1950 En torno al Museo Louisiana 1970

La tesis “1950 En torno al Museo Louisiana 1970” analiza varias obras relacionadas con el espacio doméstico, que se realizaron entre 1950 y 1970 en Dinamarca, un periodo de esplendor de la Arquitectura Moderna. Tras el aislamiento y restricciones del conflicto bélico que asoló Europa, los jóvenes arquitectos daneses, estaban deseosos por experimentar nuevas ideas de procedencia internacional, favorecidos por diferentes circunstancias, encuentran el mejor campo de ensayo en el espacio doméstico. La mejor arquitectura doméstica en Dinamarca, de aquel periodo, debe entenderse como un sistema compuesto por diferentes autores, que tienen en común muchas más similitudes que diferencias, se complementan unos a otros. Para la comprensión y el entendimiento de ello se hace necesario el estudio de varias figuras y edificios, que completen este sistema cuya investigación está escasamente desarrollada. La tesis propone un viaje para conocer los nombres de algunos de sus protagonistas, que mostraron con su trabajo, que tradición y vanguardia no estarán reñidas. El objetivo es desvelar las claves de la Modernidad Danesa, reconocer, descubrir y recuperar el legado de algunos de sus protagonistas en el ámbito doméstico, cuya lección se considera de total actualidad. Una arquitectura que asume las aportaciones extranjeras con moderación y crítica, cuya íntima relación con la tradición arquitectónica y la artesanía propias, será una de sus notas especiales. Del estudio contrastado de varios proyectos y versiones, se obtienen valores comunes entre sus autores, al igual que se descubren sus afinidades o diferencias respecto a los mismos asuntos; que permitirán comprender sus actuaciones según las referencias e influencias, y definir las variables que configuran sus espacios arquitectónicos. La línea de conexión entre los edificios elegidos será su particular relación con la naturaleza y el lugar en que se integran. La fachada, lugar donde se negociará la relación entre el interior y el paisaje, será un elemento entendido de un modo diferente en cada uno de ellos, una relación que se extenderá en todas ellas, más allá de su perímetro. La investigación se ha estructurado en seis capítulos, que van precedidos de una Introducción. En el capítulo primero, se estudian y se señalan los antecedentes, las figuras y edificios más relevantes de la Tradición Danesa, para la comprensión y el esclarecimiento de algunas de las claves de su Modernidad en el campo de la Arquitectura, que se produce con una clara intención de encontrar su propia identidad y expresión. Esta Modernidad floreciente se caracteriza por la asimilación de otras culturas extranjeras desde la moderación, con un punto de vista crítico, y encuentra sus raíces ancladas a la tradición arquitectónica y la artesanía propia, que fragua en la aparición de un ideal común con enorme personalidad y que hoy se valora como una auténtica aportación de una cultura considerada entonces periférica. Se mostrará el debate y el camino seguido por las generaciones anteriores, a las obras análizadas. Las sensibilidades por lo vernáculo y lo clásico, que aparentemente son contradictorias, dominaran el debate con la misma veracidad y respetabilidad. La llamada tercera generación por Sigfried Giedion reanudará la práctica entre lo clásico y lo vernáculo, apoyados en el espíritu del trabajo artesanal y de la tradición, con el objetivo de conocer del acto arquitectónico su “la verdad” y “la esencia original”. El capítulo segundo, analiza la casa Varming, de 1953, situada en un área residencial de Gentofte, por Eva y Nils Koppel, que reinterpreta la visión de Asplund de un paisaje interior continuación del exterior, donde rompen la caja de ladrillo macizo convencional propia de los años 30. Es el ejemplo más poderoso de la unión de tradición e innovación en su obra residencial. Sus formas sobrias entre el Funcionalismo Danés y la Modernidad se singularizarán por su abstracción y volúmenes limpios que acentúan el efecto de su geometría, prismática y brutalista. El desplazamiento de los cuerpos que lo componen, unos sobre otros, generan un ritmo, que se producirá a otras escalas, ello unido a las variaciones de sus formas y a la elección de sus materiales, ladrillo y madera, le confieren a la casa un carácter orgánico. El edificio se ancla a la tierra resolviéndose en diferentes niveles tras el estudio del lugar y su topografía. El resultado es una versión construida del paisaje, en la cual el edificio da forma al lugar y ensalza la experiencia del escenario natural. La unidad de las estructuras primitivas, parece estar presente. Constituye un ejemplo de la “La idea de Promenade de Asplund”, el proyecto ofrece diferentes recorridos, permitiendo su propia vivencia de la casa, que ofrece la posibilidad vital de decidir. El capítulo tercero trata sobre el pabellón de invitados de Niels Bohr de 1957, situado un área boscosa, en Tisvilde Hegn, fue el primer edificio del arquitecto danés Vilhelm Wohlert. Arraigado a la Tradición Danesa, representa una renovación basada en la absorción de influencias extranjeras: la Arquitectura Americana y la Tradición Japonesa. La caja de madera, posada sobre un terreno horizontal, tiene el carácter sensible de un organismo vivo, siempre cambiante según las variaciones de luz del día o temperatura. Cuando se abre, crea una prolongación del espacio interior, que se extiende a la naturaleza circundante, y se