Two enzymes responsible for the formation of herbivore-induced volatiles of maize, the methyltransferase AAMT1 and the terpene synthase TPS23, are regulated by a similar signal transduction pathway

Herbivore-induced volatiles play an important role in the indirect defense of plants. After herbivore damage, volatiles are released from the plant and can attract herbivore enemies that protect the plant from additional damage. The herbivore-induced volatile blend is complex and usually consists of mono- and sesquiterpenes, aromatic compounds, and indole. Although these classes of compounds are generally produced at different times after herbivore damage, the release of the terpene (E)-β-caryophyllene and the aromatic ester methyl anthranilate appear to be tightly coordinated. We have studied the herbivore induction patterns of two terpene synthases from Zea mays L. (Poaceae), TPS23 and TPS10, as well as S-adenosyl-L-methionine:anthranilic acid carboxyl methyltransferases (AAMT1), which are critical for the production of terpenes and anthranilate compounds, respectively. The transcript levels of tps23 and aamt1 displayed the same kinetics after damage by the larvae of Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae), and showed the same organ-specific and haplotype-specific expression patterns. Despite its close functional relation to TPS23, the terpene synthase TPS10 is not expressed in roots and does not display the haplotype-specific expression pattern. The results indicate that the same JA-mediated signaling cascade maycontrol the production of both the terpene (E)-β-caryophyllene and aromatic ester methyl anthranilate.

Bovine prion protein gene (PRNP) promoter polymorphisms modulate PRNP expression and may be responsible for differences in bovine spongiform encephalopathy susceptibility

The susceptibility of humans to the variant Creutzfeldt-Jakob disease is greatly influenced by polymorphisms within the human prion protein gene (PRNP). Similar genetic differences exist in sheep, in which PRNP polymorphisms modify the susceptibility to scrapie. However, the known coding polymorphisms within the bovine PRNP gene have little or no effect on bovine spongiform encephalopathy (BSE) susceptibility in cattle. We have recently found a tentative association between PRNP promoter polymorphisms and BSE susceptibility in German cattle (Sander, P., Hamann, H., Pfeiffer, I., Wemheuer, W., Brenig, B., Groschup, M., Ziegler, U., Distl, O., and Leeb, T. (2004) Neurogenetics 5, 19-25). A plausible hypothesis explaining this observation could be that the bovine PRNP promoter polymorphisms cause changes in PRNP expression that might be responsible for differences in BSE incubation time and/or BSE susceptibility. To test this hypothesis, we performed a functional promoter analysis of the different bovine PRNP promoter alleles by reporter gene assays in vitro and by measuring PRNP mRNA levels in calves with different PRNP genotypes in vivo. Two variable sites, a 23-bp insertion/deletion (indel) polymorphism containing a RP58-binding site and a 12-bp indel polymorphism containing an SP1-binding site, were investigated. Band shift assays indicated differences in transcription factor binding to the different alleles at the two polymorphisms. Reporter gene assays demonstrated an interaction between the two postulated transcription factors and lower expression levels of the ins/ins allele compared with the del/del allele. The in vivo data revealed substantial individual variation of PRNP expression in different tissues. In intestinal lymph nodes, expression levels differed between the different PRNP genotypes.

Capturing socially motivated linguistic change. How the use of gender-fair language afffects support for social initiatives in Austria and Poland.

Gender-fair language consists of the symmetric linguistic treatment of women and men instead of using masculine forms as generics. In this study, we examine how the use of gender-fair language affects readers' support for social initiatives in Poland and Austria. While gender-fair language is relatively novel in Poland, it is well established in Austria. This difference may lead to different perceptions of gender-fair usage in these speech communities. Two studies conducted in Poland investigate whether the evaluation of social initiatives (Study 1: quotas for women on election lists; Study 2: support for women students or students from countries troubled by war) is affected by how female proponents (lawyers, psychologists, sociologists, and academics) are referred to, with masculine forms (traditional) or with feminine forms (modern, gender-fair). Study 3 replicates Study 2 in Austria. Our results indicate that in Poland, gender-fair language has negative connotations and therefore, detrimental effects particularly when used in gender-related contexts. Conversely, in Austria, where gender-fair language has been implemented and used for some time, there are no such negative effects. This pattern of results may inform the discussion about formal policies regulating the use of gender-fair language.

