Impact studies from different factors on root development from Stevia rebaudiana bertoni

The demand for an alternative and a high potency sweetener to substitute sugar increases year in year out, more so as a high percentage of the world population becomes increasingly diabetic. The alternative natural sweetener at hand has been Stevia rebaudiana Bertoni, a plant species, native to Paraguay and a member of the family compositae. Stevia is usually propagated by stem cuttings due to low percentage (10 %) seed germination, thus limiting large scales cultivation. To cultivate this crop en mass therefore, there is need to evolve efficient rooting techniques. Influences of irradiation from light, and hormones on rooting have been reported. The rooting efficacy in stem cuttings of this crop under varying light wavelengths, dark and hormone factors was investigated. Evaluated parameters include- (i) day of root emergent, (ii) percentage of rooted cuttings, (iii) average number, (iv) length and (v) width, of roots. Analysis of variance at p<.05 revealed that the number, length and width, of roots differed significantly in each case at p<0.000. Light irradiation was highly effective and a necessary factor for rooting in stems cuttings of this crop. The red light-IBA combined factors served best in stem micro-cutting practice and facilitation of effective mass cultivation in stevia crop.

El hombre aristotélico : Una construcción retrospectiva

En la obra aristotélica la plenitud del eîdos en cada hombre no es otra cosa que el despliegue del contenido virtual de todas las posibilidades ínsitas en su realidad particular. El movimiento de su physis representa la progresiva concreción de este contenido. La vida constituye un movimiento continuo desde la generación hasta la corrupción, sólo es posible conocer las variantes, relaciones y alcances de lo que se haya en sí contenido una vez actualizado. Así, estudiar los procesos que constituyen la vida de un hombre nos llevan a revisar la tensión originaria de potencia y acto (Met. V; IX pral.; Fís. passim). La especulación respecto de dicha tensión -perdurable en una vida- introduce una particular tónica de retroactividad a las consideraciones sobre la vida humana que, a la luz de Solón (Heródoto I, xxx-xxxiii), aproximan a considerar que no es posible afirmar de un hombre que es feliz en tanto que vive; análogamente, decimos de la actualización del eîdos

El hombre aristotélico : Una construcción retrospectiva

En la obra aristotélica la plenitud del eîdos en cada hombre no es otra cosa que el despliegue del contenido virtual de todas las posibilidades ínsitas en su realidad particular. El movimiento de su physis representa la progresiva concreción de este contenido. La vida constituye un movimiento continuo desde la generación hasta la corrupción, sólo es posible conocer las variantes, relaciones y alcances de lo que se haya en sí contenido una vez actualizado. Así, estudiar los procesos que constituyen la vida de un hombre nos llevan a revisar la tensión originaria de potencia y acto (Met. V; IX pral.; Fís. passim). La especulación respecto de dicha tensión -perdurable en una vida- introduce una particular tónica de retroactividad a las consideraciones sobre la vida humana que, a la luz de Solón (Heródoto I, xxx-xxxiii), aproximan a considerar que no es posible afirmar de un hombre que es feliz en tanto que vive; análogamente, decimos de la actualización del eîdos

Compensación de energía reactiva en los servicios auxiliares de una central térmica

El proyecto tiene como objeto analizar desde el punto de vista técnico-económico las posibilidades para mejorar el factor de potencia en los consumos auxiliares de la Central térmica Litoral, perteneciente a Endesa Generación. Debido a un cambio normativo, las tarifas de acceso a las redes de transporte y distribución de energía eléctrica aplicarán ahora también a los consumos propios de las empresas productoras de electricidad dentro del ámbito de aplicación de las tarifas de acceso. Como la energía reactiva se facturará en función del factor de potencia se propone para su compensación la instalación de bancos de condensadores para mejorar el mismo y disminuir la penalización. Para el dimensionamiento de los bancos se han utilizado los consumos horarios de los años 2012 y 2013 de tres fronteras: grupos 1 y 2 de 400 kV y los transformadores auxiliares de arranque (OTAAs) de 6 kV , teniendo en cuenta las peculiaridades de una Central de Generación, ya que sólo serán objeto de estudio (a efecto de penalizaciones) las horas en las que el grupo está parado o arrancando. El resultado final muestra la diferencia en cuanto a los periodos de amortización de una instalación con generación y sin generación, siendo esta última más rentable económicamente. ABSTRACT The goal of this project is to analyse the possibilities to better the power factor in the auxiliary consumption of the Thermal Plant Litoral from an economic and technical viewpoint. Due to a normative changes, the access price rates for the transportation and distribution of electrical energy, will also apply now to the electricity productive companies own consumption within the scope of the access rate. Since reactive power is invoiced based on the potency factor, we propose the installation of capacitor banks to improve the aforementioned power factor as compensation so as to reduce sanctions. We have used the time profiles from 2012 and 2013 to dimension the banks, using 3 borders: Group 1 and 2 (400 kV) and the auxiliary starter transformers (OTAA's) of 6kV, taking into account the special characteristics of a Generation Plant , since we will only study the hours that the group was shut down or powering up (since these are what the penalties cover). The final result shows the difference in regards to repayment periods for both generating and nongenerating installation, being the aforesaid the most economically worthwile of the two