expande hacia el espacio exterior, permitiendo su movilización. Se establece una arquitectura de flujos. Hay un interés por la materia, su textura y el efecto emocional que emana. Las proporciones y dimensiones del edificio están reguladas por un módulo, que se ajusta a la medida del hombre, destacando la gran unidad del edificio. La llave se su efecto estético está en su armonía y equilibrio, que transmiten serenidad y belleza. El encuentro con la naturaleza es la lección más básica del proyecto, donde un mundo de relaciones es amable al ser humano. El capítulo cuarto, analiza el proyecto del Museo Louisiana de 1958, en Humlebæk, primer proyecto de la pareja de arquitectos daneses Jørgen Bo y Vilhelm Wohlert. La experiencia en California de Wohlert donde será visitado por Bo, será trascendental para el desarrollo de Louisiana, donde la identidad Danesa se fusiona con la asimilación de otras culturas, la arquitectura de Frank Lloyd Wright, la del área de la Bahía y la Tradición Japonesa principalmente. La idea del proyecto es la de una obra de arte integral: arquitectura, arte y paisaje, coexistirían en un mismo lugar. Diferentes recursos realzarán su carácter residencial, como el uso de los materiales propios de un entorno doméstico, la realización a la escala del hombre, el modo de usar la iluminación. Cubiertas planas que muestran su artificialidad, parecen flotar sobre galerías acristaladas, acentuarán la fuerza del plano horizontal y establecerán un recorrido en zig-zag de marcado ritmo acompasado. Ritmo que tiene que ver con la encarnación del pulso de la naturaleza, que se acompaña de juegos de luz, y de otras vibraciones materiales a diferentes escalas, imagen, que encuentra una analogía semejante en la cultura japonesa. Todo se coordina con la trama estructural, que conlleva a una construcción y proporción disciplinada. Louisiana atiende al principio de crecimiento de la naturaleza, con la que su conexión es profunda. Hay un dinamismo expresado por el despliegue del edificio, que evoca a algunos proyectos de la Tradición Japonesa. Los blancos muros tienen su propia identidad como formas en sí mismas, avanzan prolongándose fuera de la línea del vidrio, se mueven libremente siguiendo el orden estructural, acompañando al espacio que fluye, en contacto directo con la naturaleza que está en un continuo estado de flujos. Se da todo un mundo de relaciones, donde existe un dialogo entre el paisaje, arte y arquitectura. El capítulo quinto, se dedica a analizar la segunda casa del arquitecto danés Halldor Gunnløgsson, de 1959. Evoca a la Arquitectura Japonesa y Americana, pero es principalmente resultado de una fuerte voluntad y disciplina artística personal. La cubierta, plana, suspendida sobre una gran plataforma pavimentada, que continúa la sección del terreno construyendo de lugar, tiene una gran presencia y arroja una profunda sombra bajo ella. En el interior un espacio único, que se puede dividir eventualmente, discurre en torno a un cuerpo central. El espacio libre fluye, extendiéndose a través de la transparencia de sus ventanales a dos espacios contrapuestos: un patio ajardinado íntimo, que inspira calma y sosiego; y la naturaleza salvaje del mar que proyecta el color del cielo, ambos en constante estado de cambio. El proyecto se elabora de un modo rigurosamente formal, existiendo al mismo tiempo un perfecto equilibrio entre la abstracción de su estructura y su programa. La estructura de madera cuyo orden se extiende más allá de los límites de su perímetro, está formada por pórticos completos como elementos libres, queda expuesta, guardando una estrecha relación con el concepto de modernidad de Mies, equivalente a la arquitectura clásica. La preocupación por el efecto estético es máxima, nada es improvisado. Pero además la combinación de materiales y el juego de las texturas hay una cualidad táctil, cierto erotismo, que flota alrededor de ella. La precisión constructiva y su refinamiento se acercan a Mies. La experiencia del espacio arquitectónico es una vivencia global. La influencia de la arquitectura japonesa, es más conceptual que formal, revelada en un respeto por la naturaleza, la búsqueda del refinamiento a través de la moderación, la eliminación de los objetos innecesarios que distraen de la experiencia del lugar y la preocupación por la luz y la sombra, donde se establece cierto paralelismo con el oscuro mundo del invierno nórdico. Hay un entendimiento de que el espacio, en lugar de ser un objeto inmaterial definido por superficies materiales se entiende como interacciones dinámicas. El capítulo sexto. Propone un viaje para conocer algunas de las viviendas unifamiliares más interesantes que se construyeron en el periodo, que forman parte del sistema investigado. Del estudio comparado y orientado en varios temas, se obtienen diversa conclusiones propias del sistema estudiado. La maestría de la sustancia y la forma será una característica distintiva en Dinamarca, se demuestra que hay un acercamiento a la cultura de Oriente, conceptual y formal, y unos intereses comunes con cierta arquitectura Americana. Su lección nos sensibiliza hacia un sentido fortalecido de proporción, escala, materialidad, textura y peso, densidad del espacio, se valora lo táctil y lo visual, hay una sensibilidad hacia la naturaleza, hacia lo humano, hacia el paisaje, la integridad de la obra. ABSTRACT The thesis “1950 around the Louisiana Museum 1970” analyses several works related to domestic space, which