Mutation Update of the CLCN5 Gene Responsible for Dent Disease 1

Dent disease is a rare X-linked tubulopathy characterized by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis and/or nephrolithiasis, progressive renal failure, and variable manifestations of other proximal tubule dysfunctions. It often progresses over a few decades to chronic renal insufficiency, and therefore molecular characterization is important to allow appropriate genetic counseling. Two genetic subtypes have been described to date: Dent disease 1 is caused by mutations of the CLCN5 gene, coding for the chloride/proton exchanger ClC-5; and Dent disease 2 by mutations of the OCRL gene, coding for the inositol polyphosphate 5-phosphatase OCRL-1. Herein, we review previously reported mutations (n = 192) and their associated phenotype in 377 male patients with Dent disease 1 and describe phenotype and novel (n = 42) and recurrent mutations (n = 24) in a large cohort of 117 Dent disease 1 patients belonging to 90 families. The novel missense and in-frame mutations described were mapped onto a three-dimensional homology model of the ClC-5 protein. This analysis suggests that these mutations affect the dimerization process, helix stability, or transport. The phenotype of our cohort patients supports and extends the phenotype that has been reported in smaller studies.

Regulation and transcript analysis of the ceroperon responsible for quorum sensing in Rhodobacter sphaeroides 2.4.1

Rhodobacter sphaeroides 2.4.1 is a Gram negative facultative photoheterotrophic bacterium that has been shown to have an N-acyl homoserine lactone-based quorum sensing system called cer for c&barbelow;ommunity e&barbelow;scape r&barbelow;esponse. The cer ORFs are cerR, the transcriptional regulator, cerI, the autoinducer synthase and cerA , whose function is unknown. The autoinducer molecule, 7,8- cis-N-(tetradecenoyl) homoserine lactone, has been characterized. The objective of this study was to identify an environmental stimulus that influences the regulation of cerRAI and, to characterize transcription of the cer operon. ^ A cerR::lacZ transcriptional fusion was made and β-Galactosidase assays were performed in R. sphaeroides 2.4.1 strains, wild type, AP3 (CerI−) and AP4 (CerR−). The cerR::lacZ β-Galactosidase assays were used as an initial survey of the mode of regulation of the Cer system. A cerA::lacZ translational fusion was created and was used to show that cerA can be translated. The presence of 7,8-cis-N-(tetradecenoyl) homoserine lactone was detected from R. sphaeroides strains wild type and AP4 (CerR−) using a lasR::lacZ translational fusion autoinducer bioassay. The cerR::lacZ transcriptional fusion in R. sphaeroides 2.4.1 wild type was tested under different environmental stimuli, such as various carbon sources, oxygen tensions, light intensities and culture media to determine if they influence transcription of the cer ORFs. Although lacZ assay data implicated high light intensity at 100 W/m2 to stimulate cer transcription, quantitative Northern RNA data of the cerR transcript showed that low light intensity at 3 W/m2 is at least one environmental stimulus that induces cer transcription. This finding was supported by DNA microarray analysis. Northern analysis of the cerRAI transcript provided evidence that the cer ORFs are co-transcribed, and that the cer operon contains two additional genes. Bioinformatics was used to identify genes that may be regulated by the Cer system by identifying putative lux box homologue sequences in the presumed promoter region of these genes. Genes that were identified were fliQ, celB and calsymin, all implicated in interacting with plants. Primer extension was used to help localize cis-elements in the promoter region. The cerR::lacZ transcriptional fusion was monitored in a subset of different global DNA binding transcriptional regulator mutant strains of R. sphaeroides 2.4.1. Those regulators involved in maintaining an anaerobic photosynthetic lifestyle appeared to have an effect. Collectively, the data imply that R. sphaeroides 2.4.1 activates the Cer system when grown anaerobic photosynthetically at low light intensity, 3 W/m2, and it may be involved in an interaction with plants. ^

Ueber die Palmengattung Synechanthus Hrm. Wendl.

