La planta Miralles: representación y pensamiento en la arquitectura de Enric Miralles

Resulta imposible disociar la evolución de la arquitectura de Enric Miralles de lo que fue el desarrollo de un sistema de representación propio. Partiendo de una posición heredada de su formación en la Escuela de Arquitectura de Barcelona y de su práctica en el estudio Viaplana-Piñón, donde adquiere el gusto por la precisión en el dibujo técnico, la delineación sobre papel vegetal o el grafismo constituido exclusivamente a base de líneas del mismo grosor, Miralles pronto evoluciona hacia un método caracterizado por un personal uso del sistema diédrico, vinculado a una concepción fragmentaria de la planta de arquitectura y del espacio mismo. Miralles proyectará por fragmentos de planta, asignándoles una geometría característica para diferenciarlos entre sí y desarrollar su espacialidad y sección con cierta autonomía, a través de planos y maquetas independientes. Gran parte de la arquitectura que elabora con Carme Pinós, en solitario o con Benedetta Tagliabue, estará compuesta por colecciones de piezas heterogéneas herederas de los fragmentos de la planta original, que encajan entre sí no en base a esquemas clásicos de integración subordinada o jerárquica, sino a través de posiciones relativas de yuxtaposición o superposición, caracterizadas por una ausencia de compacidad en la solución de conjunto. Este sistema de representación se apoya por tanto en la geometría como mecanismo de diferenciación por piezas, se basa en la fragmentación del diédrico desde la fragmentación de la planta, y en la falta de compacidad como soporte de pensamiento separativo. Un sistema que se define como “planta Miralles”, término que incluye todas las técnicas de representación empleadas por el arquitecto, desde planos a maquetas, pero que enfatiza la importancia estratégica de la planta como origen y guía del proyecto de arquitectura. La tesis se estructura en los tres primeros capítulos como un corolario de las categorías enunciadas, explicando, en orden cronológico a través de los proyectos, la evolución de la geometría, la utilización del diédrico, y el impacto de la falta de compacidad en la obra construida. Mientras que estos capítulos son globales, se refieren a la trayectoria de este método en su totalidad, el cuarto y último es un estudio de detalle de su aplicación en un proyecto particular, el Ayuntamiento de Utrecht, a través de los dibujos originales de Miralles. Tanto en la explicación global como en el estudio de detalle de este sistema de representación, la tesis pone de manifiesto su instrumentalidad en el pensamiento de esta arquitectura, argumentando que ésta no podría haber sido desarrollada sin la existencia del mismo. La relación entre representación y pensamiento es por tanto un tema capital para explicar esta obra. No obstante, hasta la fecha, las referencias al mismo en la bibliografía disponible no han pasado de ser una colección de opiniones dispersas, incapaces de construir por sí mismas un cuerpo estructurado y coherente de conocimiento. Se ha insistido sobremanera en el análisis y contextualización de los proyectos individuales, y poco en el estudio de la técnica proyectual utilizada para pensarlos y llevarlos a cabo. En definitiva, se han priorizado los resultados frente a los procesos creativos, existiendo por tanto un inexplicable vacío teórico respecto a un tema de gran importancia. Este vacío es el marco donde se inserta la necesidad de esta tesis doctoral. La investigación que aquí se presenta explica el origen y evolución del sistema de representación de Enric Miralles, desde su etapa como estudiante en la Escuela de Arquitectura de Barcelona hasta los últimos proyectos que elabora con Benedetta Tagliabue, así como el estudio de sus consecuencias en la obra construida. Termina concluyendo que su