Arquitectura experimental en España 1965-1985

Entre los años 1965 y 1985, arquitectos españoles irrumpieron con una serie de aportaciones singulares que abrieron nuevas líneas de exploración más allá de los límites que marcaba la tradición. Se trata de un conjunto de prácticas arquitectónicas que podríamos calificar de “experimentales”3. El carácter aislado y disperso de estas manifestaciones4, ha contribuido a su desconocimiento y falta de difusión tanto dentro como fuera de nuestras fronteras. Hasta la fecha no se ha realizado una revisión del tema en su conjunto. Con este estudio se pretende analizar la producción arquitectónica experimental de este periodo en su globalidad, generar una estructura que permita incluir las distintas vías experimentales, de idear y proyectar arquitectura, que podemos encontrar dentro de nuestro territorio, inscribiéndolas en un contexto más amplio. Se han identificado cuatro nichos de experimentación en torno a los cuales se estructura la producción experimental española en el arco temporal definido: “sistemas de organización espacial”, “interacción con el medio ambiente”, “lógica constructiva e imaginación material” y “el campo expandido del lenguaje y el proceso proyectual”. La comprensión de estas manifestaciones, con una mirada global, es la que mejor puede contribuir a la construcción de un corpus propio, que sea reconocible por sus diferencias y especificidades. Se trata igualmente de un estudio comparado, que busca facilitar la inscripción de las prácticas experimentales españolas en el marco de la cultura internacional, no como una excepción encerrada en su particularismo, ni como una derivación inmediata de tendencias externas, sino como una realidad donde apoyar una historia que entrelaza lo local y lo internacional. En definitiva, con esta investigación se pretende recuperar un fragmento reciente de la arquitectura en España, en un periodo clave, en el que comienza a abrirse al intercambio de teorías y prácticas, con el resto de Europa y América. Incluyendo voces paralelas al discurso central, que habían sido desplazadas durante el proceso de construcción del mismo, se quiere participar en la transmisión de un legado completo de nuestra cultura y práctica arquitectónica. ABSTRACT Between the years 1965 and 1985, Spanish architects burst in with unique contributions that could be described as "experimental"1. The isolated and dispersed nature of these occurrences2 has contributed to the oversight and lack of dissemination of the Spanish experimental architecture both within and beyond our borders. To date, there has been no review of the topic in its entirety. This study tries to examine the experimental architectural production of this period as a whole, generating a structure that allows for the inclusion of different experimental ways of envisioning and projecting architecture within our territory, registering them in a wider context. Four niches of experimentation have been identified around which the Spanish experimental production in the time span defined: "spatial organisation systems," "interaction with the environment", "constructive logic and equipment imagination" and” expanded field of language and the design process. " Understanding these manifestations, with a global perspective, can best contribute to the construction of our own corpus, which is recognisable by their differences and specificities. At the same time, it is a comparative study that seeks to facilitate the registration of the Spanish experimental practices within the framework of international culture, not as an exception enclosed in its particularity, not as an immediate derivation of external trends, but as a reality to support a story that weaves together the local and the international. In short, it deals with recovering a recent fragment of architecture, in a key period in which Spain begins to open to the exchange of cultural and architectural theories and practices with the rest of Europe and America. Including parallel voices to the central discourse, which until recently had been displaced during the construction processes of that same discourse, this research wants to participate in the transmission of a complete legacy of the history of our culture and architectural practice.

Construcción de una teoría de "lo grande" : inteligencia acumulativa en la definición de una realidad aumentada