Vom Hofgärtner Herm. Wendland

Dinámicas de ocupación urbana del Anillo Verde metropolitano, desde sus orígenes en el Plan General del Área Metropolitana de Madrid de 1963 hasta el Plan General de Ordenación Urbana de 1997

El Anillo Verde metropolitano, definido por el Plan General de Ordenación Urbana del Área Metropolitana de Madrid en 1963 siguiendo los modelos planteados por la cultura urbanística internacional, como armadura de la estructura urbana del AMM, espacio protagonista dentro del sistema de espacios libres, lugar de uso público destinado al recreo y contacto con la naturaleza de la población madrileña, se convierte en realidad en una reserva de suelo que va entrando en juego motivado por las alianzas entre el poder institucional y la clase social dominante actuando al margen del planeamiento, poniendo en evidencia la escasez de recursos legales y culturales disponibles para la salvaguarda de los intereses comunes y, donde los condicionantes geográficos y naturales del territorio madrileño han influido decisivamente en la especialización funcional y espacial del Área Metropolitana de Madrid. Así pues considerando esta idea como HIPÓTESIS, el objetivo de la TESIS sería demostrarla, para lo cual se hace necesario primero, acotar espacial y temporalmente el objeto de estudio, es decir, del Anillo Verde metropolitano1, segundo, contextualizar histórica y disciplinarmente los presupuestos teóricos que conformaban la idea del Anillo Verde, tercero, reconocer, localizar y documentar las piezas que han ido materializando la ocupación urbana del Anillo Verde, clasificándolas según parámetros temporales, funcionales, urbanísticos y, formales, lo que permite analizar la geografía, uso, instrumentación y forma de su transformación a escala general metropolitana y, cuarto, profundizar a modo de comprobación a escala municipal y urbana en dos escenarios representativos del conjunto metropolitano: el municipio de Pozuelo de Alarcón y el distrito de Hortaleza-Barajas. El contenido del documento se divide en tres bloques, el bloque I, se centra en las bases teóricas, el bloque II sitúa el hilo argumental de la tesis a escala metropolitana y el bloque III comprueba el fenómeno a escala municipal y urbana. De esta forma, se comienza por la comprensión del significado del concepto del Anillo Verde, que va más allá de la dimensión instrumental asignada de límite y contención urbana frente al crecimiento de la ciudad industrial de principios del siglo XX, basada en la descentralización de la ciudad tradicional, para adquirir un significado más complejo, como gran espacio de reserva y salvaguarda de valores naturales y culturales que se expresaban en su territorio y que permitirían alcanzar el equilibrio entre la ciudad y sus habitantes, es decir, entre el hombre y el espacio que habita. Se hace un recorrido por las principales corrientes urbanísticas que se van nutriendo de distintas disciplinas (economía, sociología, geografía, biología, ecología) para plantear teorías que permitieran materializar un nuevo orden urbano según principios de equidad social, económica y ambiental, en una secuencia donde Europa y Estados Unidos realizaban un constante intercambio -el movimiento de la Ciudad Jardín o el Regionalismo, que dieron paso a propuestas como el Greater London o el Gran Berlín, donde la figura del Anillo Verde tenía un papel protagonista, y del que también participaría nuestro país y la ciudad de Madrid, con modelos regionales como el Plan Besteiro y urbanos como el Plan Bidagor, antecedentes directos del Plan General de Ordenación Urbana del Área Metropolitana de Madrid de 1963 que pone en marcha la ordenación del crecimiento metropolitano de Madrid. El hilo argumental de la tesis se organiza en una doble aproximación: un acercamiento a escala metropolitana a partir del reconocimiento del modelo de ciudad definido en los distintos planes generales que acompañaron el desarrollo metropolitano (municipio de Madrid y de los siete términos municipales que rodeaban a este y que tenían suelo destinado a Anillo Verde), haciendo referencia además a las relaciones con el planeamiento regional, concretando en una escala de aproximación municipal que avanza hasta la interpretación urbana detallada. El primer acercamiento tiene lugar en el bloque II y se organiza en tres capítulos. El capítulo 4 se dedica al punto obligado de partida de la geografía local, describiendo las características biofísicas de los terrenos que formaban parte del Anillo Verde, que han marcado históricamente la forma de aprovechamiento del territorio, desde las extensiones de bosques mediterráneos al norte y al oeste continuación del Monte del Pardo, a los distintos tipos de cultivo que se adaptaban al sustrato geológico y la forma del terreno (de las suaves ondulaciones de sedimentos arcósicos al norte a las extensas plataformas arenosas y yesíferas del sur), además de las zonas de huertos aprovechando las depresiones y los cursos de agua (arroyo del Monte Carmelo, arroyo de Valdebebas, arroyo del Quinto, arroyo del Santo, arroyo Butarque, arroyo Meaques y arroyo Pozuelo). Una vez reconocida la realidad física, el capítulo 5, avanza en la descripción de los distintos modelos de ciudad propuestos desde el planeamiento urbanístico, en sus distintas escalas, la regional y la municipal, como respuesta a la