Azotobacter vinelandii nitrogenase: “Kinetics of nif gene expression and insights into the roles of FdxN and NifQ in FeMo-co biosynthesis”

The Molybdenum-nitrogenase is responsible for most biological nitrogen fixation activity (BNF) in the biosphere. Due to its great agronomical importance, it has been the subject of profound genetic and biochemical studies. The Mo nitrogenase carries at its active site a unique iron-molybdenum cofactor (FeMoco) that consists of an inorganic 7 Fe, 1 Mo, 1 C, 9 S core coordinated to the organic acid homocitrate. Biosynthesis of FeMo-co occurs outside nitrogenase through a complex and highly regulated pathway involving proteins acting as molecular scaffolds, metallocluster carriers or enzymes that provide substrates in appropriate chemical forms. Specific expression regulatory factors tightly control the accumulation levels of all these other components. Insertion of FeMo-co into a P-cluster containing apo-NifDK polypeptide results in nitrogenase reconstitution. Investigation of FeMo-co biosynthesis has uncovered new radical chemistry reactions and new roles for Fe-S clusters in biology.

Al Este del Retiro

La zona de Madrid al Este del Retiro ha estado indefectiblemente condicionada en su tardío desarrollo urbano por su posición a espaldas del Real Sitio. La construcción hacia 1640 de las tapias que rodeaban los reales jardines transformó la red de caminos que partían hacia oriente, aisló los terrenos ubicados más al Este de la ciudad, con la que ya sólo se podrían comunicar por las carreteras de Aragón y Valencia, y condenó las expectativas de desarrollo urbano reduciendo los precios de las propiedades, lo cual determinó durante décadas los usos y la arquitectura de la zona. El Anteproyecto de Ensanche de Carlos María de Castro constituye el germen a partir del cual, durante un lento proceso de casi cien años, fue configurándose la ciudad que hoy conocemos. La identificación en el Archivo de Villa del primer plano general del Ensanche trazado por Castro, del cual anteriores trabajos advirtieron de su existencia aunque se desconocía su localización, es la principal aportación de esta investigación. Por un lado, este primer plano general del Ensanche manuscrito es, por sí mismo, un documento de indudable importancia en la historia del urbanismo madrileño. En segundo lugar, el análisis de su contenido arroja nueva luz sobre la propuesta original de Castro, parcialmente censurada por la Dirección General de Obras Públicas antes de la aprobación del plan en 1860. Especialmente en lo referente a la zona de Madrid al Este del Retiro, proyectada como barrio obrero del Ensanche, este documento ha aportado un enfoque desconocido hasta ahora sobre el paisaje urbano concebido por Castro para la más ambiciosa propuesta planteada en mucho tiempo al problema de la vivienda obrera. Finalmente, el análisis de la factura del plano revela la superposición de varias capas de dibujo, evidenciando que durante un tiempo fue un documento vivo, utilizado como plano de trabajo por el equipo de Castro durante aproximadamente diez años, hasta la destitución del ingeniero en 1868. Posteriores análisis del plano sobre otros ámbitos de la ciudad arrojarán sin duda nuevos datos sobre el proceso proyectual del conjunto del Ensanche. Pero la dinámica de lo real, sintetizable en múltiples factores de índole social, económica y legislativa, transformó durante las primeras décadas de andadura del Ensanche la ciudad proyectada por Castro al Este del Retiro. El dibujo de la ciudad, entendido como herramienta de análisis y empleado con éxito en trabajos de investigación realizados por otros autores en la misma línea, ha permitido deducir la reconstitución gráfica del estado de la ciudad en diferentes momentos singulares del desarrollo urbanístico de la zona, así como de la propuesta original de barrio obrero de Castro. No hay que olvidar que, a pesar del escaso interés que suscitaba entre los inversores inmobiliarios el ámbito geográfico de estudio de esta tesis, fue objeto, durante casi un siglo, de numerosas propuestas de ordenación y urbanización que, aunque no llegaron a materializarse, fueron configurando una suerte de desarrollo virtual de la ciudad paralelo al devenir de la realidad. De esta forma, el dibujo se constituye en esta tesis como fuente de información, herramienta de pensamiento y resultado de la investigación en sí mismo, ilustrando y contribuyendo al mejor conocimiento de la forma urbana. ABSTRACT The area of Madrid to the East of the Retiro has been inevitably conditioned in its late urban development by its position behind the Royal Site. The construction of the walls surrounding the royal gardens around 1640 transformed the network of roads departing eastward, isolated land located to the East of the city, with which already only could communicate by roads of Aragon and Valencia, condemned the expectations of urban development by reducing the prices of the properties, and determined for decades uses and architecture of the area. The Carlos María de Castro preliminary design of City Expansion is the germ from which, during a slow process of almost one hundred years, the city which we know today was setting up. The discovery in the City Archive of the City Expansion first drawing traced by Castro, which previous investigations warned of its existence although its location was unknown, is the main contribution of this research. Firstly, this hand drawn general plan of the city expansion is by itself a document of undoubted importance in the history of Madrid urbanism. Secondly, the analysis of its content sheds new light on Castro´s original proposal, partially censored by the Dirección General de Obras Públicas before the approval of the plan in 1860. Especially concerning the area of Madrid to the East of the Retiro, projected as a workingclass district of the City Expansion, this document has provided an unknown up to now approach on the urban landscape designed by Castro for the more ambitious proposal put forward in a long time to the problem of worker housing. Finally, analysis of hand drawn plan reveals the superposition of several layers of drawing, demonstrating that for a time it was a living document, used as a work plan by the Castro team for approximately ten years, until the dismissal of the engineer in 1868. Subsequent analysis of the drawing on other areas of the city will have no doubt new data on the design process of the whole City Expansion. But the dynamics of reality, synthesizable on multiple factors in social, economic and legislative, transformed during the first decades of existence of the City Expansion designed by Castro to the East of the Retiro. Drawing of the city, understood as a tool of analysis and used successfully in research works done by other authors on the same line, has allowed to deduct graphic reconstitution of the city status in different and singular moments in the urban development of the area, as well as the original Castro´s proposal of working-class district. It should not be forgotten that, despite the lack of interest which raised among investors the geographic scope of this thesis study, it was the object, for nearly a century, of numerous proposals for urbanization which, although they didn´t materialize, were setting up a sort of virtual development of the city parallel to the becoming of the reality. In this way, drawing is used in the thesis as a source of information, tool of thought and outcome of the research itself, illustrating and contributing to a better understanding of urban form.