Ni lo uno, ni lo otro: posibilidad de lo neutro en arquitectura

Esta investigación se ocupa de comprobar la viabilidad de un término, un concepto que se detecta como posible instrumento para nombrar algunas operaciones en arquitectura. Ese concepto es Lo Neutro. Frente al sistema de dualidades con el que enunciamos lo real, lo Neutro se sitúa, no tanto en el punto medio, como en el punto opuesto a ambos términos. No es la conjunción de contrarios, sino aquello que desvía la propia pregunta que obliga a elegir. Próximo a los conceptos de indefinición, indiferencia e inacción, lo Neutro se revela como un mecanismo fundamental de disolución. Reconociendo en la lengua varios sentidos para el término, con cada capítulo, la tesis va construyendo una definición ampliada de lo Neutro hasta configurar un conjunto de significados aplicables a la arquitectura. El primero de ellos es el que se deriva del concepto de indiferencia funcional. Tras un análisis del contexto teórico sobre el que se asienta la dialéctica bi-direccional forma-función, su consolidación en los primeros años del siglo XX y también su puesta en crisis mediante la introducción de nociones multi-relacionales como ‘entorno’ y ‘sistema’, la investigación propone tres modelos de estudio: la Planta Tipo, la Planta Única y la Planta de Equivalencias. En cada uno de estos casos, aunque el tema fundamental es la neutralidad programática, se reconoce también la presencia de los mecanismos geométricos y operaciones conceptuales que lo Neutro utiliza para generar respectivamente condiciones de ‘soporte’, de ‘marco’ o de ‘campo’. Estas tres condiciones espaciales apuntan a un entendimiento de lo Neutro que asocia su fórmula de doble negación (ni lo uno, ni lo otro) con el estado más incipiente de la materia, aquel en el que todavía no es pero que contiene ya todos los elementos de posibilidad de ser: lo Neutro como potencia. Aquello que la lógica nos impide afirmar, que una cosa es y no-es al mismo tiempo, nos lo concede la categoría aristotélica de la potencia. Por ello, en la última parte de la tesis se desarrollan de forma más autónoma aquellos temas vinculados con estos tres tipos de espacio que se basan en la renuncia, abordando la cuestión de la potencialidad desde la negación. Partiendo de dos condiciones superficiales básicas, la del muro vertical y la del suelo horizontal, se analizan algunos discursos basados en una restricción radical a estos dos elementos. Así, el muro ciego encuentra su precedente artístico en el lienzo en blanco y la tabula rasa se pone en relación con el concepto de ‘terrain vague’. Por último, la posibilidad de toda potencia de no actualizarse lleva a explorar el no-hacer como renuncia voluntaria y su capacidad de transformarse en acto de resistencia. ABSTRACT The concern of this research is to test the feasibility of a term, a concept that has been detected as a potential tool to name some architectural operations. That concept is the Neutral. In front of the dualities system with which we enounce the real, the Neutral places itself, not so much in the midpoint as in the opposite point of both terms. It is not the conjunction of the contraries, but that which deflects the question itself that compels us to make a choice. Recognizing the multiple interpretations of the term in the common language, with each chapter, the thesis builds an expanded definition of the Neutral to configure a set of meanings applicable to architecture. The first of them is the one that derives from the concept of functional indifference. After an analysis of the theoretical context in which the two-way dialectic between form and function lies, its consolidation in the early twentieth century and its call into crisis by the introduction of multi-relational concepts such as 'environment' and 'system', the research suggests three study models: the Typical Plan, the Single Plan and the Equivalents Plan. In each of these cases, although the fundamental issue is programmatic neutrality, it also recognizes the presence of geometric mechanisms and conceptual operations that the Neutral uses to generate respectively 'support', 'frame' or 'field' conditions. These three spatial conditions point to an understanding of the Neutral which associates its double negation formula (neither one nor the other) with the most incipient state of matter, that in which it not yet is, but already contains every element of the possibility of being: the neutral as potentiality. That which logic does not allow us to affirm, that one thing is and is-not at the same time, it is granted by the Aristotelian category of potentiality. Therefore, in the last part of the thesis, the topics relating these three types of space which are based on renouncement are more autonomously developed, addressing the issue of potentiality from the standpoint of denial. Starting from two basic surface conditions, the vertical wall and the horizontal floor, some speeches based on a radical restriction on these two elements are analyzed. Thus, the blind wall finds its artistic precedent in the blank canvas and the tabula rasa attitude gets in relation to the concept of ‘terrain vague’. Finally, the possibility of any potency of not being updated leads to explore the not-doing as voluntary resignation and its ability to transform itself into an act of resistance.