were carried out between 1950 and 1970 in Denmark, a golden age of modern architecture. After the isolation and limitations brought about by the war that blighted Europe, young Danish architects were keen to experiment with ideas of an international origin, encouraged by different circumstances. They find the best field of rehearsal to be the domestic space. The best architecture of that period in Denmark should be understood as a system composed of different authors, who have in common with each other many more similarities than differences, thus complimenting each other. In the interests of understanding, the study of a range of figures and buildings is necessary so that this system, the research of which is still in its infancy, can be completed. The thesis proposes a journey of discovery through the names of some of those protagonists who were showcased through their work so that tradition and avant- garde could go hand in hand. The objective is to unveil the keys to Danish Modernity; to recognise, discover and revive the legacy of some of its protagonists in the domestic field whose lessons are seen as entirely of the present. For an architect, the taking on of modern contributions with both moderation and caution, with its intimate relationship with architectural tradition and its own craft, will be one of his hallmarks. With the study set against several projects and versions, one can derive common values among their authors. In the same way their affinities and differences in respect of the same issue emerge. This will allow an understanding of their measures in line with references and influences and enable the defining of the variables of their architectural spaces. The common line between the buildings selected will be their particular relationship with nature and the space with which they integrate. The façade, the place where the relationship between the interior and the landscape would be negotiated, wouldl be the discriminating element in a distinct way for each one of them. It is through each of these facades that this relationship would extend, and far beyond their physical perimeter. The investigation has been structured into six chapters, preceded by an introduction. The first chapter outlines and analyses the backgrounds, figures and buildings most relevant to the Danish Tradition. This is to facilitate the understanding and elucidation of some of the keys to its modernity in the field of architecture, which came about with the clear intention to discover its own identity and expression. This thriving modernity is characterized by its moderate assimilation with foreign cultures with a critical eye, and finds its roots anchored in architectural tradition and its own handcraft. It is forged in the emergence of a common ideal of enormous personality which today has come to be valued as an authentic contribution to the sphere from a culture that was formerly seen as on the peripheries. What will be demonstrated is the path taken by previous generations to these works and the debate that surrounds them. The sensibilities for both the vernacular and the classic, which at first glance may seem contradictory, will dominate the debate with the same veracity and respectability. The so-called third generation of Sigfried Giedion will revive the process between the classic and the vernacular, supported in spirit by the handcraft work and by tradition, with the objective of discovering the “truth” and the “original essence” of the architectural act. The second chapter analyzes the Varming house, built by Eva and Nils Koppel 1953, which is situated in a residential area of Gentofte. This reinterprets Asplund’s vision of an interior landscape extending to the exterior, where we see a break with the conventional sturdy brick shell of the 1930s. It is the most powerful example of the union of tradition and innovation in his their residential work. Their sober forms caught between Danish Functionalism and modernity are characterized by their abstraction and clean shapes which accentuate their prismatic and brutal geometry, The displacement of the parts of which they are composed, one over the other, generate a rhythm. This is produced to varying scales and is closely linked to its forms and the selection of materials – brick and wood – that confer an organic character to the house. The building is anchored to the earth, finding solution at different levels through the study of place and topography. The result is an adaption constructed out of the landscape, in which the building gives form to the place and celebrates the experience of the natural setting. The unity of primitive structures appears to be present. It constitutes an example of “Asplund’s Promenade Idea”. Different routes of exploration within are available to the visitor, allowing for one’s own personal experience of the house, allowing in turn for the vital chance to decide. The third chapter deals with Niels Bohr’s guest pavilion. Built in 1957, it is situated in a wooded area of Tisvilde Hegn and was the architect Vilhelm Wohlert’s first building. Rooted in the Danish Tradition, it represents a renewal based on the absorption of foreign influences: American architecture and the Japanese tradition. The wooden box, perched atop a horizontal terrain, possesses the sensitive character of the living organism, ever-changing in accordance with the variations in daylight and temperature. When opened up, it creates an