desarrollo es paralelo al de la arquitectura de Miralles, poniendo de manifiesto su vinculación y mutua interdependencia. ABSTRACT It is impossible to dissociate the evolution of the architecture of Enric Miralles from the development of his own system of representation. Starting from a position inherited from his training at the Barcelona School of Architecture and his practice at the office of Viaplana-Piñón, where he acquires a liking for precision in drafting and a graphic style based exclusively on lines of the same thickness, Miralles soon moves into a method defined by a customized use of the dihedral system, connected to a fragmented conception of the floorplan and space itself. Breaking up the floorplan into multiple fragments, Miralles will design an architecture where each of them has a unique shape and geometry, developing their sections and spatial qualities with a certain degree of autonomy within the whole, through separate plans and models. Many of the projects he designs with Carme Pinós, individually or with Benedetta Tagliabue, will consist of collections of heterogeneous pieces, heirs of the original floorplan fragments, which do not fit together according to classical principles of subordinate or hierarchical integration, but based on relative positions of juxtaposition or superposition that lead to a lack of compactness in the overall scheme. This system of representation is thus based on the use of geometry as a way of differentiating architectural pieces, on the fragmentation of the dihedral system from the fragmentation of the floorplan, and on a lack of compactness as a device of separative thinking. This system is defined as “Miralles plan”, a term that includes all techniques of representation used by the architect, from plans to models, and that emphasizes the particular importance of the floorplan as the guiding force of the design process. The first three chapters of the thesis have been structured as a corollary of these categories, explaining, in chronological order through Miralles’ projects, the evolution of geometry, the customization of the dihedral system, and the impact of the lack of compactness on the built work. While these three chapters are global, for they refer to the overall evolution of this system, the fourth and last one is a case study of its application to a particular project, the Utrecht Town Hall, through Miralles’ original drawings. Both in the global and particular explanations of this system of representation, the thesis highlights its instrumentality in the process of thinking this architecture, arguing that it could not have been designed without its parallel development. The relationship between thinking and representation is therefore a key issue to explain this architecture. However, to date, existing references to it in the available literature have not evolved from a collection of scattered opinions, unable to build for themselves a structured and coherent body of knowledge. Great emphasis has been put on the critical contextualization of this architecture through the analysis of the projects themselves, but little on the study of the design technique used to think and carry them out. Results have been prioritized over creative processes, existing therefore an inexplicable theoretical void on an issue of great importance. This void is the conceptual framework where the need for this thesis is inserted. This research explains the origin and evolution of Enric Miralles’ system of representation, from his time as student at the Barcelona School of Architecture to the last projects he designed with Benedetta Tagliabue, as well as the study of its impact on the built work. It concludes that the development of this system runs parallel to that of the architecture it is used for, making it explicit its indissolubility and mutual interdependence.