Ni lo uno, ni lo otro: posibilidad de lo neutro en arquitectura

Esta investigación se ocupa de comprobar la viabilidad de un término, un concepto que se detecta como posible instrumento para nombrar algunas operaciones en arquitectura. Ese concepto es Lo Neutro. Frente al sistema de dualidades con el que enunciamos lo real, lo Neutro se sitúa, no tanto en el punto medio, como en el punto opuesto a ambos términos. No es la conjunción de contrarios, sino aquello que desvía la propia pregunta que obliga a elegir. Próximo a los conceptos de indefinición, indiferencia e inacción, lo Neutro se revela como un mecanismo fundamental de disolución. Reconociendo en la lengua varios sentidos para el término, con cada capítulo, la tesis va construyendo una definición ampliada de lo Neutro hasta configurar un conjunto de significados aplicables a la arquitectura. El primero de ellos es el que se deriva del concepto de indiferencia funcional. Tras un análisis del contexto teórico sobre el que se asienta la dialéctica bi-direccional forma-función, su consolidación en los primeros años del siglo XX y también su puesta en crisis mediante la introducción de nociones multi-relacionales como ‘entorno’ y ‘sistema’, la investigación propone tres modelos de estudio: la Planta Tipo, la Planta Única y la Planta de Equivalencias. En cada uno de estos casos, aunque el tema fundamental es la neutralidad programática, se reconoce también la presencia de los mecanismos geométricos y operaciones conceptuales que lo Neutro utiliza para generar respectivamente condiciones de ‘soporte’, de ‘marco’ o de ‘campo’. Estas tres condiciones espaciales apuntan a un entendimiento de lo Neutro que asocia su fórmula de doble negación (ni lo uno, ni lo otro) con el estado más incipiente de la materia, aquel en el que todavía no es pero que contiene ya todos los elementos de posibilidad de ser: lo Neutro como potencia. Aquello que la lógica nos impide afirmar, que una cosa es y no-es al mismo tiempo, nos lo concede la categoría aristotélica de la potencia. Por ello, en la última parte de la tesis se desarrollan de forma más autónoma aquellos temas vinculados con estos tres tipos de espacio que se basan en la renuncia, abordando la cuestión de la potencialidad desde la negación. Partiendo de dos condiciones superficiales básicas, la del muro vertical y la del suelo horizontal, se analizan algunos discursos basados en una restricción radical a estos dos elementos. Así, el muro ciego encuentra su precedente artístico en el lienzo en blanco y la tabula rasa se pone en relación con el concepto de ‘terrain vague’. Por último, la posibilidad de toda potencia de no actualizarse lleva a explorar el no-hacer como renuncia voluntaria y su capacidad de transformarse en acto de resistencia. ABSTRACT The concern of this research is to test the feasibility of a term, a concept that has been detected as a potential tool to name some architectural operations. That concept is the Neutral. In front of the dualities system with which we enounce the real, the Neutral places itself, not so much in the midpoint as in the opposite point of both terms. It is not the conjunction of the contraries, but that which deflects the question itself that compels us to make a choice. Recognizing the multiple interpretations of the term in the common language, with each chapter, the thesis builds an expanded definition of the Neutral to configure a set of meanings applicable to architecture. The first of them is the one that derives from the concept of functional indifference. After an analysis of the theoretical context in which the two-way dialectic between form and function lies, its consolidation in the early twentieth century and its call into crisis by the introduction of multi-relational concepts such as 'environment' and 'system', the research suggests three study models: the Typical Plan, the Single Plan and the Equivalents Plan. In each of these cases, although the fundamental issue is programmatic neutrality, it also recognizes the presence of geometric mechanisms and conceptual operations that the Neutral uses to generate respectively 'support', 'frame' or 'field' conditions. These three spatial conditions point to an understanding of the Neutral which associates its double negation formula (neither one nor the other) with the most incipient state of matter, that in which it not yet is, but already contains every element of the possibility of being: the neutral as potentiality. That which logic does not allow us to affirm, that one thing is and is-not at the same time, it is granted by the Aristotelian category of potentiality. Therefore, in the last part of the thesis, the topics relating these three types of space which are based on renouncement are more autonomously developed, addressing the issue of potentiality from the standpoint of denial. Starting from two basic surface conditions, the vertical wall and the horizontal floor, some speeches based on a radical restriction on these two elements are analyzed. Thus, the blind wall finds its artistic precedent in the blank canvas and the tabula rasa attitude gets in relation to the concept of ‘terrain vague’. Finally, the possibility of any potency of not being updated leads to explore the not-doing as voluntary resignation and its ability to transform itself into an act of resistance.