Configuración formal de los rosetones románicos de la ciudad de Zamora

La presente tesis estudia los rosetones románicos de la ciudad de Zamora. La elección del tema tiene como objetivo profundizar en el conocimiento de estos elementos ya que la información existente sobre ellos es muy escasa. El análisis de estos rosetones se ha realizado desde una perspectiva globalizadora que abarca aspectos tales como los geográficos, morfológicos, funcionales, compositivos, constructivos, geométricos, ornamentales, otros. Así mismo, para el desarrollo de esta investigación se ha considerado necesario el estudio de temas históricos, estilísticos, simbólicos, religiosos, culturales, etc., que aportan el marco contextual que permiten su mejor entendimiento. El estudio de cada rosetón ha permitido implementar y desarrollar un método de trabajo analítico basado en el estudio particular de una serie de aspectos como los anteriormente mencionados, así como plantear una estrategia que permite la reconstitución gráfica de los rosetones, basándose en un sistema de módulos que facilitan trabajar de acuerdo a las proporciones de los elementos; hecho que permite acercarnos con gran exactitud a la representación del objeto real cuando se carece de medidas. El desarrollo de esta investigación ha llevado a establecer entre otras cosas que la definición de “ventana circular” que se le atribuye a los rosetones románicos no es acertada, puesto que la función que cumplen en el edificio religioso es más bien de carácter simbólico. ABSTRACT This thesis studies the Romanesque rose windows of the Zamora city. The choice of topic is intended to deepen the knowledge of these elements as the existing information about them is very scarce. The analysis of these rose windows was made from a global perspective covering aspects such as geographic, morphological, functional, compositional, construction, geometric, ornamental, other. Also, for the development of this research it was considered necessary to study historical, stylistic, symbolic, religious, cultural issues, etc., that provide the contextual framework that allow for better understanding. The study of each rose windows has allowed implement and develop a method of analytical work based on the particular study a number of issues such as those mentioned above, as well as devise a strategy that allows the graphic reconstitution of the rose windows, based on a system of modules facilitate work according to the proportions of the elements; made with great precision approach allows the representation of the real thing when it lacks measures. The development of this research has led to establish among other things that the definition of "circular window" that is attributed to the Romanesque rose windows is not successful because the role in the religious building is rather symbolic.

