Materialidad poética: arquitectura suiza en el entorno de los Grisones 1992-2004

El entorno alpino de los Grisones ha sido un laboratorio donde experimentar un método arquitectónico procedente de diversas fuentes durante los años 70 en la ETHZ. Este método ha producido durante las últimas tres décadas un discurso teórico propiamente suizo alemán. La tradición continúa siendo un valor cultural para la comunidad alpina de los Grisones, que apuesta por seguir relacionando la arquitectura con su historia y su paisaje. La especificidad de cada entorno suizo alemán es un hecho obvio pero, por encima de sus rasgos característicos, hay un denominador común que les da unidad en silencio: se trata de una idea de la construcción. El fenómeno de los Grisones es territorial en su localización concreta, pero comparte el trasfondo teórico del resto de la arquitectura suizo alemana reciente. La tesis recorre transversalmente las condiciones que han dado lugar a esa trayectoria común hasta la actualidad, en que la arquitectura suizo alemana se halla precisamente en un momento de cambio generacional. La obra de Peter Märkli constituye en esta tesis un paradigma de la voluntad suizo alemana por formar parte de la continuidad cultural de la arquitectura, como una actitud de resistencia compartida frente al mundo globalizado. La tesis se articula a partir de ocho proyectos recientes, realizados por arquitectos suizo alemanes en el entorno del territorio alpino de los Grisones. Estos proyectos condensan la trayectoria arquitectónica suizo alemana durante el siglo XX, así como el fenómeno arquitectónico que ha tenido lugar en el entorno de los Grisones durante las últimas tres décadas. La tesis analiza a través de cada pareja de proyectos los episodios teóricos, artísticos y filosóficos más relevantes que se sintetizan en estas obras, así como sus referentes arquitectónicos y la dimensión trascendente de los materiales en los que la cultura suiza hunde sus raíces: la madera, la piedra y el hormigón. Las vanguardias pictóricas, el Movimiento Moderno y las corrientes humanistas y regionalistas que se fueron sucediendo durante el pasado siglo, configuran el escenario en el que tomó cuerpo la sensibilidad arquitectónica suizo alemana reciente, que emerge a través de la compleja red de relaciones establecidas entre el panorama suizo y el internacional. El mismo bagaje teórico subyace al territorio suizo alemán a nivel general, por lo que asociarlo a través de esta tesis a proyectos realizados en un entorno característico, permite acceder a una realización concreta de ese debate en un entorno tan particular como son los Grisones. La aproximación a los proyectos es de carácter fenomenológico: la experiencia directa de los mismos ha sido el sustrato fundamental del enfoque de la tesis. El trabajo de campo no se limitó a visitar los proyectos que vertebran la tesis, sino que trató de abarcar las obras más relevantes de la producción suizo alemana reciente en los Grisones, con el fin de adquirir cierto grado de sensibilidad perceptiva hacia la singularidad de su identidad territorial. La tesis hace especial hincapié en las obras de Peter Märkli y Peter Zumthor, representantes de los dos extremos del discurso suizo alemán reciente, respecto a la materialidad y al carácter de la secuencia espacial, sistemático o fluido respectivamente, pero que asimismo comparten el clasicismo formalista que subyace al fenómeno suizo alemán. Los dos proyectos que establecen el periodo 1992-2004 explorado por esta investigación constituyen dos hitos fundamentales. El Museo de La Congiunta en Giornico de Peter Märkli se construyó en 1992 y se erige a partir de entonces como un icono arquitectónico de la condensación típicamente suizo alemana de base reductiva. Este paradigma de síntesis conceptual y constructiva comenzó a forjarse en la década de 1930 de la mano del Konkrete Kunst formulado por Max Bill, que es la única manifestación artística genuinamente suiza del siglo XX y que desarrolló pictóricamente los valores de precisión, rigor, racionalismo y abstracción afines a la sensibilidad suizo alemana. En segundo lugar, el proyecto para la rehabilitación de la Villa Garbald y la construcción de la Torre Roccolo en Castasegna llevado a cabo por Miller & Maranta en 2004, supuso la consumación de la Analoge Architektur, que fue la primera corriente arquitectónica propiamente suiza, desarrollada durante la década de 1980 por Miroslav Sik. La Deutscher Tendenza es un periodo de la trayectoria suizo alemana reciente poco conocido, representado por una figura tan relevante como es Peter Zumthor. Las obras que proyectó Zumthor entre 1979 y 1985 forman parte de un periodo en que el arquitecto se encontraba profundamente influido por el neorracionalismo italiano, como ilustra la escuela de Churwalden de 1983. La arquitectura suizo alemana reciente exploró sistemáticamente los sistemas compositivos de las vanguardias pictóricas y del Movimiento Moderno, por encima de su formalismo concreto. La segunda fase de la obra de Zumthor entre 1985 y 1996 se caracteriza por la transfiguración del plano libre del sistema compositivo neoplasticista desarrollado por De Stijl, plano que adquirió grosor hasta convertirse en la caja articuladora de la fluidez de la secuencia espacial. El formalismo de Zumthor en esta fase implica la prioridad del esquema compositivo, compuesto por un perímetro regular en el que se reúnen las cajas que, como las superficies de color de las composiciones de Mondrian, articulan el espacio a su alrededor. La Residencia de Masans de Zumthor de 1993 es un