Acompañando e intensificando la información entrante en el explorar generativo

Los llamados procesos creativos en general, y los del proyectar arquitectónico en particular, mantienen aproximaciones hacia el objeto centradas principalmente en el procedimiento, es decir, en lo estratégico, lo metodológico o/y lo paradigmático. En ellas, además, el potencial de información no suele ser completo ni contemplado o, si lo ha sido, de manera inconciente, o referido de nuevo a lo procedimental. Igualmente, se centra el interés de estas aproximaciones, o en el objeto propuesto o resultado, o en lo procesal, pero sin atender a su constitución, es decir, a la información misma. Por tanto, y como reclama la física, la base constituyente informacional de estas aproximaciones, no ha sido considerada hasta ahora, ni se ha intentado sistematizar. Junto a esta omisión, estos acercamientos no permiten que cada humano configure de manera autónoma, independiente e íntegramente su propio proceso pues, los comentados procedimientos, están apoyados en marcos contextuales, culturales o/y procesales, reflejando así una orientación limitada en espacio-tiempo. Es así que se propone una potencia, o “aquellas cualidades poseídas por las cosas en cuya virtud éstas son totalmente impasibles o inmutables, o no se dejan cambiar fácilmente…”, según la define Aristóteles en “Metafísica”, como la posibilidad de, a la vez, aludir a un abanico informacional completo y apuntar hacia la íntegra elaboración de un proceso personal propio. Desde lo informacional, que a su vez es energético dependiendo de la disciplina científica considerada, se diferencian, en primer lugar, unos atributos o términos mínimos, que son unas potencias que compendian el abanico informacional completo. Es decir, son mínimos máximos. Estos atributos forman la fase cualitativa de la información a la que se llama acompañamiento. En segundo lugar, y apoyado tanto en el funcionamiento cerebral, en el espacio-tiempo cuántico, como en los nuevos experimentos e investigaciones de la biología, la física y la neurociencia en especial, se abren líneas nuevas para acceder a la información, antes contemplada de manera lineal, local y como entidad separada. Por ello, esta segunda aproximación presenta una potencia de intensificación de datos, que permite un aumento de los mismos en el cerebro y, por ello, la posibilidad de evitar el problema del “papel en blanco”. A esta fase se la nombra promoción. En tercer lugar, ambas fases constituyen la generación como propuesta de tesis, siendo la misma un cambio de cualquier tipo en el que alguien es agente de algo, específicamente, cuando un humano es mediador entre sucesos. Fusionando ambas, se añade simultáneamente una con-formación potencial global, que es sinérgicamente más que la suma de las dos anteriores. De esta manera agrupadora, y con objeto de materializar y sistematizar ahora esta generación o potencia, se presenta una puesta en práctica. Para ello, se desarrolla un modelo analítico-geométrico-paramétrico y se expone su aplicación en dicho caso práctico. Además, dicho modelo presenta un funcionamiento autorreferido u holográfico, reflejando tanto a los estudios científicos revisados, como al propio funcionamiento de los atributos o/y de todas las potencias que se presentan en esta investigación. ABSTRACT Generally speaking the so-called creative processes, and particularly those of the architectural design, keep approaches into the object oriented mainly in the process, so to speak, into the strategical, the methodological and/ or into the paradigmatic. In addition, they don’t usually take into account the potential of information neither in a complete manner nor even contemplated or, if considered, worked out unconsciously, or referred back to the procedural. Similarly, the interest of these approaches is focused either in the proposed object or the output, or in the processual, but leaving their constituent out, being it the information itself. Therefore, as physics is claiming nowadays, the constituent core of these approaches have neither been taken into account so far, nor tried to systematize. Along with this omission, these approaches do not allow each human being to set up autonomously, independently and entirely her/ his own process, because the mentioned procedures are supported by contextual, cultural and/ or procedural frameworks, reflecting then a perspective limited in space-time. Thus a potency is proposed, or "those qualities possessed by things under which they are totally impassive or immutable, or are not easily changed...", as defined by Aristotle in "Metaphysics", as the possibility to, and at the same time, alluding to a full informational range and point out to a whole development of an own personal process. From the informational stand, which in turn is energetic depending on the scientific discipline considered, it is distinguished, in the first place, a minimum set of attributes or terms, which are potencies that summarize the full informational range. That is, they are maximum minimums. These attributes build up the qualitative phase of the information being called accompaniment. Secondly, and supported in the brain functioning, in quantum space-time, as in new experiments and research carried out by biology, physics and neuroscience especially, new lines to access information are being opened, being contemplated linearly, locally and as a detached entity before. Thus, this second approach comes up with a potency of data`s intensifying that allows an increase in the brain thereof and, therefore, the possibility of avoiding the problem of "the blank paper". Promotion is how this phase is appointed. In the third place, both phases form the generation as the dissertation proposal, being it a change of any kind in which someone is the agent of something, specifically, when a human being is the mediator in between events. Fusing both of them, a global potential formation-with is added simultaneously, which is synergistically greater than the sum of the previous two. In this grouping way, and now in order to materialize and systemize this generation or potency, an implementation is displayed. To this end, an analytical-geometrical-parametrical model is developed and put into practice as a case study. In addition, this model features a self-referral or holographic functioning, being aligned to both scientific reviewed studies, and the very functioning either of the attributes and/ or all the potencies that are introduced in this research.

