GSK3, and examined the phenotype of the mutant. The mck1 mutant exhibited a NaCl stress-sensitive phenotype that was rescued by AtGSK1. Also, constitutive expression of MCK1 complemented the NaCl-sensitive phenotype of the calcineurin mutants. Therefore, these results suggest that Mck1p is involved in the NaCl stress signaling in yeast and that AtGSK1 may functionally replace Mck1p in the NaCl stress response in the calcineurin mutant. To investigate the biological function of AtGSK1 in Arabidopsis we examined the expression of AtGSK1. Northern-blot analysis revealed that the expression is differentially regulated in various tissues with a high level expression in flower tissues. In addition, the AtGSK1 expression was induced by NaCl and exogenously applied ABA but not by KCl. Taken together, these results suggest that AtGSK1 is involved in the osmotic stress response in Arabidopsis.

Tomato Ve disease resistance genes encode cell surface-like receptors

In tomato, Ve is implicated in race-specific resistance to infection by Verticillium species causing crop disease. Characterization of the Ve locus involved positional cloning and isolation of two closely linked inverted genes. Expression of individual Ve genes in susceptible potato plants conferred resistance to an aggressive race 1 isolate of Verticillium albo-atrum. The deduced primary structure of Ve1 and Ve2 included a hydrophobic N-terminal signal peptide, leucine-rich repeats containing 28 or 35 potential glycosylation sites, a hydrophobic membrane-spanning domain, and a C-terminal domain with the mammalian E/DXXXLφ or YXXφ endocytosis signals (φ is an amino acid with a hydrophobic side chain). A leucine zipper-like sequence occurs in the hydrophobic N-terminal signal peptide of Ve1 and a Pro-Glu-Ser-Thr (PEST)-like sequence resides in the C-terminal domain of Ve2. These structures suggest that the Ve genes encode a class of cell-surface glycoproteins with receptor-mediated endocytosis-like signals and leucine zipper or PEST sequences.