coyuntura social, económica y política que ha caracterizado el proceso de ocupación del Anillo Verde al compás de la construcción del AMM. Se han reunido las propuestas de planeamiento municipal de los distintos municipios que disponían de terreno calificado como Anillo Verde: Madrid, Coslada, Getafe, Leganés, Alcorcón, Boadilla del Monte y Pozuelo de Alarcón. Además se han incorporado las distintas propuestas de ordenación territorial que han servido de referencia al planeamiento municipal, en todas sus versiones, desde las sectoriales, de mayor éxito y apoyo institucional, a los distintos intentos de ordenación integral, de mayor complejidad pero de menor calado, precisamente por la dificultad de consenso entre la ordenación física y el desarrollo económico, entre los intereses privados y el beneficio público. El primer horizonte, comienza con la formulación del Plan General de Ordenación Urbana del Área Metropolitana de Madrid de 1963, su desarrollo y la puesta en marcha de los primeros planes municipales en la década de los años setenta, donde se comprueba la necesidad de un marco regional que “ordene” el territorio de forma integral y sirva de referencia a las actuaciones sectoriales que habían marcado el primer desarrollo metropolitano. El segundo, se sitúa dos décadas más tarde con la aprobación del Plan General de Ordenación Urbana de Madrid de 1985 y el conjunto de planes municipales de los términos limítrofes, que siguen su filosofía de austeridad en cuanto a crecimiento territorial. El tercero se inicia en 1997 con la siguiente generación de planes de corte neoliberal que imponen un modelo territorial basado en las grandes operaciones metropolitanas de centralidad, infraestructuras y equipamiento, que consumen de forma indiscriminada la totalidad del territorio madrileño. Será en el último capítulo del segundo bloque (capítulo 6) donde se represente gráficamente a escala metropolitana y se analicen las 229 piezas que han ido colmatando el espacio destinado a Anillo Verde, según los parámetros de estudio, en base a las cuales se plantean las primeras conclusiones generales de la tesis, poniendo de manifiesto que las alianzas entre los agentes soberanos en la construcción de la ciudad y su entorno han trasgredido sucesivamente las determinaciones del Planeamiento en su definición de modelo de ciudad y territorio, acusando la carencia de recursos instrumentales y jurídicos que alentaron el proceso de su desmantelamiento, y revelando la influencia de los condicionantes geográficos y naturales en la especialización funcional y segregación social en el conjunto del Área Metropolitana de Madrid. Se remata el discurso metropolitano con una batería de conclusiones que interpretan el fenómeno de ocupación del anillo de verdor metropolitano confirmando las hipótesis iniciales, reconociendo los valores medioambientales y culturales trasgredidos, sus diversos actores, las numerosas operaciones urbanísticas desarrolladas con distintos usos y envergadura, así como los instrumentos de planeamiento utilizados, en base a las cuales se materializa la construcción del AMM según un modelo extendido (spread), dibujando una mancha de aceite (o grase-spots según Geddes) que precisamente había querido evitarse desde el planeamiento urbanístico con la definición de un Anillo Verde, espacio inmune a la edificación, que se aleja de su papel estructurante (equilibrador entre la ciudad y sus habitantes) para convertirse en armadura de la estructura comunicativa, que una vez consolidada se convierte en la mejor aliada de la máquina inmobiliaria. El último paso, se desarrolla en el bloque III que se divide en los capítulo 7,8 y 9 y supone la comprobación de lo descrito en el conjunto de escala metropolitana, en dos aspectos fundamentales, la falta de consideración por los valores culturales y medioambientales que han modelado el territorio, imprimiéndole un carácter singular y específico y, la estructura del dominio del suelo, donde se reconoce de forma precisa el grupo social y los agentes encargados en cada momento de comercializar los suelos del anillo, que bajo el paraguas de la urgencia social y el engañoso beneficio popular, obtienen importantes beneficios económicos. Con esa intención, se da un salto hacia la escala municipal y urbana, seleccionando dos escenarios de estudio, el municipio de Pozuelo de Alarcón, que representa la materialización del crecimiento suburbano de la élite madrileña ocupando las zonas de mayor valor ecológico del anillo, y el distrito de Hortaleza-Barajas que ofrece su territorio a las grandes operaciones metropolitanas, apoyándose en el eje de actividad marcado por la conexión Madrid-Barcelona y el sistema aeroportuario de escala global, ambos situados al norte de la línea de borde entre la Sierra y la Mancha, ocupando por tanto los lugares más valiosos de la geografía madrileña (estructura funcional anticipada por Bidagor en 1946 en su modelo de ciudad adaptada al territorio madrileño) Una vez descrito este proceso trasgresor de límites, de normas, de conductas, y desde una perspectiva del fenómeno suficientemente documentada, en el capítulo 10, se realiza una reflexión sobre la incidencia real de la propuesta urbanística del Anillo Verde en la construcción del AMM, de la misma forma que se sugieren nuevos roles al planeamiento en un formato