Entre: de repente, sin fin, lo innombrable

Etiquetamos para reducir nuestra incertidumbre; clasificamos en base a términos opuestos para poder entender. El universo y el espacio construido en particular, se explican desde una arquitectónica dual: dentro – fuera, arriba – abajo, derecha – izquierda, día – noche, hombre – mujer. Lo que nos diferencia como grupo, raza o género, distinguiendo los modos de vivir, las ideologías y las teorías, es la relación que establecemos entre los términos opuestos: entre lo individual y lo colectivo, lo privado y lo público, la imaginación y la realidad, la identidad y la otredad, el orden y el caos, el deseo y la saciedad. El afán clasificatorio dicotómico de la realidad que la modernidad lleva a su extremo, hiere la vida: la mutua exclusión de los opuestos elimina la distancia, el espacio-tiempo entre ambos, que es donde la vida se sitúa. La concepción dual que rige nuestro conocimiento contamina también la relación entre los arquitectos y la sociedad, siendo la causa del ambiente tóxico que envuelve al habitante que ya no se identifica con los lugares que habita. Puede que sea oportuno pasar del pensamiento binario a una lógica de lo intersticial que abandone la dualidad para instalarse en el Entre; los arquitectos serían mediadores entre el poder y la vida, sintiendo el Entre como el transcurso de la vida y la potencia de interacción: los lugares ambiguos e intermedios, es donde sucede el encuentro entre los términos, entre arquitectos y habitantes, entre la imaginación y la realidad, entre tú y yo. ABSTRACT We label to reduce our uncertainty; we classify based on opposites to understand. The universe and all constructed space in particular, are explained from a dual architecture: inside - out, up - down, right - left, day - night, male - female, black - white. What differentiate us as a group, race, gender, distinguishing the lifestyles, ideologies and theories, is how we draw the relation between the opposites: between the individual and the collective, private and public, imagination and reality , identity and otherness, order and disorder, desire and satiety. The dichotomous classification of reality that modernity carried to its extreme, hurts life: the mutual exclusion of opposites eliminates the distance, the space-time between the two, which is where life is located. The dual conception that governs our knowledge has also contaminated the relationship between architect and society, being the cause of the toxic environment that surrounds the inhabitant who no longer identify himself with the places he inhabit. It may be appropriate pass from binary thinking to a logic of the interstitial which abandon duality to settle in Between; architect then will be the mediator between authorities and life, sensing the Between as a flowing of life a potency of interaction rather than a separation between the extremes; ambiguous, in –between, places are where the encounter between opposites happens, between the architect and the inhabitant, between imagination and reality, between you and me.

A nerve growth factor mimetic TrkA antagonist causes withdrawal of cortical cholinergic boutons in the adult rat