elongation of the interior space which extends into the surrounding nature and it expands towards the exterior space, allowing for its mobilisation. It establishes an architecture of flux. There is interest in the material, its texture and the emotional effect it inspires. The building’s proportions and dimensions are regulated by a module, which is adjusted by hand, bringing out the great unity of the building. The key to its aesthetic effect is its harmony and equilibrium, which convey serenity and beauty. The meeting with nature is the most fundamental lesson of the project, where a world of relationships softens the personality of the human being. The fourth chapter analyzes the Louisiana Museum project of 1958 in 1958. It was the first project of the Danish architects Jørgen Bo and Vilhelm Wohlert. Wohlert’s experience in California where he was visited by Bo would be essential to the development of Louisiana, where the Danish identity is fused in assimilation with other cultures, the architecture of Frank Lloyd Wright, that of the Bahía area and principally the Japanese tradition. The idea of the project was for an integrated work of art: architecture, art and landscape, which would coexist in the same space. A range of different resources would realize the residential character, such as the use of materials taken from a domestic environment, the attainment of human scale and the manner in which light was used. Flat roof plans that show their artificiality and appear to float over glassed galleries. They accentuate the strength of the horizontal plan and establish a zigzag route of marked and measured rhythm. It is a rhythm that has to do with the incarnation of nature’s pulse, which is accompanied with plays of light, as well as material vibrations of different scales, imagery which uncovers a parallel analogy with Japanese culture. Everything is coordinated along a structural frame, which involves a disciplined construction and proportion. Louisiana cherishes nature’s principle of growth, to which its connection is profound. Here is a dynamism expressed through the disposition of the building, which evokes in some projects the Japanese tradition. The white walls possess their own identity as forms in their own right. They advance, extending beyond the line of glass, moving freely along the structural line, accompanying a space that flows and in direct contact with nature that is itself in a constant state of flux. It creates a world of relationships, where dialogue exists between the landscape, art and architecture. The fifth chapter is dedicated to analyzing the Danish architect Halldor Gunnløgsson’s second house, built in 1959. It evokes both Japanese and American architecture but is principally the result of a strong will and personal artistic discipline. The flat roof suspended above a large paved platform – itself continuing the constructed terrain of the place – has great presence and casts a heavy shadow beneath. In the interior, a single space, which can at length be divided and which flows around a central space. The space flows freely, extending through the transparency of its windows which give out onto two contrasting locations: an intimate garden patio, inspiring calm and tranquillity, and the wild nature of the sea which projects the colour of sky, both in a constant state of change. The project is realized in a rigorously formal manner. A perfect balance exists between the abstraction of his structure and his project. The wooden structure, whose order extends beyond the limits of its perimeter, is formed of complete porticos of free elements. It remains exposed, maintaining a close relationship with Mies’ concept of modernity, analogous to classical architecture. The preoccupation with the aesthetic effect is paramount and nothing is improvised. But in addition to this - the combination of materials and the play of textures - there is a tactile quality, a certain eroticism, which lingers all about. The constructive precision and its refinement are close to Mies. The experience of the architectural space is universal. The influence of Japanese architecture, more conceptual than formal, is revealed in a respect for nature. It can be seen in the search for refinement through moderation, the elimination of the superfluous object that distract from the experience of place and the preoccupation with light and shade, where a certain parallel with the dark world of the Nordic winter is established. There is an understanding that space, rather than being an immaterial object defined by material surfaces, extends instead as dynamic interactions. The sixth chapter. This proposes a journey to discover some of the unfamiliar residences of most interest which were constructed in the period, and which form part of the system being investigated. Through the study of comparison and one which is geared towards various themes, diverse conclusions are drawn regarding the system being researched. The expertise in substance and form will be a distinctive characteristic in Denmark, demonstrating an approach to the culture of the Orient, both conceptual and formal, and some common interests in certain American architecture. Its teachings sensitize us to a strengthened sense of proportion, scale, materiality, texture and weight and density of space. It values both the tactile and the visual. There is a sensitivity to nature, to the human, to the landscape and to the integrity of the work.