intencionado de largo recorrido en oposición a lo inmediato y circunstancial, que permita hacer una nueva lectura de los presupuestos teóricos que conformaban la idea del Anillo Verde, espacio articulador (medioambiental, social y cultural) del territorio madrileño. ABSTRACT The Metropolitan Greenbelt was defined by the 1963 Master Plan for the Madrid Metropolitan Area (MMA), following established international models of urban development, as the structural framework of the MMA, the principal open space within its network of open spaces and a public area of recreation and contact with nature for the residents of Madrid. In reality, however, it ha become a reserve of land in which various alliances between the institutional authorities and the dominant social class have been operating on the margin of the original plan, exposing a scarcity of legal and cultural resources for the safeguarding of common interests, and in which the geographical and natural characteristics of the territory itself have come to play an influential role in the functional specialization and spatial segregation of the MMA. With that idea as its HYPOTHESIS, the aim of this THESIS is to demonstrate its reality. The first step in this is to delineate, temporally and spatially, the object of study; i.e. the Metropolitan Greenbelt2. The second is to contextualize historically and disciplinarily those theoretical ideas which conform to the greenbelt concept. The third is to acknowledge, locate and document the elements which have characterized the urban occupation of the Greenbelt and classify these according to the parameters of time, function, urban development and form, which in turn would enable the geography, use, instrumentation and form of its transformation to be analysed on a general metropolitan scale. The fourth step, as a method of verification, is an in-depth analysis of two representative settings within the metropolitan network: the municipality of Pozuelo de Alarcón and the Hortaleza-Barajas district. The content of the document is divided into three parts. Part I focuses on the study’s theoretical foundations, Part II establishes a line of argument at the metropolitan level and Part III examines the phenomenon from a municipal and urban perspective. The thesis, then, begins with a study of the greenbelt concept itself and its meaning, which is far more complex than the accepted instrumental dimension of limiting and containing urbanization in response to the growth of the industrial city of the early 20th century, and which is based on a decentralization of the traditional city. This wider purpose is the setting aside of a large reserved space to safeguard the natural and cultural values of the region and thereby achieve a balance between the city and its residents; that is to say, between man and the space he inhabits. The principal currents of thought in urban planning will then be examined. These have drawn upon a variety of disciplines (economics, sociology, geography, biology, ecology) to develop theories for establishing a new urban order according to the principles of social, economic and environmental equity, and have involved a constant interchange between Europe and the United States. Thus, the City Garden and Regionalist movements would clear the way for proposals such as Greater London and Great Berlin, Chicago and Washington, in which the greenbelt would play a fundamental role. The participation of our own country and the city of Madrid is also discussed, through regional models such as the Besteiro Plan and urban ones like the Bidagor Plan, direct forerunners of 1963’s General Organizational Plan for the Madrid Metropolitan Area, which would set into motion the organization of Madrid’s metropolitan growth. The line of argument followed in this thesis is two-fold: first, an examination of metropolitan development in keeping with the city model as defined in the various General Plans for the development of both the municipality of Madrid and the seven surrounding municipalities which have land designated for its Greenbelt; and second, an examination of this growth in relation to Regional Planning measures, is detailed on a smaller scale (municipal and district), where the conditioning factors affecting the land property structure and the network of biophysical units may be analysed in depth. The first of these is dealt with in Part II and organized into three chapters (4, 5 and 6). Chapter 4 is dedicated to the obligatory starting point of the geographical setting itself. The biophysical characteristics of the territories set aside for the Greenbelt, and which historically have played a role in the area’s exploitation, are described here. These range from expanses of Mediterranean woodland to the north and west of Monte del Pardo to the various types of farmland that have been adapted to the geological substratum and the contours of the terrain (gentle undulations of arkosic sediment in the north, and wide sandy and gypsiferous tableland in the south), as well as orchards planted in low valleys and along watercourses (the creeks of Monte Carmelo, Valdebebas, Quinto, Santo, Butarque, Meaques and Pozuelo). Once this physical reality ha been detailed, in Chapter 5 will examine the various city models proposed by urban planners, both regionally and municipally, in response