La tesis analiza los antecedentes y el contexto arquitectónico de aquellas obras que se pueden enmarcar dentro del concepto de lo Grande. Arrancando en los movimientos utópicos, megaestructuralistas o radicales de los años sesenta, nos conduce hasta conceptos, proyectos y teorías contemporáneos. Para ello es preciso, definir lo Grande no solo a través de cuestiones dimensionales, sino también de cambio de dimensión espacial y conceptual. Se estudian arquitecturas, que, una vez superado un cierto volumen, magnitud o grado de complejidad, se convierten en un ‘edificio grande’, adquiriendo propiedades de otra naturaleza, generando un cambio o salto entre escalas, o un desplazamiento entre campos, rompiendo los límites habituales de la escala, el tamaño u otras codificaciones con las que se categorizan los proyectos entre la ‘arquitectura’ y la ‘ciudad’. El cuerpo de tesis principal se estructura en dos secciones, ‘Genealogías y asociaciones’ y ‘Desplazamiento de conceptos’, y una Conclusión, que trata de compilar las consideraciones o conclusiones extraídas de lo anterior, construyendo así la propia teoría de lo Grande. Genealogías y asociaciones revisa las formas de conocimiento que contextualizan lo Grande, haciendo un barrido de las estructuras sociales, económicas y políticas, desde una perspectiva y un marco con influencia directa en la arquitectura. Recorre teorías, tesis y proyectos que reflexionan o trabajan en lo Grande y en los límites entre las escalas desde la década de los sesenta hasta hoy día. En ese barrido examina conceptos y condiciones capaces de abrir nuevos paradigmas y escenarios arquitectónicos. El contexto sociocultural que lo enmarca resulta crucial para definir posteriormente conceptos, en un barrido desde la cultura de masas a la sociedad de la información, repasando la influencia y repercusión que ha tenido la tecnología y la globalización en el desarrollo de la arquitectura. Por último, se estudian conceptos entre el espacio y a la política, mediante una análisis que enmarque las posiciones de apoyo, refuerzo o rechazo en la relación de la obra de arquitectura y la naturaleza política que esta adquiere por la mera condición de ser Grande. Desplazamiento de conceptos, ofrece condiciones y ejemplos que permiten definir las ‘propiedades’ de lo Grande, a través de una investigación teórica y analítica de las herramientas y estrategias que operan en estas escalas, para confirmar cómo, desarrollos aparentemente cuantitativos, pueden dar lugar a avances cualitativos o a mutaciones que permitan superar los paradigmas anteriores. El objetivo es la definición del conjunto de instrumentos, estrategias y operaciones que sintetizan los valores de cada concepto, con las que construir una ‘cosmogonía’ sobre el tema, apoyándose para ello en textos de referencia, proyectos paradigmáticos y conceptos derivados de los análisis. Los conceptos a estudio devienen directamente de ciertos atributos y características detectadas en las genealogías de lo Grande. Así, consideramos Grande a aquella arquitectura cuya escala y tamaño ha experimentado lo que definiremos como ‘cambio de dimensiones’, no solo a nivel dimensional, sino también perceptivo, técnico y contextual; su tamaño no solo depende de sus magnitudes sino también de la cantidad de materia que contiene el sistema o que circula por él; que tiene trazas, dotes y propiedades de infraestructura; que está instalada, y por tanto, es reactiva con el medio; por ello, es al mismo tiempo autónoma respecto al todo, aunque relacionada con él; un espacio capaz de aceptar la indeterminación de ciertas partes frente a la especificidad de otras, como forma de admitir la pérdida del control total y el detalle; por tanto, que ha desplazado la planificación y el diseño por la investigación programática y la estrategia operativa. En definitiva una arquitectura que desplaza o muta conceptos, atributos y estrategias de otras disciplinas, de los saltos entre escalas y de los nuevos programas. ABSTRACT The thesis will analyze the architectural background and context of those works which can be framed within the concept of Bigness. From the utopian, megastructuralism and radical movements of the sixties, it will lead to contemporary concepts, projects and theories, and ultimately define a theory of Bigness. It will be necessary to define Bigness or the Large, not only in terms of size, but also considering change in spatial and conceptual dimensions. Its case studies are architectures which, surpassing a certain volume, magnitude or degree of complexity, become a ‘large building’ and acquire characteristics of another nature. This generates a change or ‘leap’ in scale, or a displacement in fields, breaking the conventional limits of scale, size and other codes used to categorise projects within ‘architecture’ or the ‘city’. The main body of the thesis is structured into two sections, Genealogies and Associations and Displacement of Concepts, and the Conclusion, which will compile the considerations and conclusions extracted from the previous parts, building the actual theory of Bigness. The first section, Genealogies and Associations, will review the forms of knowledge which contextualize the Large, covering a spectrum of social, economic and political structures from the framework and perspective of their direct influence on architecture. It will account theories, thesis and projects which reflect or work on Bigness and on the limits between scales from the sixties to today, examining those concepts and conditions which may be useful to unfold new settings and paradigms. As the sociocultural context will also prove crucial in order to later define concepts, a broad sweep will cover from the mass society to the information society, revising the influence and repercussions of technology and globalization on the development of architecture. Lastly, it will study concepts in-between space and politics, by means of a study framing positions of support, assistance or rejection towards the work of architecture, and the political nature the work is given only due to its condition of being Big. The following section, Displacement of Concepts, provides conditions and examples which allow the definition of the ‘characteristics’ of Bigness by means of a theoretical and analytical investigation of the tools and strategies which operate on these scales. This is in order to confirm how apparently quantitative developments can lead to qualitative advances or to mutations that enable the overcoming of previous paradigms. The objective is to define the set of strategies, operations and tools, which synthesize the values of each concept; the grounding for a “cosmogony” of the subject, underpinned by relevant reference texts, paradigmatic projects and concepts stemming from the analyses. The concepts of study arise directly from certain attributes and characteristics detected in Bigness. For this purpose, it is that architecture whose dimensions and size have experienced what we will define as a ‘change of dimensions’, not only of size, but also perceptual, technical and contextual;its size depends not only on its magnitude but also the amount of material that contains or flows through system; which possesses traits, faculties and features of an infrastructure; which is installed, and is therefore reactive with its surroundings; due to this, it will be autonomous with respect to the whole and yet related to it; a space which can accept the undetermined nature of some of its parts as well as the specificity of other parts, acknowledging the loss of total control and detail; and thus, which has displaced planning and design with operational strategies. In conclusion, an architecture that displaces or mutates concepts, attributes and strategies stemming from other disciplines, from the jumps between scales, and from the new programs.