Distribution of haplotypes from a chromosome 21 region distinguishes multiple prehistoric human migrations

Despite mounting genetic evidence implicating a recent origin of modern humans, the elucidation of early migratory gene-flow episodes remains incomplete. Geographic distribution of haplotypes may show traces of ancestral migrations. However, such evolutionary signatures can be erased easily by recombination and mutational perturbations. A 565-bp chromosome 21 region near the MX1 gene, which contains nine sites frequently polymorphic in human populations, has been found. It is unaffected by recombination and recurrent mutation and thus reflects only migratory history, genetic drift, and possibly selection. Geographic distribution of contemporary haplotypes implies distinctive prehistoric human migrations: one to Oceania, one to Asia and subsequently to America, and a third one predominantly to Europe. The findings with chromosome 21 are confirmed by independent evidence from a Y chromosome phylogeny. Loci of this type will help to decipher the evolutionary history of modern humans.

Structural homologies with ATP- and folate-binding enzymes in the crystal structure of folylpolyglutamate synthetase

Folylpolyglutamate synthetase, which is responsible for the addition of a polyglutamate tail to folate and folate derivatives, is an ATP-dependent enzyme isolated from eukaryotic and bacterial sources, where it plays a key role in the retention of the intracellular folate pool. Here, we report the 2.4-Å resolution crystal structure of the MgATP complex of the enzyme from Lactobacillus casei. The structural analysis reveals that folylpolyglutamate synthetase is a modular protein consisting of two domains, one with a typical mononucleotide-binding fold and the other strikingly similar to the folate-binding enzyme dihydrofolate reductase. We have located the active site of the enzyme in a large interdomain cleft adjacent to an ATP-binding P-loop motif. Opposite this site, in the C domain, a cavity likely to be the folate binding site has been identified, and inspection of this cavity and the surrounding protein structure suggests that the glutamate tail of the substrate may project into the active site. A further feature of the structure is a well defined Ω loop, which contributes both to the active site and to interdomain interactions. The determination of the structure of this enzyme represents the first step toward the elucidation of the molecular mechanism of polyglutamylation of folates and antifolates.

Mass spectrometric evidence for the enzymatic mechanism of the depolymerization of heparin-like glycosaminoglycans by heparinase II

Heparin-like glycosaminoglycans, acidic complex polysaccharides present on cell surfaces and in the extracellular matrix, regulate important physiological processes such as anticoagulation and angiogenesis. Heparin-like glycosaminoglycan degrading enzymes or heparinases are powerful tools that have enabled the elucidation of important biological properties of heparin-like glycosaminoglycans in vitro and in vivo. With an overall goal of developing an approach to sequence heparin-like glycosaminoglycans using the heparinases, we recently have elaborated a mass spectrometry methodology to elucidate the mechanism of depolymerization of heparin-like glycosaminoglycans by heparinase I. In this study, we investigate the mechanism of depolymerization of heparin-like glycosaminoglycans by heparinase II, which possesses the broadest known substrate specificity of the heparinases. We show here that heparinase II cleaves heparin-like glycosaminoglycans endolytically in a nonrandom manner. In addition, we show that heparinase II has two distinct active sites and provide evidence that one of the active sites is heparinase I-like, cleaving at hexosamine–sulfated iduronate linkages, whereas the other is presumably heparinase III-like, cleaving at hexosamine–glucuronate linkages. Elucidation of the mechanism of depolymerization of heparin-like glycosaminoglycans by the heparinases and mutant heparinases could pave the way to the development of much needed methods to sequence heparin-like glycosaminoglycans.