proyecto canónico de este sistema compositivo, que culmina en el esquema radial de las Termas de Vals de 1996. La imagen poética se convirtió en el lenguaje propio del pensamiento de la arquitectura suizo alemana, que permitía una reflexión no verbal, a partir de unos pocos arquetipos fundamentales. Los arquitectos del Ticino buscaron en la Tendenza italiana durante la década de 1970 una alternativa al insostenible modelo territorial que estaba destruyendo su paisaje cultural. Algunos de estos arquitectos comenzaron a dar clase durante esa década en la ETHZ, por lo que transmitieron sus inquietudes al alumnado e introdujeron a Aldo Rossi en la facultad, que era uno de los máximos representantes de la Tendenza. El método analógico de Rossi, basado en la psicología analítica de Carl Jung, fue una influencia fundamental en la ETHZ durante los años 70. Rossi partía de una teoría afín al colectivo intelectual suizo que los arquitectos de la ETHZ pronto comprendieron que debían independizar del contexto italiano, desarrollando su propio método a partir de 1985 con la Analoge Architektur. El valor de la preexistencia de acuerdo a este método incipiente se hallaba en su papel articulador de la continuidad cultural de una comunidad, que Miroslav Sik definió a partir de su concepto de Altneue (literalmente, lo Nuevo-Viejo). La fusión entre lo nuevo y lo viejo era la base de la espontaneidad con la que habían crecido los asentamientos históricamente. Sik consideraba que la continuidad dependía de la comprensión de los patrones preexistentes, con el objeto de mantener su vigencia mediante la incorporación de estrategias contemporáneas, que renovarían su significado sin perder su esencia. La Casa Gugalun en Versam de Zumthor de 1994 supone una aproximación a escala doméstica a este planteamiento. La arquitectura suizo alemana reciente a menudo busca en sus referentes plásticos una manera de explorar ámbitos poco accesibles para la disciplina arquitectónica. El pensamiento arquitectónico basado en la imagen poética se implementa a partir de la tectónica de la construcción, que es el medio para comunicar una idea vertebradora del proyecto de principio a fin. La construcción como medio introduce el concepto clave de la apariencia de la arquitectura, estrechamente relacionado con la filosofía idealista de Friedrich Schiller. La apariencia debe expresar el sentido conceptual de la obra, que los arquitectos suizo alemanes denominan idea, por lo que la construcción no tiene un valor por sí misma, sino en cuanto a la apariencia. La Capilla de Oberrealta de Christian Kerez de 1993 puede ser considerada una obra escultórica en el paisaje, más próxima a las artes plásticas que a la arquitectura. La tensión que surge entre la figura ideal y la pieza sometida a una sutil deformación confiere un carácter dinámico a la composición, que intensifica el efecto perceptivo, de acuerdo a la teoría del pensamiento visual de Rudolph Arnheim. La deformación al servicio de la psicología de la percepción es un fenómeno que caracteriza la arquitectura suizo alemana reciente. El concepto de Forme Forte (forma fuerte) fue introducido por Martin Steinmann para caracterizar el objeto arquitectónico denominado internacionalmente Swiss Box. Este concepto se caracteriza por una predilección por lo monolítico, lo unitario y lo arquetípico. La condición más sustancial de una Forme Forte es, sin embargo, la de configurar un elemento estructurador del entorno. Tanto la base morfológica de una Forme Forte, como su capacidad para estructurar el lugar, se corresponden con los conceptos de arquetipo y monumento de la teoría de Rossi. La tesis explora el sentido de la deformación en la producción suizo alemana reciente, a partir de la Escuela de Paspels de Valerio Olgiati de 1998 y la Torre Roccolo de Villa Garbald de Miller&Maranta de 1998. La teoría tectónica de Gottfried Semper y la razón de la forma de Adolf Loos constituyen el sustrato teórico, que relaciona la actividad existencial de habitar con la materialidad de la arquitectura suizo alemana reciente. La teoría tectónica de Semper fundamenta las revisiones posteriores de los sistemas constructivos tradicionales suizo alemanes. Esta influencia sirvió como base a la vanguardia suiza de la Neues Bauen, que desarrolló un racionalismo funcionalista durante las décadas de 1920 y 1930, que constituye una gramática de base no simbólica. Esta gramática constituye el fundamento de la transición del orden clásico a la materialidad como instrumento compositivo durante la segunda mitad del siglo XX, consolidando el concepto de la base material de la forma. La revisión de la tradición regionalista y racionalista llevada a cabo por la generación de arquitectos suizo alemanes de la década de 1950 y 1960, constituyó una investigación lingüística sustancial. Rudolph Olgiati es la figura determinante de este periodo. La revisión de esta investigación se codificó en clave estructuralista durante los años 70 en la ETHZ, influenciada por la presencia de Roland Barthes como profesor invitado. Esta revisión constituyó la base de la articulación tectónica propiamente suizo alemana todavía vigente, como ilustra expresamente la obra de Burkhalter&Sumi en esta tesis. La obra de Gion Caminada en Vrin entre 1995 y 2002 aporta una intensa investigación sobre el Strickbau, que implementa una y otra vez variaciones sobre un sistema tradicional, ilustrando las infinitas posibilidades que ofrece un lenguaje, paradójicamente limitado por los medios disponibles en un entorno alpino aislado. Para terminar Jürg Conzett es el ingeniero con el