O EVANGELHO SEGUNDO DOSTOIÉVSKI UMA ABORDAGEM INTERTEXTUAL DA IMAGEM DE CRISTO NO ROMANCE O IDIOTA

No romance O Idiota, Dostoiévski cria, por meio do príncipe Míchkin, uma personagem com as características do Cristo. Sabe-se que a Bíblia, principalmente o Novo Testamento, acompanhou o escritor desde sua infância até o momento de sua morte. O primeiro capítulo, dedicado ao referencial teórico da pesquisa, lida com o universo da linguagem. Tanto o texto literário quanto a literatura bíblica procedem do mito. Neste sen-tido, religião e literatura se tocam e se aproximam. O segundo capítulo foi escrito na intenção de mostrar como o Cristo e os Evangelhos são temas, motivos e imagens recorrentes na obra de Dostoiévski. A literatura bíblica está presente, com mais ou menos intensidade, em diversas das principais obras do escritor russo e não somente em O Idiota. A hipótese de que Dostoiévski cria um Cristo e um Evangelho por meio de O Idiota é demonstrada na análise do romance, no terceiro capítulo. A tese proposta é: Dostoiévski desenvolve um evangelho literário, por meio de Míchkin, misto de um Cristo russo, ao mesmo tempo divino e humano, mas também idiota e quixotesco. Na dinâmica intertextual entre os Evangelhos bíblicos e O Idiota, entre Cristo e Míchkin, a literatura e o sagrado se revelam, como uma presença divina. Nas cenas e na estruturação do enredo que compõe o romance, Cristo se manifesta nas ações de Míchkin, na luz, na beleza, mas também na tragicidade de uma trajetória deslocada e antinômica. O amor e a compaixão ganham forma e vida na presen-ça do príncipe, vazio de si, servo de todos.

A cobalt complex that selectively disrupts the structure and function of zinc fingers

Zinc finger domains are structures that mediate sequence recognition for a large number of DNA-binding proteins. These domains consist of sequences of amino acids containing cysteine and histidine residues tetrahedrally coordinated to a zinc ion. In this report, we present a means to selectively inhibit a zinc finger transcription factor with cobalt(III) Schiff-base complexes. 1H NMR spectroscopy confirmed that the structure of a zinc finger peptide is disrupted by axial ligation of the cobalt(III) complex to the nitrogen of the imidazole ring of a histidine residue. Fluorescence studies reveal that the zinc ion is displaced from the model zinc finger peptide in the presence of the cobalt complex. In addition, gel-shift and filter-binding assays reveal that cobalt complexes inhibit binding of a complete zinc finger protein, human transcription factor Sp1, to its consensus sequence. Finally, a DNA-coupled conjugate of the cobalt complexes selectively inhibited Sp1 in the presence of several other transcription factors.