Cloning of inversion breakpoints in the Anopheles gambiae complex traces a transposable element at the inversion junction

Anopheles arabiensis, one of the two most potent malaria vectors of the gambiae complex, is characterized by the presence of chromosomal paracentric inversions. Elucidation of the nature and the dynamics of these inversions is of paramount importance for the understanding of the population genetics and evolutionary biology of this mosquito and of the impact on malaria epidemiology. We report here the cloning of the breakpoints of the naturally occurring polymorphic inversion 2Rd′ of A. arabiensis. A cDNA clone that cytologically mapped on the proximal breakpoint was the starting material for the isolation of a cosmid clone that spanned the breakpoint. Analysis of the surrounding sequences demonstrated that adjacent to the distal breakpoint lies a repetitive element that exhibits distinct distribution in different A. arabiensis strains. Sequencing analysis of that area revealed elements characteristic of transposable element terminal repeats. We called this presumed transposable element Odysseus. The presence of Odysseus at the junction of the naturally occuring inversion 2Rd′ suggests that the inversion may be the result of the transposable element’s activity. Characteristics of Odysseus’ terminal region as well as its cytological distribution in different strains may indicate a relatively recent activity of Odysseus.

A family of fibrinogen-related proteins that precipitates parasite-derived molecules is produced by an invertebrate after infection

After infection with the digenetic trematode Echinostoma paraensei, hemolymph of the snail Biomphalaria glabrata contains lectins comprised of 65-kDa subunits that precipitate polypeptides secreted by E. paraensei intramolluscan larvae. Comparable activity is lacking in hemolymph of uninfected snails. Three different cDNAs with sequence similarities to peptides derived from the 65-kDa lectins were obtained and unexpectedly found to encode fibrinogen-related proteins (FREPs). These FREPs also contained regions with sequence similarity to Ig superfamily members. B. glabrata has at least five FREP genes, three of which are expressed at increased levels after infection. Elucidation of components of the defense system of B. glabrata is relevant because this snail is an intermediate host for Schistosoma mansoni, the most widely distributed causative agent of human schistosomiasis. These results are novel in suggesting a role for invertebrate FREPs in recognition of parasite-derived molecules and also provide a model for investigating the diversity of molecules functioning in nonself-recognition in an invertebrate.

Substitution of a mutant α2a-adrenergic receptor via “hit and run” gene targeting reveals the role of this subtype in sedative, analgesic, and anesthetic-sparing responses in vivo

Norepinephrine contributes to antinociceptive, sedative, and sympatholytic responses in vivo, and α2 adrenergic receptor (α2AR) agonists are used clinically to mimic these effects. Lack of subtype-specific agonists has prevented elucidation of the role that each α2AR subtype (α2A, α2B, and α2C) plays in these central effects. Here we demonstrate that α2AR agonist-elicited sedative, anesthetic-sparing, and analgesic responses are lost in a mouse line expressing a subtly mutated α2AAR, D79N α2AAR, created by two-step homologous recombination. These functional changes are accompanied by failure of the D79N α2AAR to inhibit voltage-gated Ca2+ currents and spontaneous neuronal firing, a measure of K+ current activation. These results provide definitive evidence that the α2AAR subtype is the primary mediator of clinically important central actions of α2AR agonists and suggest that the D79N α2AAR mouse may serve as a model for exploring other possible α2AAR functions in vivo.

Smooth muscle myosin mutants containing a single tryptophan reveal molecular interactions at the actin-binding interface

Elucidation of the molecular details of the cyclic actomyosin interaction requires the ability to examine structural changes at specific sites in the actin-binding interface of myosin. To study these changes dynamically, we have expressed two mutants of a truncated fragment of chicken gizzard smooth muscle myosin, which includes the motor domain and essential light chain (MDE). These mutants were engineered to contain a single tryptophan at (Trp-546) or near (Trp-625) the putative actin-binding interface. Both 546- and 625-MDE exhibited actin-activated ATPase and actin-binding activities similar to wild-type MDE. Fluorescence emission spectra and acrylamide quenching of 546- and 625-MDE suggest that Trp-546 is nearly fully exposed to solvent and Trp-625 is less than 50% exposed in the presence and absence of ATP, in good agreement with the available crystal structure data. The spectrum of 625-MDE bound to actin was quite similar to the unbound spectrum indicating that, although Trp-625 is located near the 50/20-kDa loop and the 50-kDa cleft of myosin, its conformation does not change upon actin binding. However, a 10-nm blue shift in the peak emission wavelength of 546-MDE observed in the presence of actin indicates that Trp-546, located in the A-site of the lower 50-kDa subdomain of myosin, exists in a more buried environment and may directly interact with actin in the rigor acto-S1 complex. This change in the spectrum of Trp-546 constitutes direct evidence for a specific molecular interaction between residues in the A-site of myosin and actin.