Cholinergic neurons respond to the administration of nerve growth factor (NGF) in vivo with a prominent and selective increase of choline acetyl transferase activity. This suggests the possible involvement of endogenous NGF, acting through its receptor TrkA, in the maintenance of central nervous system cholinergic synapses in the adult rat brain. To test this hypothesis, a small peptide, C(92-96), that blocks NGF-TrkA interactions was delivered stereotactically into the rat cortex over a 2-week period, and its effect and potency were compared with those of an anti-NGF monoclonal antibody (mAb NGF30). Two presynaptic antigenic sites were studied by immunoreactivity, and the number of presynaptic sites was counted by using an image analysis system. Synaptophysin was used as a marker for overall cortical synapses, and the vesicular acetylcholine transporter was used as a marker for cortical cholinergic presynaptic sites. No significant variations in the number of synaptophysin-immunoreactive sites were observed. However, both mAb NGF30 and the TrkA antagonist C(92-96) provoked a significant decrease in the number and size of vesicular acetylcholine transporter–IR sites, with the losses being more marked in the C(92-96) treated rats. These observations support the notion that endogenously produced NGF acting through TrkA receptors is involved in the maintenance of the cholinergic phenotype in the normal, adult rat brain and supports the idea that NGF normally plays a role in the continual remodeling of neural circuits during adulthood. The development of neurotrophin mimetics with antagonistic and eventually agonist action may contribute to therapeutic strategies for central nervous system degeneration and trauma.

Modulation of nicotinic acetylcholine receptors by strychnine

Strychnine, a potent and selective antagonist at glycine receptors, was found to inhibit muscle (α1β1γδ, α1β1γ, and α1β1δ) and neuronal (α2β2 and α2β4) nicotinic acetylcholine receptors (AcChoRs) expressed in Xenopus oocytes. Strychnine alone (up to 500 μM) did not elicit membrane currents in oocytes expressing AcChoRs, but, when applied before, concomitantly, or during superfusion of acetylcholine (AcCho), it rapidly and reversibly inhibited the current elicited by AcCho (AcCho-current). Although in the three cases the AcCho-current was reduced to the same level, its recovery was slower when the oocytes were preincubated with strychnine. The amount of AcCho-current inhibition depended on the receptor subtype, and the order of blocking potency by strychnine was α1β1γδ > α2β4 > α2β2. With the three forms of drug application, the Hill coefficient was close to one, suggesting a single site for the receptor interaction with strychnine, and this interaction appears to be noncompetitive. The inhibitory effects on muscle AcChoRs were voltage-independent, and the apparent dissociation constant for AcCho was not appreciably changed by strychnine. In contrast, the inhibitory effects on neuronal AcChoRs were voltage-dependent, with an electrical distance of ≈0.35. We conclude that strychnine regulates reversibly and noncompetitively the embryonic type of muscle AcChoR and some forms of neuronal AcChoRs. In the former case, strychnine presumably inhibits allosterically the receptor by binding at an external domain whereas, in the latter case, it blocks the open receptor-channel complex.