Heteroduplex joint formation in Escherichia coli recombination is initiated by pairing of a 3′-ending strand

The formation of heteroduplex joints in Escherichia coli recombination is initiated by invasion of double-stranded DNA by a single-stranded homologue. To determine the polarity of the invasive strand, linear molecules with direct terminal repeats were released by in vivo restriction of infecting chimeric phage DNA and heteroduplex products of intramolecular recombination were analyzed. With this substrate, the invasive strand is expected to be incorporated into the circular crossover product and the complementary strand is expected to be incorporated into the reciprocal linear product. Strands of both polarities were incorporated into heteroduplex structures, but only strands ending 3′ at the break were incorporated into circular products. This result indicates that invasion of the 3′-ending strand initiates the heteroduplex joint formation and that the complementary 5′-ending strand is incorporated into heteroduplex structures in the process of reciprocal strand exchange. The polarity of the invasive strand was not affected by recD, recJ, or xonA mutations. However, xonA and recJ mutations increased the proportion of heteroduplexes containing 5′-ending strands. This observation suggests that RecJ exonuclease and exonuclease I may enhance recombination by degrading the displaced strands during branch migration and thereby causing strand exchange to be unidirectional.

Buoyancy-driven, rapid exhumation of ultrahigh-pressure metamorphosed continental crust

Preservation of ultrahigh-pressure (UHP) minerals formed at depths of 90–125 km require unusual conditions. Our subduction model involves underflow of a salient (250 ± 150 km wide, 90–125 km long) of continental crust embedded in cold, largely oceanic crust-capped lithosphere; loss of leading portions of the high-density oceanic lithosphere by slab break-off, as increasing volumes of microcontinental material enter the subduction zone; buoyancy-driven return toward midcrustal levels of a thin (2–15 km thick), low-density slice; finally, uplift, backfolding, normal faulting, and exposure of the UHP terrane. Sustained over ≈20 million years, rapid (≈5 mm/year) exhumation of the thin-aspect ratio UHP sialic sheet caught between cooler hanging-wall plate and refrigerating, downgoing lithosphere allows withdrawal of heat along both its upper and lower surfaces. The intracratonal position of most UHP complexes reflects consumption of an intervening ocean basin and introduction of a sialic promontory into the subduction zone. UHP metamorphic terranes consist chiefly of transformed, yet relatively low-density continental crust compared with displaced mantle material—otherwise such complexes could not return to shallow depths. Relatively rare metabasaltic, metagabbroic, and metacherty lithologies retain traces of phases characteristic of UHP conditions because they are massive, virtually impervious to fluids, and nearly anhydrous. In contrast, H2O-rich quartzofeldspathic, gneissose/schistose, more permeable metasedimentary and metagranitic units have backreacted thoroughly, so coesite and other UHP silicates are exceedingly rare. Because of the initial presence of biogenic carbon, and its especially sluggish transformation rate, UHP paragneisses contain the most abundantly preserved crustal diamonds.

In vitro selection of RNA molecules that displace cocaine from the membrane-bound nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (AChR) controls signal transmission between cells in the nervous system. Abused drugs such as cocaine inhibit this receptor. Transient kinetic investigations indicate that inhibitors decrease the channel-opening equilibrium constant [Hess, G. P. & Grewer, C. (1998) Methods Enzymol. 291, 443–473]. Can compounds be found that compete with inhibitors for their binding site but do not change the channel-opening equilibrium? The systematic evolution of RNA ligands by exponential enrichment methodology and the AChR in Torpedo californica electroplax membranes were used to find RNAs that can displace inhibitors from the receptor. The selection of RNA ligands was carried out in two consecutive steps: (i) a gel-shift selection of high-affinity ligands bound to the AChR in the electroplax membrane, and (ii) subsequent use of nitrocellulose filters to which both the membrane-bound receptor and RNAs bind strongly, but from which the desired RNA can be displaced from the receptor by a high-affinity AChR inhibitor, phencyclidine. After nine selection rounds, two classes of RNA molecules that bind to the AChR with nanomolar affinities were isolated and sequenced. Both classes of RNA molecules are displaced by phencyclidine and cocaine from their binding site on the AChR. Class I molecules are potent inhibitors of AChR activity in BC3H1 muscle cells, as determined by using the whole-cell current-recording technique. Class II molecules, although competing with AChR inhibitors, do not affect receptor activity in this assay; such compounds or derivatives may be useful for alleviating the toxicity experienced by millions of addicts.