Identification and characterization of differentially expressed cDNAs of the vector mosquito, Anopheles gambiae

The isolation and study of Anopheles gambiae genes that are differentially expressed in development, notably in tissues associated with the maturation and transmission of the malaria parasite, is important for the elucidation of basic molecular mechanisms underlying vector–parasite interactions. We have used the differential display technique to screen for mRNAs specifically expressed in adult males, females, and midgut tissues of blood-fed and unfed females. We also screened for mRNAs specifically induced upon bacterial infection of larval stage mosquitoes. We have characterized 19 distinct cDNAs, most of which show developmentally regulated expression specificity during the mosquito life cycle. The most interesting are six new sequences that are midgut-specific in the adult, three of which are also modulated by blood-feeding. The gut-specific sequences encode a maltase, a V-ATPase subunit, a GTP binding protein, two different lectins, and a nontrypsin serine protease. The latter sequence is also induced in larvae subjected to bacterial challenge. With the exception of a mitochondrial DNA fragment, the other 18 sequences constitute expressed genomic sequence tags, 4 of which have been mapped cytogenetically.

A mammalian model for Laron syndrome produced by targeted disruption of the mouse growth hormone receptor/binding protein gene (the Laron mouse)

Laron syndrome [growth hormone (GH) insensitivity syndrome] is a hereditary dwarfism resulting from defects in the GH receptor (GHR) gene. GHR deficiency has not been reported in mammals other than humans. Many aspects of GHR dysfunction remain unknown because of ethical and practical limitations in studying humans. To create a mammalian model for this disease, we generated mice bearing a disrupted GHR/binding protein (GHR/BP) gene through a homologous gene targeting approach. Homozygous GHR/BP knockout mice showed severe postnatal growth retardation, proportionate dwarfism, absence of the GHR and GH binding protein, greatly decreased serum insulin-like growth factor I and elevated serum GH concentrations. These characteristics represent the phenotype typical of individuals with Laron syndrome. Animals heterozygous for the GHR/BP defect show only minimal growth impairment but have an intermediate biochemical phenotype, with decreased GHR and GH binding protein expression and slightly diminished insulin-like growth factor I levels. These findings indicate that the GHR/BP-deficient mouse (Laron mouse) is a suitable model for human Laron syndrome that will prove useful for the elucidation of many aspects of GHR/BP function that cannot be obtained in humans.

Bok is a pro-apoptotic Bcl-2 protein with restricted expression in reproductive tissues and heterodimerizes with selective anti-apoptotic Bcl-2 family members

In the intracellular death program, hetero- and homodimerization of different anti- and pro-apoptotic Bcl-2-related proteins are critical in the determination of cell fate. From a rat ovarian fusion cDNA library, we isolated a new pro-apoptotic Bcl-2 gene, Bcl-2-related ovarian killer (Bok). Bok had conserved Bcl-2 homology (BH) domains 1, 2, and 3 and a C-terminal transmembrane region present in other Bcl-2 proteins, but lacked the BH4 domain found only in anti-apoptotic Bcl-2 proteins. In the yeast two-hybrid system, Bok interacted strongly with some (Mcl-1, BHRF1, and Bfl-1) but not other (Bcl-2, Bcl-xL, and Bcl-w) anti-apoptotic members. This finding is in direct contrast to the ability of other pro-apoptotic members (Bax, Bak, and Bik) to interact with all of the anti-apoptotic proteins. In addition, negligible interaction was found between Bok and different pro-apoptotic members. In mammalian cells, overexpression of Bok induced apoptosis that was blocked by the baculoviral-derived cysteine protease inhibitor P35. Cell killing induced by Bok was also suppressed following coexpression with Mcl-1 and BHRF1 but not with Bcl-2, further indicating that Bok heterodimerized only with selective anti-apoptotic Bcl-2 proteins. Northern blot analysis indicated that Bok was highly expressed in the ovary, testis and uterus. In situ hybridization analysis localized Bok mRNA in granulosa cells, the cell type that underwent apoptosis during follicle atresia. Identification of Bok as a new pro-apoptotic Bcl-2 protein with restricted tissue distribution and heterodimerization properties could facilitate elucidation of apoptosis mechanisms in reproductive tissues undergoing hormone-regulated cyclic cell turnover.