NADH-quinone oxidoreductase: PSST subunit couples electron transfer from iron–sulfur cluster N2 to quinone

The proton-translocating NADH-quinone oxidoreductase (EC 1.6.99.3) is the largest and least understood enzyme complex of the respiratory chain. The mammalian mitochondrial enzyme (also called complex I) contains more than 40 subunits, whereas its structurally simpler bacterial counterpart (NDH-1) in Paracoccus denitrificans and Thermus thermophilus HB-8 consists of 14 subunits. A major unsolved question is the location and mechanism of the terminal electron transfer step from iron–sulfur cluster N2 to quinone. Potent inhibitors acting at this key region are candidate photoaffinity probes to dissect NADH-quinone oxidoreductases. Complex I and NDH-1 are very sensitive to inhibition by a variety of structurally diverse toxicants, including rotenone, piericidin A, bullatacin, and pyridaben. We designed (trifluoromethyl)diazirinyl[3H]pyridaben ([3H]TDP) as our photoaffinity ligand because it combines outstanding inhibitor potency, a suitable photoreactive group, and tritium at high specific activity. Photoaffinity labeling of mitochondrial electron transport particles was specific and saturable. Isolation, protein sequencing, and immunoprecipitation identified the high-affinity specifically labeled 23-kDa subunit as PSST of complex I. Immunoprecipitation of labeled membranes of P. denitrificans and T. thermophilus established photoaffinity labeling of the equivalent bacterial NQO6. Competitive binding and enzyme inhibition studies showed that photoaffinity labeling of the specific high-affinity binding site of PSST is exceptionally sensitive to each of the high-potency inhibitors mentioned above. These findings establish that the homologous PSST of mitochondria and NQO6 of bacteria have a conserved inhibitor-binding site and that this subunit plays a key role in electron transfer by functionally coupling iron–sulfur cluster N2 to quinone.

Proteinase-activated receptor 2 (PAR2)-activating peptides: Identification of a receptor distinct from PAR2 that regulates intestinal transport

The effects of PAR2-activating PAR2-activating peptides, SLIGRL (SL)-NH2, and trans-cinnamoyl-LIGRLO (tc)-NH2 were compared with the action of trypsin, thrombin, and the PAR1 selective-activating peptide: Ala-parafluoroPhe-Arg-cyclohexylAla-Citrulline-Tyr (Cit)-NH2 for stimulating intestinal ion transport. These agonists were added to the serosa of stripped rat jejunum segments mounted in Ussing chambers, and short circuit current (Isc) was used to monitor active ion transport. The relative potencies of these agonists also were evaluated in two bioassays specific for the activation of rat PAR2: a cloned rat PAR2 cell calcium-signaling assay (PAR2-KNRK cells) and an aorta ring relaxation (AR) assay. In the Isc assay, all agonists, except thrombin, induced an Isc increase. The SL-NH2-induced Isc changes were blocked by indomethacin but not by tetrodotoxin. The relative potencies of the agonists in the Isc assay (trypsin≫SL-NH2>tc-NH2>Cit-NH2) were strikingly different from their relative potencies in the cloned PAR2-KNRK cell calcium assay (trypsin≫>tc-NH2 ≅ SL-NH2≫>Cit-NH2) and in the AR assay (trypsin≫>tc-NH2 ≅ SL-NH2). Furthermore, all agonists were maximally active in the PAR2-KNRK cell and AR assays at concentrations that were one (PAR2 -activating peptides) or two (trypsin) orders of magnitude lower than those required to activate intestinal transport. Based on the distinct potency profile for these agonists and the considerable differences in the concentration ranges required to induce an Isc effect in the intestinal assay compared with the PAR2-KNRK and AR assays, we conclude that a proteinase-activated receptor, pharmacologically distinct from PAR2 and PAR1, is present in rat jejunum and regulates intestinal transport via a prostanoid-mediated mechanism.