Yeast chorismate mutase in the R state: Simulations of the active site

The isomerization of chorismate to prephenate by chorismate mutase in the biosynthetic pathway that forms Tyr and Phe involves C5—O (ether) bond cleavage and C1—C9 bond formation in a Claisen rearrangement. Development of negative charge on the ether oxygen, stabilized by Lys-168 and Glu-246, is inferred from the structure of a complex with a transition state analogue (TSA) and from the pH-rate profile of the enzyme and the E246Q mutant. These studies imply a protonated Glu-246 well above pH 7. Here, several 500-ps molecular dynamics simulations test the stability of enzyme–TSA complexes by using a solvated system with stochastic boundary conditions. The simulated systems are (i) protonated Glu-246 (stable), (ii) deprotonated Glu-246 (unstable), (iii) deprotonated Glu-246 plus one H2O between Glu-246 and the ether oxygen (unstable), (iv) the E246Q mutant (stable), and (v) addition of OH− between protonated Glu-246 and the ether oxygen. In (v), a local conformational change of Lys-168 displaced the OH− into the solvent region, suggesting a possible rate-determining step that precedes the catalytic step. In a 500-ps simulation of the enzyme complexed with the reactant chorismate or the product prephenate, no water molecule remained near the oxygen of the ligand. Calculations using the linearized Poisson–Boltzmann equation show that the effective pKa of Glu-246 is shifted from 5.8 to 8.1 as the negative charge on the ether oxygen of the TSA is changed from −0.56 electron to −0.9 electron. Altogether, these results support retention of a proton on Glu-246 to high pH and the absence of a water molecule in the catalytic steps.

Analysis of three structurally related antiviral compounds in complex with human rhinovirus 16

Rhinoviruses are a frequent cause of the common cold. A series of antirhinoviral compounds have been developed that bind into a hydrophobic pocket in the viral capsid, stabilizing the capsid and interfering with cell attachment. The structures of a variety of such compounds, complexed with rhinovirus serotypes 14, 16, 1A, and 3, previously have been examined. Three chemically similar compounds, closely related to a drug that is undergoing phase III clinical trials, were chosen to determine the structural impact of the heteroatoms in one of the three rings. The compounds were found to have binding modes that depend on their electronic distribution. In the compound with the lowest efficacy, the terminal ring is displaced by 1 Å and rotated by 180° relative to the structure of the other two. The greater polarity of the terminal ring in one of the three compounds leads to a small displacement of its position relative to the other compounds in the hydrophobic end of the antiviral compound binding pocket to a site where it makes fewer interactions. Its lower efficacy is likely to be the result of the reduced number of interactions. A region of conserved residues has been identified near the entrance to the binding pocket where there is a corresponding conservation of the mode of binding of these compounds to different serotypes. Thus, variations in residues lining the more hydrophobic end of the pocket are primarily responsible for the differences in drug efficacies.

Vimentin Dephosphorylation by Protein Phosphatase 2A Is Modulated by the Targeting Subunit B55

The intermediate filament protein vimentin is a major phosphoprotein in mammalian fibroblasts, and reversible phosphorylation plays a key role in its dynamic rearrangement. Selective inhibition of type 2A but not type 1 protein phosphatases led to hyperphosphorylation and concomitant disassembly of vimentin, characterized by a collapse into bundles around the nucleus. We have analyzed the potential role of one of the major protein phosphatase 2A (PP2A) regulatory subunits, B55, in vimentin dephosphorylation. In mammalian fibroblasts, B55 protein was distributed ubiquitously throughout the cytoplasm with a fraction associated to vimentin. Specific depletion of B55 in living cells by antisense B55 RNA was accompanied by disassembly and increased phosphorylation of vimentin, as when type 2A phosphatases were inhibited using okadaic acid. The presence of B55 was a prerequisite for PP2A to efficiently dephosphorylate vimentin in vitro or to induce filament reassembly in situ. Both biochemical fractionation and immunofluorescence analysis of detergent-extracted cells revealed that fractions of PP2Ac, PR65, and B55 were tightly associated with vimentin. Furthermore, vimentin-associated PP2A catalytic subunit was displaced in B55-depleted cells. Taken together these data show that, in mammalian fibroblasts, the intermediate filament protein vimentin is dephosphorylated by PP2A, an event targeted by B55.