Pleiotropic Alterations in Lipid Metabolism in Yeast sac1 Mutants: Relationship to “Bypass Sec14p” and Inositol Auxotrophy

SacIp dysfunction results in bypass of the requirement for phosphatidylinositol transfer protein (Sec14p) function in yeast Golgi processes. This effect is accompanied by alterations in inositol phospholipid metabolism and inositol auxotrophy. Elucidation of how sac1 mutants effect “bypass Sec14p” will provide insights into Sec14p function in vivo. We now report that, in addition to a dramatic accumulation of phosphatidylinositol-4-phosphate, sac1 mutants also exhibit a specific acceleration of phosphatidylcholine biosynthesis via the CDP-choline pathway. This phosphatidylcholine metabolic phenotype is sensitive to the two physiological challenges that abolish bypass Sec14p in sac1 strains; i.e. phospholipase D inactivation and expression of bacterial diacylglycerol (DAG) kinase. Moreover, we demonstrate that accumulation of phosphatidylinositol-4-phosphate in sac1 mutants is insufficient to effect bypass Sec14p. These data support a model in which phospholipase D activity contributes to generation of DAG that, in turn, effects bypass Sec14p. A significant fate for this DAG is consumption by the CDP-choline pathway. Finally, we determine that CDP-choline pathway activity contributes to the inositol auxotrophy of sac1 strains in a novel manner that does not involve obvious defects in transcriptional expression of the INO1 gene.

The chemistry of the S-nitrosoglutathione/glutathione system

S-Nitrosothiols have generated considerable interest due to their ability to act as nitric oxide (NO) donors and due to their possible involvement in bioregulatory systems—e.g., NO transfer reactions. Elucidation of the reaction pathways involved in the modification of the thiol group by S-nitrosothiols is important for understanding the role of S-nitroso compounds in vivo. The modification of glutathione (GSH) in the presence of S-nitrosoglutathione (GSNO) was examined as a model reaction. Incubation of GSNO (1 mM) with GSH at various concentrations (1–10 mM) in phosphate buffer (pH 7.4) yielded oxidized glutathione, nitrite, nitrous oxide, and ammonia as end products. The product yields were dependent on the concentrations of GSH and oxygen. Transient signals corresponding to GSH conjugates, which increased by one mass unit when the reaction was carried out with 15N-labeled GSNO, were identified by electrospray ionization mass spectrometry. When morpholine was present in the reaction system, N-nitrosomorpholine was formed. Increasing concentrations of either phosphate or GSH led to lower yields of N-nitrosomorpholine. The inhibitory effect of phosphate may be due to reaction with the nitrosating agent, nitrous anhydride (N2O3), formed by oxidation of NO. This supports the release of NO during the reaction of GSNO with GSH. The products noted above account quantitatively for virtually all of the GSNO nitrogen consumed during the reaction, and it is now possible to construct a complete set of pathways for the complex transformations arising from GSNO + GSH.

Lipidic cubic phases: A novel concept for the crystallization of membrane proteins

Understanding the mechanisms of action of membrane proteins requires the elucidation of their structures to high resolution. The critical step in accomplishing this by x-ray crystallography is the routine availability of well-ordered three-dimensional crystals. We have devised a novel, rational approach to meet this goal using quasisolid lipidic cubic phases. This membrane system, consisting of lipid, water, and protein in appropriate proportions, forms a structured, transparent, and complex three-dimensional lipidic array, which is pervaded by an intercommunicating aqueous channel system. Such matrices provide nucleation sites (“seeding”) and support growth by lateral diffusion of protein molecules in the membrane (“feeding”). Bacteriorhodopsin crystals were obtained from bicontinuous cubic phases, but not from micellar systems, implying a critical role of the continuity of the diffusion space (the bilayer) on crystal growth. Hexagonal bacteriorhodopsin crystals diffracted to 3.7 Å resolution, with a space group P63, and unit cell dimensions of a = b = 62 Å, c = 108 Å; α = β = 90° and γ = 120°.

On the molecular basis and regulation of cellular capacitative calcium entry: Roles for Trp proteins

During the last 2 years, our laboratory has worked on the elucidation of the molecular basis of capacitative calcium entry (CCE) into cells. Specifically, we tested the hypothesis that CCE channels are formed of subunits encoded in genes related to the Drosophila trp gene. The first step in this pursuit was to search for mammalian trp genes. We found not one but six mammalian genes and cloned several of their cDNAs, some in their full length. As assayed in mammalian cells, overexpression of some mammalian Trps increases CCE, while expression of partial trp cDNAs in antisense orientation can interfere with endogenous CCE. These findings provided a firm connection between CCE and mammalian Trps. This article reviews the known forms of CCE and highlights unanswered questions in our understanding of intracellular Ca2+ homeostasis and the physiological roles of CCE.