El dibujo como herramienta en el proceso creativo del cine y la arquitectura

El director de cine y el arquitecto, exploran las posibilidades que les ofrece la imagen, en el sentido de la visualización de ciertas relaciones que son catalizadoras de las emociones. La obra cinematográfica y la obra arquitectónica son producto del pensamiento y contienen todos los procesos que las idearon, así como aquellos mecanismos necesarios para generar el espacio y la secuencia. El objetivo general de la tesis consiste en conocer las analogías existentes en el modo en que el arquitecto y el cineasta afrontan el proceso de creación de sus proyectos desde la acción gráfica. Si consideramos el medio gráfico como un recurso creativo en el proyecto arquitectónico y cinematográfico, el boceto, croquis o “storyboard” se convierten en documentos fundamentales, objeto de estudio, para extraer las claves y la manera en que se desarrolla dicho proceso. El pensamiento gráfico se encuentra íntimamente unido a su modo de expresión. El estudio de ese proceso de pensamiento basado principalmente en imágenes, permite establecer analogías en la manera en que ambos autores hacen uso del dibujo para imaginar acontecimientos evocadores de emociones que definan el carácter dramático de sus ideas. La conexión y yuxtaposición de imágenes mentales, como operaciones de montaje que alientan la construcción de ideas, conceptos y sensaciones, son claves en el curso de la concepción arquitectónica y cinematográfica, y el dibujo, una herramienta que permite a ambos autores su desarrollo. La aproximación al modo en que el arquitecto y el cineasta emprenden el proceso de ideación de sus proyectos a través del dibujo, se aborda desde las estrategias gráficas de cuatro autores: Sergei Eisenstein, Le Corbusier, Akira Kurosawa y Rem Koolhaas. La estructura del trabajo se desarrolla en dos bloques. El primero, compuesto por los primeros cuatro capítulos, afronta desde un punto de vista general, la potencialidad de la imagen en el pensamiento gráfico y el papel de la acción gráfica en el curso proyectual que realiza el arquitecto, por un lado, y el director de cine por otro, tratando de extraer las analogías y los puntos de encuentro de ambos autores durante el proceso. El segundo bloque, correspondiente a los cuatro últimos capítulos, aborda el uso del medio gráfico de manera más concreta, en la figura de dos de los arquitectos y dos de los cineastas más influyentes del pasado y del presente siglo, tratando de comprender el papel del dibujo en el desarrollo conceptual de su obra. ABSTRACT Both the movie director and the architect explore the potential offered by the image, as the visualization of certain relations that are catalyst of emotions. Cinematography and architecture works are a result of a thought and they include all the processes that created them, as much as the essential tools to generate the space and the sequence. This thesis aims to get an understanding of the analogies underlying in which architects and movie directors face the creation process of theirs projects from the graphic action. If we consider graphic media as a creative resource in the architectural and the cinematographic project, the sketch or storyboard becomes the fundamental documents, the study object, to decode the clues and the way in which the process unfolds. Graphic thinking comes across its way of expression. The study of this thinking, based mainly in images, let set up analogies in a way in which both authors use a drawing to imagine events of emotions that define the dramatic nature of their ideas. The connection and juxtaposition of mental images, as editing or montages that encourage the creation of ideas, concepts and sensations, are key in the course of the architectural and the cinematographic conception, and the drawing, a tool that allows both authors their development. The approach to the way the architect and the movie director get to the process of the creation of their projects through the drawing is addressed from the graphic strategies of these four authors: Sergei Eisenstein, Le Corbusier, Akira Kurosawa and Rem Koolhaas. The structure of the work is developed in two blocks. The first one, the first four chapters, face up, from a general point of view, the potential of the image in the graphic thinking and the role of the graphic action in the course of the project, that architects, as well as movie directors, are making trying to find the analogies and common points during the process. The second block, the last four chapters, deal with the use of the graphic media in a more detail manner, taking as example two of the architects and two of the movie directors more influential of the past and the present centuries, trying to understand the role of the drawing in the conceptual development of the their work.

CRIAÇÃO E RECRIAÇÕES COSMOGÔNICAS EM ISAÍAS 51, 9-11: Um estudo do uso do mito cosmogônico pelo Dêutero-Isaías para o entendimento da comunidade dos cativos judaítas em Babilônia

Como estudar uma cultura ou uma comunidade perdida nos tempos bíblicos? Esta é um questão motriz para o autor. Foi dessa maneira que surgiu o seu interesse em discutir a possibilidade do uso do mito cosmogônico para o entendimento da comunidade dos cativos judaítas em Babilônia. É uma iniciativa, que precisava ser trilhada pelos pesquisadores que se dispusessem ao estudo das culturas do mundo bíblico. Assim se elegeu o tema Mito Cosmogônico no Primeiro Testamento como instrumento de aprofundamento da pesquisa bíblica. O mito é uma escolha mais ou menos óbvia, pela sua capacidade de funcionar como paradigma, pragmática e traditiva contra-hegemônica dentro de um contexto social interétnico. Estas eram ponderações vindas de matrizes como a do fenomenólogo Mircea Eliade, do Antropólogo Roger Bastide e do teólogo e fenomenólogo José Severino Croatto. É por isto que um paralelo é traçado entre o mito de Marduk e o texto de Isaías 51, 9-11, que fala de Javé como sendo criador do mundo e que luta contra as forças do caos. Isto é feito, com vistas à percepção da profecia do Isaías do exílio, como parentesco e sua justaposição com a mitologia babilônica, e ambos se aproximam bastante de forma sintagmática e histórico-social. Coube ainda saber se a profecia do Dêutero-Isaías atuava da mesma maneira que o poema Enuma elish funcionava para os babilônicos. Ou seja, fazia-se surgir modelos sociais às comunidades de escravos dentro do Império Neobabilônico; se com base nestes cânticos, os cativos conseguiam construir um ordenamento para as suas comunidades, que gozavam de uma relativa autonomia, tais como colônias e guetos ; se de posse dessa ousada profecia, os judeus da golah eram capazes de elaborar uma desobediência cívil nos termos de um nutrir nos corações, uma utopia que rompesse com o status quo do passado, comprometendo-os com a esperança no Javé criador.(AU)