Architectural specificity in chromatin structure at the TATA box in vivo: Nucleosome displacement upon β-phaseolin gene activation

Extensive studies of the β-phaseolin (phas) gene in transgenic tobacco have shown that it is highly active during seed embryogenesis but is completely silent in leaf and other vegetative tissues. In vivo footprinting revealed that the lack of even basal transcriptional activity in vegetative tissues is associated with the presence of a nucleosome that is rotationally positioned with base pair precision over three phased TATA boxes present in the phas promoter. Positioning is sequence-dependent because an identical rotational setting is obtained upon nucleosome reconstitution in vitro. A comparison of DNase I and dimethyl sulfate footprints in vivo and in vitro strongly suggests that this repressive chromatin architecture is remodeled concomitant with gene activation in the developing seed. This leads to the disruption of histone-mediated DNA wrapping and the assembly of the TATA boxes into a transcriptionally competent nucleoprotein complex.

trans/cis (Z/E) photoisomerization of the chromophore of photoactive yellow protein is not a prerequisite for the initiation of the photocycle of this photoreceptor protein

The chromophore of photoactive yellow protein (PYP) (i.e., 4-hydroxycinnamic acid) has been replaced by an analogue with a triple bond, rather than a double bond (by using 4-hydroxyphenylpropiolic acid in the reconstitution, yielding hybrid I) and by a “locked” chromophore (through reconstitution with 7-hydroxycoumarin-3-carboxylic acid, in which a covalent bridge is present across the vinyl bond, resulting in hybrid II). These hybrids absorb maximally at 464 and 443 nm, respectively, which indicates that in both hybrids the deprotonated chromophore does fit into the chromophore-binding pocket. Because the triple bond cannot undergo cis/trans (or E/Z) photoisomerization and because of the presence of the lock across the vinyl double bond in hybrid II, it was predicted that these two hybrids would not be able to photocycle. Surprisingly, both are able. We have demonstrated this ability by making use of transient absorption, low-temperature absorption, and Fourier-transform infrared (FTIR) spectroscopy. Both hybrids, upon photoexcitation, display authentic photocycle signals in terms of a red-shifted intermediate; hybrid I, in addition, goes through a blue-shifted-like intermediate state, with very slow kinetics. We interpret these results as further evidence that rotation of the carbonyl group of the thioester-linked chromophore of PYP, proposed in a previous FTIR study and visualized in recent time-resolved x-ray diffraction experiments, is of critical importance for photoactivation of PYP.

Absence of cytokine receptor-dependent specificity in red blood cell differentiation in vivo

Erythropoietin (EPO) is required for red blood cell development, but whether EPO-specific signals directly instruct erythroid differentiation is unknown. We used a dominant system in which constitutively active variants of the EPO receptor were introduced into erythroid progenitors in mice. Chimeric receptors were constructed by replacing the cytoplasmic tail of constitutively active variants of the EPO receptor with tails of diverse cytokine receptors. Receptors linked to granulocyte or platelet production supported complete erythroid development in vitro and in vivo, as did the growth hormone receptor, a nonhematopoietic receptor. Therefore, EPOR-specific signals are not required for terminal differentiation of erythrocytes. Furthermore, we found that cellular context can influence cytokine receptor signaling.

Interaction of the human androgen receptor transactivation function with the general transcription factor TFIIF

The human androgen receptor (AR) is a ligand-activated transcription factor that regulates genes important for male sexual differentiation and development. To better understand the role of the receptor as a transcription factor we have studied the mechanism of action of the N-terminal transactivation function. In a protein–protein interaction assay the AR N terminus (amino acids 142–485) selectively bound to the basal transcription factors TFIIF and the TATA-box-binding protein (TBP). Reconstitution of the transactivation activity in vitro revealed that AR142–485 fused to the LexA protein DNA-binding domain was competent to activate a reporter gene in the presence of a competing DNA template lacking LexA binding sites. Furthermore, consistent with direct interaction with basal transcription factors, addition of recombinant TFIIF relieved squelching of basal transcription by AR142–485. Taken together these results suggest that one mechanism of transcriptional activation by the AR involves binding to TFIIF and recruitment of the transcriptional machinery.

Nrf2 is essential for protection against acute pulmonary injury in mice

Nrf2 is a member of the “cap ‘n’ collar” family of transcription factors. These transcription factors bind to the NF-E2 binding sites (GCTGAGTCA) that are essential for the regulation of erythroid-specific genes. Nrf2 is expressed in a wide range of tissues, many of which are sites of expression for phase 2 detoxification genes. Nrf2−/− mice are viable and have a normal phenotype under normal laboratory conditions. The NF-E2 binding site is a subset of the antioxidant response elements that have the sequence GCNNNGTCA. The antioxidant response elements are regulatory sequences found on promoters of several phase 2 detoxification genes that are inducible by xenobiotics and antioxidants. We report here that Nrf2−/− mice are extremely susceptible to the administration of the antioxidant butylated hydroxytoluene. With doses of butylated hydroxytoluene that are tolerated by wild-type mice, the Nrf2−/− mice succumb from acute respiratory distress syndrome. Gene expression studies show that the expression of several detoxification enzymes is altered in the Nrf2−/− mice. The Nrf2−/− mice may prove to be a good in vivo model for toxicological studies. As oxidative damage causes DNA breakage, these mice may also be useful for testing carcinogenic agents.