que han desarrollado sus proyectos la mayoría de los arquitectos en el entorno de los Grisones, formado durante 8 años con Zumthor y colaborador habitual de Caminada entre otros. Conzett es el referente que relaciona la ingeniería con la arquitectura en el ámbito de esta tesis, imprescindible en el contexto de esta investigación. ABSTRACT The Alpine setting of Grisons has been a laboratory for architectural experimentation with an approach forged in the 1970s from various sources in the Swiss Federal Institute of Technology in Zurich (ETHZ). This method has produced a uniquely German-Swiss theoretical discourse over the past three decades. Tradition continues to be a cultural value for Grisons, a region committed to continue linking its architecture with the region’s history and landscape. The specificity of the German-Swiss environment is obvious, but beyond its characteristic features, there is a common denominator that gives these settings a silent unity: it is an idea of building. The phenomenon of Grisons is geographic in its specific location, but it shares the theoretical basis of the rest of the new German-Swiss architecture. This thesis transversely delves into the conditions that have shaped this common path up to the present, just as German-Swiss architecture is going through a time of generational change. In this thesis, Märkli Peter's work constitutes a paradigm of the German-Swiss will to uphold the cultural and architectural continuity of the region, exemplifying an attitude of shared resistance to the globalized world. The thesis is structured around eight recent projects carried out by German-Swiss architects in the Alpine region of Grisons. These projects represent the trajectory of twentieth century German-Swiss architectural history, as well as the architectural phenomenon that has taken place in the environs of Grisons over the past three decades. Through these projects the thesis analyzes the most relevant theoretical, artistic and philosophical references that are synthesized in the works, as well as their architectural references and the transcendental dimension of the materials in which Swiss culture is rooted: wood, stone and concrete. The artistic avant-garde, together with Modernism and the humanistic and regional movements that occurred during the last century, set the stage for present-day German-Swiss architectural sensitivity, which emerges from a complex web of relationships established between the Swiss and international panorama. This same theoretical background and experience underlies all of the German-Swiss territory in general, so associating it through this thesis to a particular context allows the description of a specific embodiment of this debate, within the unique environment of Grisons. The methodological approach to analyzing the projects was phenomenological: direct experience is the main substrate underpinning the focus of the thesis. Field work was not limited to visiting the projects featured in the thesis, but rather encompassed the most important works of recent German-Swiss construction in the Grisons in order to gain some degree of perceptual sensitivity to the uniqueness of its territorial identity. The present paper puts special emphasis on the works of Peter Märkli and Peter Zumthor, who share the formal classicist perspective of the German-Swiss context but stand on opposite sides of the philosophical spectrum with regard to the notion of materiality and the nature of space, which they conceive as systematic or fluid, respectively. The two projects that establish the boundaries of the time period 1992–2004 explored by this research represent two key milestones. The Museum La Congiunta in Giornico, by Peter Märkli, was built in 1992 and quickly established itself as an architectural icon of German-Swiss reductionism. This paradigm of conceptual and constructive synthesis began to take shape in the 1930s under the banner of Konkrete Kunst (Concrete Art), led by Max Bill. The only genuinely Swiss artistic movement of the twentieth century, Konkrete Kunst was characterized by the artistic values of precision, rigor, rationalism and abstraction, sentiments that were very close to the German-Swiss tradition. Secondly, the project for the rehabilitation of Villa Garbald and the construction of the Roccolo Tower in Castasegna, conducted by Miller&Maranta in 2004, represented the consummation of the Analoge Architektur, which was the first truly Swiss architectural movement, spearheaded in the 1980s by Miroslav Sik. The Deutscher Tendenza is a little-known period of recent German-Swiss history, represented by the important figure of Peter Zumthor. The projects that Zumthor led between 1979 and 1985 are part of a period when Italian Neo-Rationalism exercised a profound influence on the architect, as illustrated by the Churwalden School, circa 1983. Recent German-Swiss architecture systematically explored the compositional systems of the artistic avant-garde and Modernism, beyond its specific formal aspects. The second phase of Zumthor's work, between 1985 and 1996, is characterized by the transfiguration of the free plane, neoplastic compositional system developed by De Stijl, a plane that thickened until it became the box articulating fluidity from the spatial sequence. Zumthor's formalism in this phase prioritizes the compositional scheme, consisting of a regular perimeter in which the boxes that -like Mondrian’s colored surfaces- arrange the space around it. Zumthor’s Masans Residence, circa 1993, is a canonical project of this compositional system, which