CRIAÇÃO E RECRIAÇÕES COSMOGÔNICAS EM ISAÍAS 51, 9-11: Um estudo do uso do mito cosmogônico pelo Dêutero-Isaías para o entendimento da comunidade dos cativos judaítas em Babilônia

Como estudar uma cultura ou uma comunidade perdida nos tempos bíblicos? Esta é um questão motriz para o autor. Foi dessa maneira que surgiu o seu interesse em discutir a possibilidade do uso do mito cosmogônico para o entendimento da comunidade dos cativos judaítas em Babilônia. É uma iniciativa, que precisava ser trilhada pelos pesquisadores que se dispusessem ao estudo das culturas do mundo bíblico. Assim se elegeu o tema Mito Cosmogônico no Primeiro Testamento como instrumento de aprofundamento da pesquisa bíblica. O mito é uma escolha mais ou menos óbvia, pela sua capacidade de funcionar como paradigma, pragmática e traditiva contra-hegemônica dentro de um contexto social interétnico. Estas eram ponderações vindas de matrizes como a do fenomenólogo Mircea Eliade, do Antropólogo Roger Bastide e do teólogo e fenomenólogo José Severino Croatto. É por isto que um paralelo é traçado entre o mito de Marduk e o texto de Isaías 51, 9-11, que fala de Javé como sendo criador do mundo e que luta contra as forças do caos. Isto é feito, com vistas à percepção da profecia do Isaías do exílio, como parentesco e sua justaposição com a mitologia babilônica, e ambos se aproximam bastante de forma sintagmática e histórico-social. Coube ainda saber se a profecia do Dêutero-Isaías atuava da mesma maneira que o poema Enuma elish funcionava para os babilônicos. Ou seja, fazia-se surgir modelos sociais às comunidades de escravos dentro do Império Neobabilônico; se com base nestes cânticos, os cativos conseguiam construir um ordenamento para as suas comunidades, que gozavam de uma relativa autonomia, tais como colônias e guetos ; se de posse dessa ousada profecia, os judeus da golah eram capazes de elaborar uma desobediência cívil nos termos de um nutrir nos corações, uma utopia que rompesse com o status quo do passado, comprometendo-os com a esperança no Javé criador.(AU)

"Mídia, Cultura e Desenvolvimento: Estudo Comparado de Revistas Regionais"