The newt ribozyme is part of a riboprotein complex

Strand-specific transcripts of a satellite DNA of the newts, Notophthalmus and Triturus, are present in cells in monomeric and multimeric sizes. These transcripts undergo self-catalyzed, site-specific cleavage in vitro: the reaction requires Mg2+ and is mediated by a “hammerhead” domain. Transcription of the newt ribozyme appears to be performed by RNA polymerase II under the control of a proximal sequence element and a distal sequence element. In vitro, the newt ribozyme can cleave in trans an RNA substrate, suggesting that in vivo it might be involved in RNA processing events, perhaps as a riboprotein complex. Here we show that the newt ribozyme is in fact present as a riboprotein particle of about 12 S in the oocytes of Triturus. In addition, reconstitution experiments and gel-shift analyses show that a complex is assembled in vitro on the monomeric ribozyme molecules. UV cross-linking studies identify a few polypeptide species, ranging from 31 to 65 kDa, associated to the newt ribozyme with different affinities. Finally, we find that an appropriate oligoribonucleotide substrate is specifically cleaved by the riboproteic activity in S-100 ovary extracts.

Expansion in vitro of transplantable human cord blood stem cells demonstrated using a quantitative assay of their lympho-myeloid repopulating activity in nonobese diabetic–scid/scid mice

Human hematopoiesis originates in a population of stem cells with transplantable lympho-myeloid reconstituting potential, but a method for quantitating such cells has not been available. We now describe a simple assay that meets this need. It is based on the ability of sublethally irradiated immunodeficient nonobese diabetic–scid/scid (NOD/SCID) mice to be engrafted by intravenously injected human hematopoietic cells and uses limiting dilution analysis to measure the frequency of human cells that produce both CD34−CD19+ (B-lymphoid) and CD34+ (myeloid) colony-forming cell progeny in the marrow of such recipients 6 to 8 weeks post-transplant. Human cord blood (CB) contains ≈5 of these competitive repopulating units (CRU) per ml that have a similar distribution between the CD38− and CD38+ subsets of CD34+ CB cells as long-term culture-initiating cells (LTC-IC) (4:1 vs. 2:1). Incubation of purified CD34+CD38− human CB cells in serum-free medium containing flt-3 ligand, Steel factor, interleukin 3, interleukin 6, and granulocyte colony-stimulating factor for 5–8 days resulted in a 100-fold expansion of colony-forming cells, a 4-fold expansion of LTC-IC, and a 2-fold (but significant, P < 0.02) increase in CRU. The culture-derived CRU, like the original CB CRU, generated pluripotent, erythroid, granulopoietic, megakaryopoietic, and pre-B cell progeny upon transplantation into NOD/SCID mice. These findings demonstrate an equivalent phenotypic heterogeneity amongst human CB cells detectable as CRU and LTC-IC. In addition, their similarly modest response to stimulation by a combination of cytokines that extensively amplify LTC-IC from normal adult marrow underscores the importance of ontogeny-dependent changes in human hematopoietic stem cell proliferation and self-renewal.

Glutamic acid 286 in subunit I of cytochrome bo3 is involved in proton translocation

Glutamic acid 286 (E286; Escherichia coli cytochrome bo3 numbering) in subunit I of the respiratory heme-copper oxidases is highly conserved and has been suggested to be involved in proton translocation. We report a technique of enzyme reconstitution that yields essentially unidirectionally oriented cytochrome bo3 vesicles in which proton translocation can be measured. Such experiments are not feasible in the E286Q mutant due to strong inhibition of respiration, but this is not the case for the mutants E286D and E286C. The reconstituted E286D mutant enzyme readily translocates protons whereas E286C does not. Loss of proton translocation in the D135N mutant, but not in D135E or D407N, also is verified using proteoliposomes. Stopped-flow experiments show that the peroxy intermediate accumulates in the reaction of the E286Q and E286C mutant enzymes with O2. We conclude that an acidic function of the 286 locus is essential for the mechanism of proton translocation.