culminates in the radial pattern of the Therme Vals, circa 1996. The poetic image became the appropriate language of thought for German-Swiss architecture, which invited a nonverbal reflection inspired by a few fundamental archetypes. The architects of Ticino sought, through the Italian Tendenza of the 1970s, an alternative to the unsustainable territorial model that was destroying their cultural landscape. Some of these architects began to teach during that decade at ETHZ, and naturally they transmitted their unease to their students. These architects also introduced Aldo Rossi, one of the leading representatives of the Tendenza, to the school. Rossi’s analogue method, based on the analytical psychology of Carl Jung, was a major influence on the ETHZ during the 1970s. Rossi’s theoretical grounding was very much in tune with Swiss intellectualism, and it did not take long for architects from ETHZ to realize that they should break away from the Italian context, developing their own method from 1985 on with the Analoge Architektur. The value of the pre-existing conformity of this emerging method, stemmed from its role in facilitating the cultural continuity of a community, which Miroslav Sik defined in his concept of Altneue (literally, Old-new). This fusion of old and new was the basis for the spontaneity with which settlements had historically grown. Sik considered that continuity depended on understanding the existing patterns and sustaining their relevance through the incorporation of contemporary strategies, which would renew their meaning without losing their essence. Zumthor’s Gugalun House in Versam, circa 1994, is a domestic-scale approach to this philosophy. Modern German-Swiss architecture often looks to its references in visual art for a way to explore areas that are normally inaccessible to the architectural discipline. Architectural thinking based on the poetic image is achieved through a building’s tectonics, which communicate the core idea of a project from start to finish. The understanding of construction as a medium introduces the key concept of the appearance in architecture, closely related to the idealistic philosophy of Friedrich Schiller. The appearance should express the conceptual meaning of the work, which German-Swiss architects call the idea, so that the building does not have value in and of itself, but only in terms of its appearance. The Oberrealta Chapel by Christian Kerez, circa 1993, can be considered a sculpture in the landscape, closer to the visual arts than to architecture. The tension that arises between the ideal figure and the piece subjected to a subtle deformation confers a dynamic character onto the composition, intensifying the perceptual effect, according to Rudolf Arnheim’s theory of visual thought. The deformation in the service of the psychology of perception is a phenomenon that characterizes recent German-Swiss architecture. Martin Steinmann introduced the Forme Forte (Strong Form) concept to describe the architectural object known internationally as the Swiss Box. This concept is characterized by a predilection for all things monolithic, unitary and archetypal. The most substantial condition of a Forme Forte, however, is the configuration of a structuring element in the environment. Both the morphological basis of a Forme Forte and its ability to frame the place, correspond to the concepts of archetype and monument put forward by Rossi’s theory. The present thesis explores the sense of deformation in recent German-Swiss production, based on the School of Paspels by Valerio Olgiati, circa 1998 and the Roccolo Tower in Villa Garbald, by Miller&Maranta, circa 1998. Gottfried Semper’s tectonic theory and Adolf Loos’s reason for form constitute the theoretical foundation that links the existential activity of dwelling to the materiality of recent German-Swiss architecture. Semper’s tectonic theory laid the foundation for subsequent revisions of the German-Swiss traditional building systems. This influence was the basis for the Swiss avant-garde of Neues Bauen, which developed a functional rationalism during the 1920s and 30s that served as a non-symbolic grammatical foundation for the transition from classical order to materiality as a compositional tool. During the second half of the twentieth century, this architectural grammar helped consolidate the concept of the material base of the form. The revision of the regionalist and rationalist tradition, carried out by the generation of German-Swiss architects of the 1950s and 60s, constituted a substantial linguistic investigation. Rudolph Olgiati is the key figure of this period. The research was codified in terms of structuralism in the 1970s in ETHZ and influenced by the presence of Roland Barthes as a visiting professor. This revision was the basis of the uniquely German-Swiss tectonic design still in use today, as specifically illustrated by the work of Burkhalter & Sumi in this thesis. Gion Caminada's work in Vrin between 1995 and 2002 offers an extensive study on the Strickbau, which implements variations on a traditional system again and again, illustrating the endless possibilities of a language that paradoxically seems limited by the available resources in a remote Alpine setting. Finally, Jürg Conzett is the engineer with whom most architects in the Grisons region have developed their projects. Trained under Zumthor for 8 years, and a regular collaborator of Caminada, among others, Conzett is the reference point linking engineering with architecture in this thesis, essential in the context of this research.