Historicamente tido como nacional, o meio revista está sendo regionalmente reinventado. Seus moldes tradicionais passam por uma readaptação e os esquematismos dominantes na produção jornalística já não são homogêneos. Há um movimento setorial à procura de um novo mercado, incrustado nas especificidades regionais e no desenvolvimento socioeconômico que está vicejando num cotidiano desconhecido pela chamada grande imprensa. O mercado de revistas no Brasil cresce consecutivamente e de forma organizada há anos. Embora não haja registros nas fontes de autoridade, as revistas regionais e as tiragens têm se multiplicado velozmente, contrariando os revezes econômico-financeiros sentidos por outros setores da indústria cultural. Este fenômeno é o objetivo desta pesquisa: mapear a nova geografia do meio revista nas cinco macrorregiões brasileiras para entender como as identidades regionais são processadas em favor da comunicação com públicos específicos, característica que está na essência da revista. Métodos mistos de pesquisa qualitativa e quantitativa traçam o caminho da justaposição necessária para descrever este surto de publicações regionais. Estudo de casos múltiplos e análise de conteúdo envolvendo cinco revistas de cada uma das regiões políticoadministrativas, descrevem e discutem as tendências da segmentação no mercado editorial além do eixo Rio-São Paulo. Como resultado desta investigação, chega-se às seguintes conclusões: a consolidação de um novo campo jornalístico regional, profissionalizado, competente e criativo, distante do amadorismo, do bairrismo e da mimetização simplista; os mais expressivos veículos de cada uma das cinco macro-regiões infra-nacionais, segundo o construto metodológico criado para esta pesquisa, trabalham as relações, modos e demandas de produção simbólica sem artificialismos; as identidades regionais instituídas estão intimamente ligadas às regiões de influência e as redes urbanas; o retrato do estilo de vida urbano estampado nas revistas do corpus reforça tanto o poder desta como veículo de comunicação, como retroalimenta os sotaques regionais nos níveis sociais onde são produzidas e digeridas.(AU)

"Mídia, Cultura e Desenvolvimento: Estudo Comparado de Revistas Regionais"

Historicamente tido como nacional, o meio revista está sendo regionalmente reinventado. Seus moldes tradicionais passam por uma readaptação e os esquematismos dominantes na produção jornalística já não são homogêneos. Há um movimento setorial à procura de um novo mercado, incrustado nas especificidades regionais e no desenvolvimento socioeconômico que está vicejando num cotidiano desconhecido pela chamada grande imprensa. O mercado de revistas no Brasil cresce consecutivamente e de forma organizada há anos. Embora não haja registros nas fontes de autoridade, as revistas regionais e as tiragens têm se multiplicado velozmente, contrariando os revezes econômico-financeiros sentidos por outros setores da indústria cultural. Este fenômeno é o objetivo desta pesquisa: mapear a nova geografia do meio revista nas cinco macrorregiões brasileiras para entender como as identidades regionais são processadas em favor da comunicação com públicos específicos, característica que está na essência da revista. Métodos mistos de pesquisa qualitativa e quantitativa traçam o caminho da justaposição necessária para descrever este surto de publicações regionais. Estudo de casos múltiplos e análise de conteúdo envolvendo cinco revistas de cada uma das regiões políticoadministrativas, descrevem e discutem as tendências da segmentação no mercado editorial além do eixo Rio-São Paulo. Como resultado desta investigação, chega-se às seguintes conclusões: a consolidação de um novo campo jornalístico regional, profissionalizado, competente e criativo, distante do amadorismo, do bairrismo e da mimetização simplista; os mais expressivos veículos de cada uma das cinco macro-regiões infra-nacionais, segundo o construto metodológico criado para esta pesquisa, trabalham as relações, modos e demandas de produção simbólica sem artificialismos; as identidades regionais instituídas estão intimamente ligadas às regiões de influência e as redes urbanas; o retrato do estilo de vida urbano estampado nas revistas do corpus reforça tanto o poder desta como veículo de comunicação, como retroalimenta os sotaques regionais nos níveis sociais onde são produzidas e digeridas.(AU)