Human deoxycytidine kinase is located in the cell nucleus

Human deoxyribonucleoside kinases are required for the pharmacological activity of several clinically important anticancer and antiviral nucleoside analogs. Human deoxycytidine kinase and thymidine kinase 1 are described as cytosolic enzymes in the literature, whereas human deoxyguanosine kinase and thymidine kinase 2 are believed to be located in the mitochondria. We expressed the four human deoxyribonucleoside kinases as fusion proteins with the green fluorescent protein to study their intracellular locations in vivo. Our data showed that the human deoxycytidine kinase is located in the cell nucleus and the human deoxyguanosine kinase is located in the mitochondria. The fusion proteins between green fluorescent protein and thymidine kinases 1 and 2 were both predominantly located in the cytosol. Site-directed mutagenesis of a putative nuclear targeting signal, identified in the primary structure of deoxycytidine kinase, completely abolished nuclear import of the protein. Reconstitution of a deoxycytidine kinase-deficient cell line with the wild-type nuclear or the mutant cytosolic enzymes both restored sensitivity toward anticancer nucleoside analogs. This paper reports that a deoxyribonucleoside kinase is located in the cell nucleus and we discuss the implications for deoxyribonucleotide synthesis and phosphorylation of nucleoside analogs.

Distal enhancer regulation by promoter derepression in topologically constrained DNA in vitro

Long-range promoter–enhancer interactions are a crucial regulatory feature of many eukaryotic genes yet little is known about the mechanisms involved. Using cloned chicken βA-globin genes, either individually or within the natural chromosomal locus, enhancer-dependent transcription is achieved in vitro at a distance of 2 kb with developmentally staged erythroid extracts. This occurs by promoter derepression and is critically dependent upon DNA topology. In the presence of the enhancer, genes must exist in a supercoiled conformation to be actively transcribed, whereas relaxed or linear templates are inactive. Distal protein–protein interactions in vitro may be favored on supercoiled DNA because of topological constraints. In this system, enhancers act primarily to increase the probability of rapid and efficient transcription complex formation and initiation. Repressor and activator proteins binding within the promoter, including erythroid-specific GATA-1, mediate this process.

Cloning, expression, and characterization of a cDNA encoding a novel human growth factor for primitive hematopoietic progenitor cells

Multiple growth factors synergistically stimulate proliferation of primitive hematopoietic progenitor cells. A human myeloid cell line, KPB-M15, constitutively produces a novel hematopoietic cytokine, termed stem cell growth factor (SCGF), possessing species-specific proliferative activities. Here we report the molecular cloning, expression, and characterization of a cDNA encoding human SCGF using a newly developed λSHDM vector that is more efficient for differential and expression cloning. cDNA for SCGF encodes a 29-kDa polypeptide without N-linked glycosylation. SCGF transiently produced by COS-1 cells supports growth of hematopoietic progenitor cells through a short-term liquid culture of bone marrow cells and exhibits promoting activities on erythroid and granulocyte/macrophage progenitor cells in primary semisolid culture with erythropoietin and granulocyte/macrophage colony-stimulating factor, respectively. Expression of SCGF mRNA is restricted to myeloid cells and fibroblasts, suggesting that SCGF is a growth factor functioning within the hematopoietic microenvironment. SCGF could disclose some human-specific mechanisms as yet unidentified from studies on the murine hematopoietic system.

Redistribution of phosphatidylethanolamine at the cleavage furrow of dividing cells during cytokinesis

Ro09-0198 is a tetracyclic polypeptide of 19 amino acids that recognizes strictly the structure of phosphatidylethanolamine (PE) and forms a tight equimolar complex with PE on biological membranes. Using the cyclic peptide coupled with fluorescence-labeled streptavidin, we have analyzed the cell surface localization of PE in dividing Chinese hamster ovary cells. We found that PE was exposed on the cell surface specifically at the cleavage furrow during the late telophase of cytokinesis. PE was exposed on the cell surface only during the late telophase and no alteration in the distribution of the plasma membrane-bound cyclic peptide was observed during the cytokinesis, suggesting that the surface exposure of PE reflects the enhanced scrambling of PE at the cleavage furrow. Furthermore, cell surface immobilization of PE induced by adding the cyclic peptide coupled with streptavidin to prometaphase cells effectively blocked the cytokinesis at late telophase. The peptide-streptavidin complex treatment had no effect on furrowing, rearrangement of microtubules, and nuclear reconstitution, but specifically inhibited both actin filament disassembly at the cleavage furrow and subsequent membrane fusion. These results suggest that the redistribution of the plasma membrane phospholipids is a crucial step for cytokinesis and the cell surface PE may play a pivotal role in mediating a coordinate movement between the contractile ring and plasma membrane to achieve successful cell division.