Quorum sensing in Escherichia coli and Salmonella typhimurium

Escherichia coli and Salmonella typhimurium strains grown in Luria–Bertani medium containing glucose secrete a small soluble heat labile organic molecule that is involved in intercellular communication. The factor is not produced when the strains are grown in Luria–Bertani medium in the absence of glucose. Maximal secretion of the substance occurs in midexponential phase, and the extracellular activity is degraded as the glucose is depleted from the medium or by the onset of stationary phase. Destruction of the signaling molecule in stationary phase indicates that, in contrast to other quorum-sensing systems, quorum sensing in E. coli and S. typhimurium is critical for regulating behavior in the prestationary phase of growth. Our results further suggest that the signaling factor produced by E. coli and S. typhimurium is used to communicate both the cell density and the metabolic potential of the environment. Several laboratory and clinical strains of E. coli and S. typhimurium were screened for production of the signaling molecule, and most strains make it under conditions similar to those shown here for E. coli AB1157 and S. typhimurium LT2. However, we also show that E. coli strain DH5α does not make the soluble factor, indicating that this highly domesticated strain has lost the gene(s) or biosynthetic machinery necessary to produce the signaling substance. Implications for the involvement of quorum sensing in pathogenesis are discussed.

Perturbation of the T cell repertoire in rheumatoid arthritis

Aberrations in the T cell repertoire with the emergence of oligoclonal populations have been described in patients with rheumatoid arthritis (RA). However, the extent of the repertoire perturbations as well as the underlying mechanisms are not known. We now have examined the diversity of the peripheral CD4 T cell repertoire by determining the frequencies of arbitrarily selected T cell receptor (TCR) β-chain sequences. Healthy individuals displayed a highly diverse repertoire, with a median frequency of individual TCR β-chain sequences of 1 in 2.4 × 107 CD4 T cells. In RA patients, the median TCR β-chain frequency was increased 10-fold, indicating marked contraction of the repertoire (P < 0.001). The loss in TCR diversity was not limited to CD4 memory T cells but also involved the compartment of naive T cells, suggesting that it reflected an abnormality in T cell repertoire formation and not a consequence of antigen recognition in the synovium. Also, control patients with chronic inflammatory disease such as hepatitis C expressed a diverse repertoire indistinguishable from that of normals. Telomere length studies indicated an increased replicative history of peripheral CD4 T cells in RA patients, suggesting an enhanced turnover within the CD4 compartment. Compared with age-matched controls, terminal restriction fragment sizes were 1.7 kilobases shorter (P < 0.001). These data demonstrate an altered CD4 T cell homeostasis in RA that may contribute to the autoimmune response as well as to the immunodeficiency in these patients.

Duplicated genes evolve independently after polyploid formation in cotton

Of the many processes that generate gene duplications, polyploidy is unique in that entire genomes are duplicated. This process has been important in the evolution of many eukaryotic groups, and it occurs with high frequency in plants. Recent evidence suggests that polyploidization may be accompanied by rapid genomic changes, but the evolutionary fate of discrete loci recently doubled by polyploidy (homoeologues) has not been studied. Here we use locus-specific isolation techniques with comparative mapping to characterize the evolution of homoeologous loci in allopolyploid cotton (Gossypium hirsutum) and in species representing its diploid progenitors. We isolated and sequenced 16 loci from both genomes of the allopolyploid, from both progenitor diploid genomes and appropriate outgroups. Phylogenetic analysis of the resulting 73.5 kb of sequence data demonstrated that for all 16 loci (14.7 kb/genome), the topology expected from organismal history was recovered. In contrast to observations involving repetitive DNAs in cotton, there was no evidence of interaction among duplicated genes in the allopolyploid. Polyploidy was not accompanied by an obvious increase in mutations indicative of pseudogene formation. Additionally, differences in rates of divergence among homoeologues in polyploids and orthologues in diploids were indistinguishable across loci, with significant rate deviation restricted to two putative pseudogenes. Our results indicate that most duplicated genes in allopolyploid cotton evolve independently of each other and at the same rate as those of their diploid progenitors. These indications of genic stasis accompanying polyploidization provide a sharp contrast to recent examples of rapid genomic evolution in allopolyploids.