to the confluence of social, economic and political interests that have characterized the process of occupation in the Greenbelt area during the construction of the MMA. Municipal planning proposals will be collected and examined for the various municipalities which have land designated for the Greenbelt: Madrid, Coslada, Getafe, Leganés, Alcorcón, Boadilla del Monte and Pozuelo de Alarcón. Furthermore, the various territorial organization proposals which have served as references for municipal planning will also be addressed here, in all of their versions –from the sectorial, which have met with more success and institutional approval, to the many attempts at integration, which have been more complex but less influential, precisely for the difficulty of reconciling physical organization with economic development, and private interest with public benefit. The first period in this process was the development of the General Plan of 1963, followed by the first municipal development plans of the 1970s, in which the need for a regional framework that “organized” the territory in an integral fashion was defined. This would serve as a reference for the sectorial actions that marked the metropolitan area’s initial development. The second came two decades later with the approval of the General Plan of 1985, and the network of municipal plans for the surrounding communities, which followed the same philosophy of austerity with regard to territorial growth. The third would begin to take form in 1997, as a new generation of neo-liberal development plans imposed a territorial model based on centralized large-scale metropolitan operations of infrastructure and equipment, which would indiscriminately consume the totality of Madrid’s land. At the end of the Part II, in Chapter 6, the metropolitan area will be represented graphically and the 229 pieces that have been gradually encroaching upon land designated for the Greenbelt will be analysed. This analysis will be carried out according to the parameters defined for the study, and the first general conclusions of the thesis will be based on its findings. It will show how alliances between the various governing authorities in the construction of the city and its environment have successively violated established plans with regard to the definitions of city and territory, how shortages of instrumental and judicial resources have accentuated the dismantling process, and how natural and geographical factors have influenced functional specialization and social segregation in the Madrid Metropolitan Area. The final step, detailed in Part III, will address two fundamental aspects of what has just been described: the lack of consideration for the cultural and environmental values which have shaped this territory and imprinted upon it a specific and unique character; and the structure of land domination, with a precise identification of the social group and agents responsible at each stage of the Greenbelt’s commercialization, who, under an umbrella of social urgency and deceptive public benefit, have used it to obtain substantial financial rewards. For this purpose, a closer look is taken at two specific areas: the municipality of Pozuelo de Alarcón, representative of the suburban growth of an elite population which has occupied the Greenbelt areas of the greatest ecological value; and the Hortaleza-Barajas district, which has offered its territory to large metropolitan business interests, based on activities centred on the connection between Madrid and Barcelona and the system of international air travel. Both of these settings are located to the north of the line which divides the Sierra from La Mancha, and thus occupy the most valuable land in the Madrid region (a functional structure anticipated by Bidagor in 1946, with his city model adapted to the territory of Madrid). Finally, an attempt will be made to interpret the phenomenon of metropolitan Greenbelt occupation, confirming initial hypotheses, specifying the environmental and cultural values that have been violated, and identifying the various players involved, as well as numerous urbanization operations of varying sizes and interests, and the instruments of planning they have used. It will be seen from this that the construction of the MMA has in fact followed a “spread” model, a “grease spot” (as Geddes calls it) which, from the outset of the planning process and according to the definition of a greenbelt as a construction-free zone, was precisely to be avoided. This structural role (to provide a balance between a city and its residents) has thus been abandoned and the Greenbelt converted instead into a communicative framework which, once consolidated, has become the greatest ally of the real estate machine. After this process of violating limits, norms and established behaviour has been described and solidly documented, a reflection will be made on the real influence of the Greenbelt proposal in the construction of the MMA. At the same time, new roles will be suggested for future planning, roles which are deliberate and long term, in opposition to the immediate and circumstantial. This will enable a new interpretation of the theoretical principles behind the greenbelt concept, a space designed to connect the territory of Madrid environmentally, socially and culturally.