Stimulation of neurite outgrowth using an electrically conducting polymer

Damage to peripheral nerves often cannot be repaired by the juxtaposition of the severed nerve ends. Surgeons have typically used autologous nerve grafts, which have several drawbacks including the need for multiple surgical procedures and loss of function at the donor site. As an alternative, the use of nerve guidance channels to bridge the gap between severed nerve ends is being explored. In this paper, the electrically conductive polymer—oxidized polypyrrole (PP)—has been evaluated for use as a substrate to enhance nerve cell interactions in culture as a first step toward potentially using such polymers to stimulate in vivo nerve regeneration. Image analysis demonstrates that PC-12 cells and primary chicken sciatic nerve explants attached and extended neurites equally well on both PP films and tissue culture polystyrene in the absence of electrical stimulation. In contrast, PC-12 cells interacted poorly with indium tin oxide (ITO), poly(l-lactic acid) (PLA), and poly(lactic acid-co-glycolic acid) surfaces. However, PC-12 cells cultured on PP films and subjected to an electrical stimulus through the film showed a significant increase in neurite lengths compared with ones that were not subjected to electrical stimulation through the film and tissue culture polystyrene controls. The median neurite length for PC-12 cells grown on PP and subjected to an electrical stimulus was 18.14 μm (n = 5643) compared with 9.5 μm (n = 4440) for controls. Furthermore, animal implantation studies reveal that PP invokes little adverse tissue response compared with poly(lactic acid-co-glycolic acid).

Comparative analyses of the secondary structures of synthetic and intracellular yeast MFA2 mRNAs

The overall folded (global) structure of mRNA may be critical to translation and turnover control mechanisms, but it has received little experimental attention. Presented here is a comparative analysis of the basic features of the global secondary structure of a synthetic mRNA and the same intracellular eukaryotic mRNA by dimethyl sulfate (DMS) structure probing. Synthetic MFA2 mRNA of Saccharomyces cerevisiae first was examined by using both enzymes and chemical reagents to determine single-stranded and hybridized regions; RNAs with and without a poly(A) tail were compared. A folding pattern was obtained with the aid of the mfold program package that identified the model that best satisfied the probing data. A long-range structural interaction involving the 5′ and 3′ untranslated regions and causing a juxtaposition of the ends of the RNA, was examined further by a useful technique involving oligo(dT)-cellulose chromatography and antisense oligonucleotides. DMS chemical probing of A and C nucleotides of intracellular MFA2 mRNA was then done. The modification data support a very similar intracellular structure. When low reactivity of A and C residues is found in the synthetic RNA, ≈70% of the same sites are relatively more resistant to DMS modification in vivo. A slightly higher sensitivity to DMS is found in vivo for some of the A and C nucleotides predicted to be hybridized from the synthetic structural model. With this small mRNA, the translation process and mRNA-binding proteins do not block DMS modifications, and all A and C nucleotides are modified the same or more strongly than with the synthetic RNA.

Structural basis of DNA bending and oriented heterodimer binding by the basic leucine zipper domains of Fos and Jun

Interactions among transcription factors that bind to separate sequence elements require bending of the intervening DNA and juxtaposition of interacting molecular surfaces in an appropriate orientation. Here, we examine the effects of single amino acid substitutions adjacent to the basic regions of Fos and Jun as well as changes in sequences flanking the AP-1 site on DNA bending. Substitution of charged amino acid residues at positions adjacent to the basic DNA-binding domains of Fos and Jun altered DNA bending. The change in DNA bending was directly proportional to the change in net charge for all heterodimeric combinations between these proteins. Fos and Jun induced distinct DNA bends at different binding sites. Exchange of a single base pair outside of the region contacted in the x-ray crystal structure altered DNA bending. Substitution of base pairs flanking the AP-1 site had converse effects on the opposite directions of DNA bending induced by homodimers and heterodimers. These results suggest that Fos and Jun induce DNA bending in part through electrostatic interactions between amino acid residues adjacent to the basic region and base pairs flanking the AP-1 site. DNA bending by Fos and Jun at inverted binding sites indicated that heterodimers bind to the AP-1 site in a preferred orientation. Mutation of a conserved arginine within the basic regions of Fos and transversion of the central C:G base pair in the AP-1 site to G:C had complementary effects on the orientation of heterodimer binding and DNA bending. The conformational variability of the Fos–Jun–AP-1 complex may contribute to its functional versatility at different promoters.