(4-Aminomethyl)phenylguanidine derivatives as nonpeptidic highly selective inhibitors of human urokinase

Increased expression of the serine protease urokinase-type plasminogen activator (uPA) in tumor tissues is highly correlated with tumor cell migration, invasion, proliferation, progression, and metastasis. Thus inhibition of uPA activity represents a promising target for antimetastatic therapy. So far, only the x-ray crystal structure of uPA inactivated by H-Glu-Gly-Arg-chloromethylketone has been reported, thus limited data are available for a rational structure-based design of uPA inhibitors. Taking into account the trypsin-like arginine specificity of uPA, (4-aminomethyl)phenylguanidine was selected as a potential P1 residue and iterative derivatization of its amino group with various hydrophobic residues, and structure–activity relationship-based optimization of the spacer in terms of hydrogen bond acceptor/donor properties led to N-(1-adamantyl)-N′-(4-guanidinobenzyl)urea as a highly selective nonpeptidic uPA inhibitor. The x-ray crystal structure of the uPA B-chain complexed with this inhibitor revealed a surprising binding mode consisting of the expected insertion of the phenylguanidine moiety into the S1 pocket, but with the adamantyl residue protruding toward the hydrophobic S1′ enzyme subsite, thus exposing the ureido group to hydrogen-bonding interactions. Although in this enzyme-bound state the inhibitor is crossing the active site, interactions with the catalytic residues Ser-195 and His-57 are not observed, but their side chains are spatially displaced for steric reasons. Compared with other trypsin-like serine proteases, the S2 and S3/S4 pockets of uPA are reduced in size because of the 99-insertion loop. Therefore, the peculiar binding mode of the new type of uPA inhibitors offers the possibility of exploiting optimized interactions at the S1′/S2′ subsites to further enhance selectivity and potency. Because crystals of the uPA/benzamidine complex allow inhibitor exchange by soaking procedures, the structure-based design of new generations of uPA inhibitors can rely on the assistance of x-ray analysis.

Microtubule reorganization is obligatory for growth cone turning

To examine the role of microtubules in growth cone turning, we have compared the microtubule organization in growth cones advancing on uniform laminin substrates with their organization in growth cones turning at a laminin–tenascin border. The majority (82%) of growth cones on laminin had a symmetrical microtubule organization, in which the microtubules entering the growth cone splay out toward the periphery of the growth cone. Growth cones at tenascin borders had symmetrically arranged microtubules in only 34% of cases, whereas in the majority of cases the microtubules were displaced toward one-half of the growth cone, presumably stabilizing in the direction of the turn along the tenascin border. These results suggest that reorganization of microtubules could underlie growth cone turning. Further evidence for the involvement of microtubule rearrangement in growth cone turning was provided by experiments in which growth cones approached tenascin borders in the presence of nanomolar concentrations of the microtubule stabilizing compound, Taxol. Taxol altered the organization of microtubules in growth cones growing on laminin by restricting their distribution to the proximal regions of the growth cone and increasing their bundling. Taxol did not stop growth cone advance on laminin. When growing in the presence of Taxol, growth cones at tenascin borders were not able to turn and grow along the laminin–tenascin border, and consequently stopped at the border. Growth cones were arrested at borders for as long as Taxol was present (up to 6 h) without showing any signs of drug toxicity. These effects of Taxol were reversible. Together, these results suggest that microtubule reorganization in growth cones is a necessary event in growth cone turning.