M Phase Phosphoprotein 10 Is a Human U3 Small Nucleolar Ribonucleoprotein Component

We have previously developed a novel technique for isolation of cDNAs encoding M phase phosphoproteins (MPPs). In the work described herein, we further characterize MPP10, one of 10 novel proteins that we identified, with regard to its potential nucleolar function. We show that by cell fractionation, almost all MPP10 was found in isolated nucleoli. By immunofluorescence, MPP10 colocalized with nucleolar fibrillarin and other known nucleolar proteins in interphase cells but was not detected in the coiled bodies stained for either fibrillarin or p80 coilin, a protein found only in the coiled body. When nucleoli were separated into fibrillar and granular domains by treatment with actinomycin D, almost all the MPP10 was found in the fibrillar caps, which contain proteins involved in rRNA processing. In early to middle M phase of the cell cycle, MPP10 colocalized with fibrillarin to chromosome surfaces. At telophase, MPP10 was found in cellular structures that resembled nucleolus-derived bodies and prenucleolar bodies. Some of these bodies lacked fibrillarin, a previously described component of nucleolus-derived bodies and prenucleolar bodies, however, and the bulk of MPP10 arrived at the nucleolus later than fibrillarin. To further examine the properties of MPP10, we immunoprecipitated it from cell sonicates. The resulting precipitates contained U3 small nucleolar RNA (snoRNA) but no significant amounts of other box C/D snoRNAs. This association of MPP10 with U3 snoRNA was stable to 400 mM salt and suggested that MPP10 is a component of the human U3 small nucleolar ribonucleoprotein.

Katanin Is Responsible for the M-Phase Microtubule-severing Activity in Xenopus Eggs

Microtubules are dynamic structures whose proper rearrangement during the cell cycle is essential for the positioning of membranes during interphase and for chromosome segregation during mitosis. The previous discovery of a cyclin B/cdc2-activated microtubule-severing activity in M-phase Xenopus egg extracts suggested that a microtubule-severing protein might play an important role in cell cycle-dependent changes in microtubule dynamics and organization. However, the isolation of three different microtubule-severing proteins, p56, EF1α, and katanin, has only confused the issue because none of these proteins is directly activated by cyclin B/cdc2. Here we use immunodepletion with antibodies specific for a vertebrate katanin homologue to demonstrate that katanin is responsible for the majority of M-phase severing activity in Xenopus eggs. This result suggests that katanin is responsible for changes in microtubules occurring at mitosis. Immunofluorescence analysis demonstrated that katanin is concentrated at a microtubule-dependent structure at mitotic spindle poles in Xenopus A6 cells and in human fibroblasts, suggesting a specific role in microtubule disassembly at spindle poles. Surprisingly, katanin was also found in adult mouse brain, indicating that katanin may have other functions distinct from its mitotic role.

Effects of Genome Position and the DNA Damage Checkpoint on the Structure and Frequency of sod2 Gene Amplification in Fission Yeast

The Schizosaccharomyces pombe sod2 gene, located near the telomere on the long arm of chromosome I, encodes a Na+ (or Li+)/H+ antiporter. Amplification of sod2 has previously been shown to confer resistance to LiCl. We analyzed 20 independent LiCl-resistant strains and found that the only observed mechanism of resistance is amplification of sod2. The amplicons are linear, extrachromosomal elements either 225 or 180 kb long, containing both sod2 and telomere sequences. To determine whether proximity to a telomere is necessary for sod2 amplification, a strain was constructed in which the gene was moved to the middle of the same chromosomal arm. Selection of LiCl-resistant strains in this genetic background also yielded amplifications of sod2, but in this case the amplified DNA was exclusively chromosomal. Thus, proximity to a telomere is not a prerequisite for gene amplification in S. pombe but does affect the mechanism. Relative to wild-type cells, mutants with defects in the DNA damage aspect of the rad checkpoint control pathway had an increased frequency of sod2 amplification, whereas mutants defective in the S-phase completion checkpoint did not. Two models for generating the amplified DNA are presented.

Overexpression of a Novel Rho Family GTPase, RacC, Induces Unusual Actin-based Structures and Positively Affects Phagocytosis in Dictyostelium discoideum

Rho family proteins have been implicated in regulating various cellular processes, including actin cytoskeleton organization, endocytosis, cell cycle, and gene expression. In this study, we analyzed the function of a novel Dictyostelium discoideum Rho family protein (RacC). A cell line was generated that conditionally overexpressed wild-type RacC three- to fourfold relative to endogenous RacC. Light and scanning electron microscopy indicated that the morphology of the RacC-overexpressing cells [RacC WT(+) cells] was significantly altered compared with control cells. In contrast to the cortical F-actin distribution normally observed, RacC WT(+) cells displayed unusual dorsal and peripheral F-actin–rich surface blebs (petalopodia, for flower-like). Furthermore, phagocytosis in the RacC WT(+) cells was induced threefold relative to control Ax2 cells, whereas fluid-phase pinocytosis was reduced threefold, primarily as the result of an inhibition of macropinocytosis. Efflux of fluid-phase markers was also reduced in the RacC WT(+) cells, suggesting that RacC may regulate postinternalization steps along the endolysosomal pathway. Treatment of cells with Wortmannin and LY294002 (phosphatidylinositol 3-kinase inhibitors) prevented the RacC-induced morphological changes but did not affect phagocytosis, suggesting that petalopodia are probably not required for RacC-induced phagocytosis. In contrast, inactivating diacylglycerol-binding motif–containing proteins by treating cells with the drug calphostin C completely inhibited phagocytosis in control and RacC WT(+) cells. These results suggest that RacC plays a role in actin cytoskeleton organization and phagocytosis in Dictyostelium.