Evidence for a structural motif in toxins and interleukin-2 that may be responsible for binding to endothelial cells and initiating vascular leak syndrome

The dose-limiting toxicity of interleukin-2 (IL-2) and immunotoxin (IT) therapy in humans is vascular leak syndrome (VLS). VLS has a complex etiology involving damage to vascular endothelial cells (ECs), extravasation of fluids and proteins, interstitial edema, and organ failure. IL-2 and ITs prepared with the catalytic A chain of the plant toxin, ricin (RTA), and other toxins, damage human ECs in vitro and in vivo. Damage to ECs may initiate VLS; if this damage could be avoided without losing the efficacy of ITs or IL-2, larger doses could be administered. In this paper, we provide evidence that a three amino acid sequence motif, (x)D(y), in toxins and IL-2 damages ECs. Thus, when peptides from RTA or IL-2 containing this sequence motif are coupled to mouse IgG, they bind to and damage ECs both in vitro and, in the case of RTA, in vivo. In contrast, the same peptides with a deleted or mutated sequence do not. Furthermore, the peptide from RTA attached to mouse IgG can block the binding of intact RTA to ECs in vitro and vice versa. In addition, RTA, a fragment of Pseudomonas exotoxin A (PE38-lys), and fibronectin also block the binding of the mouse IgG-RTA peptide to ECs, suggesting that an (x)D(y) motif is exposed on all three molecules. Our results suggest that deletions or mutations in this sequence or the use of nondamaging blocking peptides may increase the therapeutic index of both IL-2, as well as ITs prepared with a variety of plant or bacterial toxins.