Unusual 1H NMR chemical shifts support (His) Cɛ1—H⋅⋅⋅O⩵C H-bond: Proposal for reaction-driven ring flip mechanism in serine protease catalysis

13C-selective NMR, combined with inhibitor perturbation experiments, shows that the Cɛ1—H proton of the catalytic histidine in resting α-lytic protease and subtilisin BPN′ resonates, when protonated, at 9.22 ppm and 9.18 ppm, respectively, which is outside the normal range for such protons and ≈0.6 to 0.8 ppm further downfield than previously reported. They also show that the previous α-lytic protease assignments [Markley, J. L., Neves, D. E., Westler, W. M., Ibanez, I. B., Porubcan, M. A. & Baillargeon, M. W. (1980) Front. Protein Chem. 10, 31–61] were to signals from inactive or denatured protein. Simulations of linewidth vs. pH demonstrate that the true signal is more difficult to detect than corresponding signals from inactive derivatives, owing to higher imidazole pKa values and larger chemical shift differences between protonated and neutral forms. A compilation and analysis of available NMR data indicates that the true Cɛ1—H signals from other serine proteases are similarly displaced downfield, with past assignments to more upfield signals probably in error. The downfield displacement of these proton resonances is shown to be consistent with an H-bond involving the histidine Cɛ1—H as donor, confirming the original hypothesis of Derewenda et al. [Derewenda, Z. S., Derewenda, U. & Kobos, P. M. (1994) J. Mol. Biol. 241, 83–93], which was based on an analysis of literature x-ray crystal structures of serine hydrolases. The invariability of this H-bond among enzymes containing Asp-His-Ser triads indicates functional importance. Here, we propose that it enables a reaction-driven imidazole ring flip mechanism, overcoming a major dilemma inherent in all previous mechanisms, namely how these enzymes catalyze both the formation and productive breakdown of tetrahedral intermediates.

Multiple independent origins of mitochondrial gene order in birds

Mitochondrial genomes of all vertebrate animals analyzed to date have the same 37 genes, whose arrangement in the circular DNA molecule varies only in the relative position of a few genes. This relative conservation suggests that mitochondrial gene order characters have potential utility as phylogenetic markers for higher-level vertebrate taxa. We report discovery of a mitochondrial gene order that has had multiple independent originations within birds, based on sampling of 137 species representing 13 traditionally recognized orders. This provides evidence of parallel evolution in mitochondrial gene order for animals. Our results indicate operation of physical constraints on mitochondrial gene order changes and support models for gene order change based on replication error. Bird mitochondria have a displaced OL (origin of light-strand replication site) as do various other Reptilia taxa prone to gene order changes. Our findings point to the need for broad taxonomic sampling in using mitochondrial gene order for phylogenetic analyses. We found, however, that the alternative mitochondrial gene orders distinguish the two primary groups of songbirds (order Passeriformes), oscines and suboscines, in agreement with other molecular as well as morphological data sets. Thus, although mitochondrial gene order characters appear susceptible to some parallel evolution because of mechanistic constraints, they do hold promise for phylogenetic studies.

The dhp1+ gene, encoding a putative nuclear 5′→3′ exoribonuclease, is required for proper chromosome segregation in fission yeast

The Schizosaccharomyces pombe dhp1+ gene is an ortholog of the Saccharomyces cerevisiae RAT1 gene, which encodes a nuclear 5′→3′ exoribonuclease, and is essential for cell viability. To clarify the cellular functions of the nuclear 5′→3′ exoribonuclease, we isolated and characterized a temperature-sensitive mutant of dhp1 (dhp1-1 mutant). The dhp1-1 mutant showed nuclear accumulation of poly(A)+ RNA at the restrictive temperature, as was already reported for the rat1 mutant. Interestingly, the dhp1-1 mutant exhibited aberrant chromosome segregation at the restrictive temperature. The dhp1-1 cells frequently contained condensed chromosomes, most of whose sister chromatids failed to separate during mitosis despite normal mitotic spindle elongation. Finally, chromosomes were displaced or unequally segregated. As similar mitotic defects were also observed in Dhp1p-depleted cells, we concluded that dhp1+ is required for proper chromosome segregation as well as for poly(A)+ RNA metabolism in fission yeast. Furthermore, we isolated a multicopy suppressor of the dhp1-1 mutant, referred to as din1+. We found that the gene product of dhp1-1 was unstable at high temperatures, but that reduced levels of Dhp1-1p could be suppressed by overexpressing Din1p at the restrictive temperature. Thus, Din1p may physically interact with Dhp1p and stabilize Dhp1p and/or restore its activity.