The cdr2+ Gene Encodes a Regulator of G2/M Progression and Cytokinesis in Schizosaccharomyces pombe

Schizosaccharomyces pombe cells respond to nutrient deprivation by altering G2/M cell size control. The G2/M transition is controlled by activation of the cyclin-dependent kinase Cdc2p. Cdc2p activation is regulated both positively and negatively. cdr2+ was identified in a screen for regulators of mitotic control during nutrient deprivation. We have cloned cdr2+ and have found that it encodes a putative serine-threonine protein kinase that is related to Saccharomyces cerevisiae Gin4p and S. pombe Cdr1p/Nim1p. cdr2+ is not essential for viability, but cells lacking cdr2+ are elongated relative to wild-type cells, spending a longer period of time in G2. Because of this property, upon nitrogen deprivation cdr2+ mutants do not arrest in G1, but rather undergo another round of S phase and arrest in G2 from which they are able to enter a state of quiescence. Genetic evidence suggests that cdr2+ acts as a mitotic inducer, functioning through wee1+, and is also important for the completion of cytokinesis at 36°C. Defects in cytokinesis are also generated by the overproduction of Cdr2p, but these defects are independent of wee1+, suggesting that cdr2+ encodes a second activity involved in cytokinesis.

CD8+ T-cell-derived soluble factor(s), but not β-chemokines RANTES, MIP-1α, and MIP-1β, suppress HIV-1 replication in monocyte/macrophages

It has been demonstrated that CD8+ T cells produce a soluble factor(s) that suppresses human immunodeficiency virus (HIV) replication in CD4+ T cells. The role of soluble factors in the suppression of HIV replication in monocyte/macrophages (M/M) has not been fully delineated. To investigate whether a CD8+ T-cell-derived soluble factor(s) can also suppress HIV infection in the M/M system, primary macrophages were infected with the macrophage tropic HIV-1 strain Ba-L. CD8+ T-cell-depleted peripheral blood mononuclear cells were also infected with HIV-1 IIIB or Ba-L. HIV expression from the chronically infected macrophage cell line U1 was also determined in the presence of CD8+ T-cell supernatants or β-chemokines. We demonstrate that: (i) CD8+ T-cell supernatants did, but β-chemokines did not, suppress HIV replication in the M/M system; (ii) antibodies to regulated on activation normal T-cell expressed and Secreted (RANTES), macrophage inflammatory protein 1α (MIP-1α) and MIP-1β did not, whereas antibodies to interleukin 10, interleukin 13, interferon α, or interferon γ modestly reduced anti-HIV activity of the CD8+ T-cell supernatants; and (iii) the CD8+ T-cell supernatants did, but β-chemokines did not, suppress HIV-1 IIIB replication in peripheral blood mononuclear cells as well as HIV expression in U1 cells. These results suggest that HIV-suppressor activity of CD8+ T cells is a multifactorial phenomenon, and that RANTES, MIP-1α, and MIP-1β do not account for the entire scope of CD8+ T-cell-derived HIV-suppressor factors.

Adaptive evolution of a candidate gene for aging in Drosophila

Examination of the phenotypic effects of specific mutations has been extensively used to identify candidate genes affecting traits of interest. However, such analyses do not reveal anything about the evolutionary forces acting at these loci, or whether standing allelic variation contributes to phenotypic variance in natural populations. The Drosophila gene methuselah (mth) has been proposed as having major effects on organismal stress response and longevity phenotype. Here, we examine patterns of polymorphism and divergence at mth in population level samples of Drosophila melanogaster, D. simulans, and D. yakuba. Mth has experienced an unusually high level of adaptive amino acid divergence concentrated in the intra- and extracellular loop domains of the receptor protein, suggesting the historical action of positive selection on those regions of the molecule that modulate signal transduction. Further analysis of single nucleotide polymorphisms (SNPs) in D. melanogaster provided evidence for contemporary and spatially variable selection at the mth locus. In ten surveyed populations, the most common mth haplotype exhibited a 40% cline in frequency that coincided with population level differences in multiple life-history traits including lifespan. This clinal pattern was not associated with any particular SNP in the coding region, indicating that selection is operating at a closely linked site that may be involved in gene expression. Together, these consistently nonneutral patterns of inter- and intraspecific variation suggest adaptive evolution of a signal transduction pathway that may modulate lifespan in nature.