Crystal structure of the ribosomal RNA domain essential for binding elongation factors

The structure of a 29-nucleotide RNA containing the sarcin/ricin loop (SRL) of rat 28 S rRNA has been determined at 2.1 Å resolution. Recognition of the SRL by elongation factors and by the ribotoxins, sarcin and ricin, requires a nearly universal dodecamer sequence that folds into a G-bulged cross-strand A stack and a GAGA tetraloop. The juxtaposition of these two motifs forms a distorted hairpin structure that allows direct recognition of bases in both grooves as well as recognition of nonhelical backbone geometry and two 5′-unstacked purines. Comparisons with other RNA crystal structures establish the cross-strand A stack and the GNRA tetraloop as defined and modular RNA structural elements. The conserved region at the top is connected to the base of the domain by a region presumed to be flexible because of the sparsity of stabilizing contacts. Although the conformation of the SRL RNA previously determined by NMR spectroscopy is similar to the structure determined by x-ray crystallography, significant differences are observed in the “flexible” region and to a lesser extent in the G-bulged cross-strand A stack.

Initiation of Assembly of the Cell Envelope Barrier Structure of Stratified Squamous Epithelia

The cell envelope (CE) is a specialized structure that is important for barrier function in terminally differentiated stratified squamous epithelia. The CE is formed inside the plasma membrane and becomes insoluble as a result of cross-linking of constituent proteins by isopeptide bonds formed by transglutaminases. To investigate the earliest stages of assembly of the CE, we have studied human epidermal keratinocytes induced to terminally differentiate in submerged liquid culture as a model system for epithelia in general. CEs were harvested from 2-, 3-, 5-, or 7-d cultured cells and examined by 1) immunogold electron microscopy using antibodies to known CE or other junctional proteins and 2) amino acid sequencing of cross-linked peptides derived by proteolysis of CEs. Our data document that CE assembly is initiated along the plasma membrane between desmosomes by head-to-tail and head-to-head cross-linking of involucrin to itself and to envoplakin and perhaps periplakin. Essentially only one lysine and two glutamine residues of involucrin and two glutamines of envoplakin were used initially. In CEs of 3-d cultured cells, involucrin, envoplakin, and small proline-rich proteins were physically located at desmosomes and had become cross-linked to desmoplakin, and in 5-d CEs, these three proteins had formed a continuous layer extending uniformly along the cell periphery. By this time >15 residues of involucrin were used for cross-linking. The CEs of 7-d cells contain significant amounts of the protein loricrin, typically expressed at a later stage of CE assembly. Together, these data stress the importance of juxtaposition of membranes, transglutaminases, and involucrin and envoplakin in the initiation of CE assembly of stratified squamous epithelia.

The genesis of avian neural crest cells: a classic embryonic induction.

Neural crest cells arise from the ectoderm and are first recognizable as discrete cells in the chicken embryo when they emerge from the neural tube. Despite the classical view that neural crest precursors are a distinct population lying between epidermis and neuroepithelium, our results demonstrate that they are not a segregated population. Cell lineage analyses have demonstrated that individual precursor cells within the neural folds can give rise to epidermal, neural crest, and neural tube derivatives. Interactions between the neural plate and epidermis can generate neural crest cells, since juxtaposition of these tissues at early stages results in the formation of neural crest cells at the interface. Inductive interactions between the epidermis and neural plate can also result in "dorsalization" of the neural plate, as assayed by the expression of the Wnt transcripts characteristic of the dorsal neural tube. The competence of the neural plate changes with time, however, such that interaction of early neural plate with epidermis generates only neural crest cells, whereas interaction of slightly older neural plate with epidermis generates neural crest cells and Wnt-expressing cells. At cranial levels, neuroepithelial cells can regulate to generate neural crest cells when the endogenous neural folds are removed, probably via interaction of the remaining neural tube with the epidermis. Taken together, these experiments demonstrate that: (i) progenitor cells in the neural folds are multipotent, having the ability to form multiple ectodermal derivatives, including epidermal, neural crest, and neural tube cells; (ii) the neural crest is an induced population that arises by interactions between the neural plate and the epidermis; and (iii) the competence of the neural plate to respond to inductive interactions changes as a function of embryonic age.