Unusual phenotypic alteration of β amyloid precursor protein (βAPP) maturation by a new Val-715 → Met βAPP-770 mutation responsible for probable early-onset Alzheimer’s disease

We have identified a novel β amyloid precursor protein (βAPP) mutation (V715M-βAPP770) that cosegregates with early-onset Alzheimer’s disease (AD) in a pedigree. Unlike other familial AD-linked βAPP mutations reported to date, overexpression of V715M-βAPP in human HEK293 cells and murine neurons reduces total Aβ production and increases the recovery of the physiologically secreted product, APPα. V715M-βAPP significantly reduces Aβ40 secretion without affecting Aβ42 production in HEK293 cells. However, a marked increase in N-terminally truncated Aβ ending at position 42 (x-42Aβ) is observed, whereas its counterpart x-40Aβ is not affected. These results suggest that, in some cases, familial AD may be associated with a reduction in the overall production of Aβ but may be caused by increased production of truncated forms of Aβ ending at the 42 position.

Human PEX1 cloned by functional complementation on a CHO cell mutant is responsible for peroxisome-deficient Zellweger syndrome of complementation group I

The peroxisome biogenesis disorders (PBDs), including Zellweger syndrome (ZS) and neonatal adrenoleukodystrophy (NALD), are autosomal recessive diseases caused by defects in peroxisome assembly, for which at least 10 complementation groups have been reported. We have isolated a human PEX1 cDNA (HsPEX1) by functional complementation of peroxisome deficiency of a mutant Chinese hamster ovary (CHO) cell line, ZP107, transformed with peroxisome targeting signal type 1-tagged “enhanced” green fluorescent protein. This cDNA encodes a hydrophilic protein (Pex1p) comprising 1,283 amino acids, with high homology to the AAA-type ATPase family. A stable transformant of ZP107 with HsPEX1 was morphologically and biochemically restored for peroxisome biogenesis. HsPEX1 expression restored peroxisomal protein import in fibroblasts from three patients with ZS and NALD of complementation group I (CG-I), which is the highest-incidence PBD. A CG-I ZS patient (PBDE-04) possessed compound heterozygous, inactivating mutations: a missense point mutation resulting in Leu-664 → Pro and a deletion of the sequence from Gly-634 to His-690 presumably caused by missplicing (splice site mutation). Both PBDE-04 PEX1 cDNAs were defective in peroxisome-restoring activity when expressed in the patient fibroblasts as well as in ZP107 cells. These results demonstrate that PEX1 is the causative gene for CG-I peroxisomal disorders.

Identification by mass spectrometry of the phosphorylated residue responsible for activation of the catalytic domain of myosin I heavy chain kinase, a member of the PAK/STE20 family

Myosin I heavy chain kinase from Acanthamoeba castellanii is activated in vitro by autophosphorylation (8–10 mol of P per mol). The catalytically active C-terminal domain produced by trypsin cleavage of the phosphorylated kinase contains 2–3 mol of P per mol. However, the catalytic domain expressed in a baculovirus–insect cell system is fully active as isolated without autophosphorylation in vitro. We now show that the expressed catalytic domain is inactivated by incubation with acid phosphatase and regains activity upon autophosphorylation. The state of phosphorylation of all of the hydroxyamino acids in the catalytic domain were determined by mass spectrometry of unfractionated protease digests. Ser-627 was phosphorylated in the active, expressed catalytic domain, lost its phosphate when the protein was incubated with phosphatase, and was rephosphorylated when the dephosphorylated protein was incubated with ATP. No other residue was significantly phosphorylated in any of the three samples. Thus, phosphorylation of Ser-627, which is in the same position as the Ser and Thr residues that are phosphorylated in many other kinases, is necessary and sufficient for full activity of the catalytic domain. Ser-627 is also phosphorylated when full-length, native kinase is activated by autophosphorylation.

Direct interaction of Gβγ with a C-terminal Gβγ-binding domain of the Ca2+ channel α1 subunit is responsible for channel inhibition by G protein-coupled receptors

Several classes of voltage-gated Ca2+ channels (VGCCs) are inhibited by G proteins activated by receptors for neurotransmitters and neuromodulatory peptides. Evidence has accumulated to indicate that for non-L-type Ca2+ channels the executing arm of the activated G protein is its βγ dimer (Gβγ). We report below the existence of two Gβγ-binding sites on the A-, B-, and E-type α1 subunits that form non-L-type Ca2+ channels. One, reported previously, is in loop 1 connecting transmembrane domains I and II. The second is located approximately in the middle of the ca. 600-aa-long C-terminal tails. Both Gβγ-binding regions also bind the Ca2+ channel β subunit (CCβ), which, when overexpressed, interferes with inhibition by activated G proteins. Replacement in α1E of loop 1 with that of the G protein-insensitive and Gβγ-binding-negative loop 1 of α1C did not abolish inhibition by G proteins, but the exchange of the α1E C terminus with that of α1C did. This and properties of α1E C-terminal truncations indicated that the Gβγ-binding site mediating the inhibition of Ca2+ channel activity is the one in the C terminus. Binding of Gβγ to this site was inhibited by an α1-binding domain of CCβ, thus providing an explanation for the functional antagonism existing between CCβ and G protein inhibition. The data do not support proposals that Gβγ inhibits α1 function by interacting with the site located in the loop I–II linker. These results define the molecular mechanism by which presynaptic G protein-coupled receptors inhibit neurotransmission.