Selectively enhanced contextual fear conditioning in mice lacking the transcriptional regulator CCAAT/enhancer binding protein δ

CCAAT/enhancer binding protein δ (C/EBPδ) is a transcriptional regulator implicated in the hepatic acute phase response and in adipogenic and myeloid cell differentiation. We found that C/EBPδ is widely expressed in the peripheral and central nervous systems, including neurons of the hippocampal formation, indicating a role in neural functions. To examine the role of C/EBPδ in vivo, we generated mice with a targeted deletion of the C/EBPδ gene. This mutation does not interfere with normal embryonic and postnatal development. Performance in a battery of behavioral tests indicates that basic neurological functions are normal. Furthermore, performance in a Morris water maze task suggests that C/EBPδ mutant mice have normal spatial learning. However, in the contextual and auditory-cue-conditioned fear task, C/EBPδ null mice displayed significantly more conditioned freezing to the test context than did wild-type controls, but equivalent conditioning to the auditory cue. These data demonstrate a selectively enhanced contextual fear response in mice carrying a targeted genomic mutation and implicate C/EBPδ in the regulation of a specific type of learning and memory.

Combined transcriptome and proteome analysis as a powerful approach to study genes under glucose repression in Bacillus subtilis

We used 2D protein gel electrophoresis and DNA microarray technologies to systematically analyze genes under glucose repression in Bacillus subtilis. In particular, we focused on genes expressed after the shift from glycolytic to gluconeogenic at the middle logarithmic phase of growth in a nutrient sporulation medium, which remained repressed by the addition of glucose. We also examined whether or not glucose repression of these genes was mediated by CcpA, the catabolite control protein of this bacterium. The wild-type and ccpA1 cells were grown with and without glucose, and their proteomes and transcriptomes were compared. 2D gel electrophoresis allowed us to identify 11 proteins, the synthesis of which was under glucose repression. Of these proteins, the synthesis of four (IolA, I, S and PckA) was under CcpA-independent control. Microarray analysis enabled us to detect 66 glucose-repressive genes, 22 of which (glmS, acoA, C, yisS, speD, gapB, pckA, yvdR, yxeF, iolA, B, C, D, E, F, G, H, I, J, R, S and yxbF ) were at least partially under CcpA-independent control. Furthermore, we found that CcpA and IolR, a repressor of the iol divergon, were involved in the glucose repression of the synthesis of inositol dehydrogenase encoded by iolG included in the above list. The CcpA-independent glucose repression of the iol genes appeared to be explained by inducer exclusion.

Constraints on nebular dynamics and chemistry based on observations of annealed magnesium silicate grains in comets and in disks surrounding Herbig Ae/Be stars

Understanding dynamic conditions in the Solar Nebula is the key to prediction of the material to be found in comets. We suggest that a dynamic, large-scale circulation pattern brings processed dust and gas from the inner nebula back out into the region of cometesimal formation—extending possibly hundreds of astronomical units (AU) from the sun—and that the composition of comets is determined by a chemical reaction network closely coupled to the dynamic transport of dust and gas in the system. This scenario is supported by laboratory studies of Mg silicates and the astronomical data for comets and for protoplanetary disks associated with young stars, which demonstrate that annealing of nebular silicates must occur in conjunction with a large-scale circulation. Mass recycling of dust should have a significant effect on the chemical kinetics of the outer nebula by introducing reduced, gas-phase species produced in the higher temperature and pressure environment of the inner nebula, along with freshly processed grains with “clean” catalytic surfaces to the region of cometesimal formation. Because comets probably form throughout the lifetime of the Solar Nebula and processed (crystalline) grains are not immediately available for incorporation into the first generation of comets, an increasing fraction of dust incorporated into a growing comet should be crystalline olivine and this fraction can serve as a crude chronometer of the relative ages of comets. The formation and evolution of key organic and biogenic molecules in comets are potentially of great consequence to astrobiology.

Overexpression of p27Kip1 lengthens the G1 phase in a mouse model that targets inducible gene expression to central nervous system progenitor cells

We describe a mouse model in which p27Kip1 transgene expression is spatially restricted to the central nervous system neuroepithelium and temporally controlled with doxycycline. Transgene-specific transcripts are detectable within 6 h of doxycycline administration, and maximum nonlethal expression is approached within 12 h. After 18–26 h of transgene expression, the G1 phase of the cell cycle is estimated to increase from 9 to 13 h in the neocortical neuroepithelium, the maximum G1 phase length attainable in this proliferative population in normal mice. Thus our data establish a direct link between p27Kip1 and control of G1 phase length in the mammalian central nervous system and unveil intrinsic mechanisms that constrain the G1 phase length to a putative physiological maximum despite ongoing p27Kip1 transgene expression.