Microsatellite spreading in the human genome: evolutionary mechanisms and structural implications.

Microsatellites are tandem repeat sequences abundant in the genomes of higher eukaryotes and hitherto considered as "junk DNA." Analysis of a human genome representative data base (2.84 Mb) reveals a distinct juxtaposition of A-rich microsatellites and retroposons and suggests their coevolution. The analysis implies that most microsatellites were generated by a 3'-extension of retrotranscripts, similar to mRNA polyadenylylation, and that they serve in turn as "retroposition navigators," directing the retroposons via homology-driven integration into defined sites. Thus, they became instrumental in the preservation and extension of primordial genomic patterns. A role is assigned to these reiterating A-rich loci in the higher-order organization of the chromatin. The disease-associated triplet repeats are mostly found in coding regions and do not show an association with retroposons, constituting a unique set within the family of microsatellite sequences.

The wing of the enhancer-binding domain of Mu phage transposase is flexible and is essential for efficient transposition.

A tetramer of the Mu transposase (MuA) pairs the recombination sites, cleaves the donor DNA, and joins these ends to a target DNA by strand transfer. Juxtaposition of the recombination sites is accomplished by the assembly of a stable synaptic complex of MuA protein and Mu DNA. This initial critical step is facilitated by the transient binding of the N-terminal domain of MuA to an enhancer DNA element within the Mu genome (called the internal activation sequence, IAS). Recently we solved the three-dimensional solution structure of the enhancer-binding domain of Mu phage transposase (residues 1-76, MuA76) and proposed a model for its interaction with the IAS element. Site-directed mutagenesis coupled with an in vitro transposition assay has been used to assess the validity of the model. We have identified five residues on the surface of MuA that are crucial for stable synaptic complex formation but dispensable for subsequent events in transposition. These mutations are located in the loop (wing) structure and recognition helix of the MuA76 domain of the transposase and do not seriously perturb the structure of the domain. Furthermore, in order to understand the dynamic behavior of the MuA76 domain prior to stable synaptic complex formation, we have measured heteronuclear 15N relaxation rates for the unbound MuA76 domain. In the DNA free state the backbone atoms of the helix-turn-helix motif are generally immobilized whereas the residues in the wing are highly flexible on the pico- to nanosecond time scale. Together these studies define the surface of MuA required for enhancement of transposition in vitro and suggest that a flexible loop in the MuA protein required for DNA recognition may become structurally ordered only upon DNA binding.

A structural motif for the voltage-gated potassium channel pore.

Mutation studies have identified a region of the S5-S6 loop of voltage-gated K+ channels (P region) responsible for teraethylammonium (TEA) block and permeation/selectivity properties. We previously modeled a similar region of the Na+ channel as four beta-hairpins with the C strands from each of the domains forming the external vestibule and with charged residues at the beta-turns forming the selectivity filter. However, the K+ channel P region amino acid composition is much more hydrophobic in this area. Here we propose a structural motif for the K+ channel pore based on the following postulates (Kv2.1 numbering). (i) The external TEA binding site is formed by four Tyr-380 residues; P loop residues participating in the internal TEA binding site are four Met-371 and Thr-372 residues. (ii) P regions form extended hairpins with beta-turns in sequence ITMT. (iii) only C ends of hairpins form the inner walls of the pore. (iv) They are extended nonregular strands with backbone carbonyl oxygens of segment VGYGD facing the pore with the conformation BRLRL. (v) Juxtaposition of P loops of the four subunits forms the pore. Fitting the external and internal TEA sites to TEA molecules predicts an hourglass-like pore with the narrowest point (GYG) as wide as 5.5 A, suggesting that selectivity may be achieved by interactions of carbonyls with partially hydrated K+. Other potential cation binding sites also exist in the pore.