A hypothalamic follicle-stimulating hormone-releasing decapeptide in the rat

Previous studies indicated that there is a separate hypothalamic control of follicle-stimulating hormone (FSH) release distinct from that of luteinizing hormone (LH). An FSH-releasing factor (FSHRF) was purified from rat and sheep hypothalami, but has not been isolated. We hypothesized that FSHRF might be an analogue of mammalian luteinizing hormone-releasing hormone (m-LHRH) and evaluated the activity of many analogues of m-LHRH and of the known LHRHs found in lower forms. Here we demonstrate that lamprey (l) LHRH-III has a potent, dose-related FSH- but not LH-releasing action on incubated hemipituitaries of male rats. l-LHRH-I on the other hand, had little activity to release either FSH or LH. m-LHRH was equipotent to l-LHRH-III to release FSH, but also had a high potency to release LH in contrast to l-LHRH-III that selectively released FSH. Chicken LHRH-II had considerable potency to release both LH and FSH, but no selectivity in its action. Salmon LHRH had much less potency than the others tested, except for l-LHRH-I, and no selectivity in its action. Because ovariectomized, estrogen, progesterone-treated rats are a sensitive in vivo assay for FSH- and LH-releasing activity, we evaluated l-LHRH-III in this assay and found that it had a completely selective stimulatory effect on FSH release at the two doses tested (10 and 100 pmols). Therefore, l-LHRH-III is a highly potent and specific FSH-releasing peptide that may enhance fertility in animals and humans. It may be the long sought after m-FSHRF.

Inhibition of dipeptidyl peptidase IV by fluoroolefin-containing N-peptidyl-O-hydroxylamine peptidomimetics

Dipeptidyl peptidase IV (EC 3.4.14.5; DPP IV), also known as the leukocyte differentiation antigen CD26 when found as an extracellular membrane-bound proline specific serine protease, cleaves a dipeptide from the N terminus of a polypeptide chain containing a proline residue in the penultimate position. Here we report that known (Z)-Ala-ψ[CF=C]-Pro dipeptide isosteres 1 and 2, which contain O-acylhydroxylamines, were isolated as diastereomeric pairs u-1, l-1, and l-2. The effect of each diastereomeric pair as an inhibitor of human placental dipeptidyl peptidase DPP IV has been examined. The inhibition of DPP IV by these compounds is rapid and efficient. The diastereomeric pair u-1 exhibits very potent inhibitory activity with a Ki of 188 nM. Fluoroolefin containing N-peptidyl-O-hydroxylamine peptidomimetics, by virtue of their inhibitory potency and stability, are superior to N-peptidyl-O-hydroxylamine inhibitors derived from an Ala-Pro dipeptide.

Activation of the orphan nuclear receptor steroidogenic factor 1 by oxysterols

Steroidogenic factor 1 (SF-1), an orphan member of the intracellular receptor superfamily, plays an essential role in the development and function of multiple endocrine organs. It is expressed in all steroidogenic tissues where it regulates the P450 steroidogenic genes to generate physiologically active steroids. Although many of the functions of SF-1 in vivo have been defined, an unresolved question is whether a ligand modulates its transcriptional activity. Here, we show that 25-, 26-, or 27-hydroxycholesterol, known suppressors of cholesterol biosynthesis, enhance SF-1-dependent transcriptional activity. This activation is dependent upon the SF-1 activation function domain, and, is specific for SF-1 as several other receptors do not respond to these molecules. The oxysterols activate at concentrations comparable to those previously shown to inhibit cholesterol biosynthesis, and, can be derived from cholesterol by P450c27, an enzyme expressed within steroidogenic tissues. Recent studies have shown that the nuclear receptor LXR also is activated by oxysterols. We demonstrate that different oxysterols differ in their rank order potency for these two receptors, with 25-hydroxycholesterol preferentially activating SF-1 and 22(R)-hydroxycholesterol preferentially activating LXR. These results suggest that specific oxysterols may mediate transcriptional activation via different intracellular receptors. Finally, ligand-dependent transactivation of SF-1 by oxysterols may play an important role in enhancing steroidogenesis in vivo.