Prolonged amelioration of acute lung allograft rejection by overexpression of human interleukin-10 under control of a long acting ubiquitin C promoter in rats

BACKGROUND: The prolonged effect of electroporation-mediated human interleukin-10 (hIL-10) overexpression in skeletal muscle under the control of the constitutional polyubiquitin C promoter (pUb hIL-10) on rat lung allograft rejection was evaluated. METHODS: Left lung allotransplantation was performed from Brown-Norway to Fischer-F344 rats. Either 2.5 mug pCIK hIL-10 (hIL-10/cytomegalovirus early promoter enhancer) alone (Group I/sacrifice Day 5 and II/sacrifice Day 10) or in combination with 2.5 mug pUb hIL-10 (hIL-10/UbC promoter; Group III/sacrifice Day 10) were injected into the tibialis anterior muscle of the recipient, followed by electroporation 24 hours before transplantation. Animals in Control Groups IV and V without gene transfer were euthanized on Day 5 and 10, respectively. All animals received a daily non-therapeutic dose of cyclosporine A (2.5 mg/kg). RESULTS: In Control Group IV, complete rejection (median A3B3) was noted on Day 5 with a Pao(2) of 43 +/- 9 mm Hg. In recipients of Control Group V, measurement of gas exchange on Day 10 and rejection grading was impossible because of complete destruction of the allograft. Group I animals on Day 5 (233 +/- 123 mm Hg; p = 0.02 vs Group IV) and Group II animals on Day 10 (150 +/- 139 mm Hg; p = 0.15 vs Group IV) demonstrated improved graft function. Graft function in Group III was further improved on Day 10 (299 +/- 123 mm Hg; p = 0.002 vs Group IV; p = 0.05 vs Group II; p = 0.36 vs Group I). Rejection was significantly reduced in Group III (median, A2B2) compared with Group II (median, A4B3; p < 0.05). CONCLUSIONS: Interleukin-10 overexpression under control of the constitutive ubiquitin C promoter ameliorates acute rejection and preserves lung graft function for a prolonged time.

TA-RE: An Exchange Language for Mining Software Repositories

Software repositories have been getting a lot of attention from researchers in recent years. In order to analyze software repositories, it is necessary to first extract raw data from the version control and problem tracking systems. This poses two challenges: (1) extraction requires a non-trivial effort, and (2) the results depend on the heuristics used during extraction. These challenges burden researchers that are new to the community and make it difficult to benchmark software repository mining since it is almost impossible to reproduce experiments done by another team. In this paper we present the TA-RE corpus. TA-RE collects extracted data from software repositories in order to build a collection of projects that will simplify extraction process. Additionally the collection can be used for benchmarking. As the first step we propose an exchange language capable of making sharing and reusing data as simple as possible.

Hsp110 chaperones control client fate determination in the hsp70-Hsp90 chaperone system.

Heat shock protein 70 (Hsp70) plays a central role in protein homeostasis and quality control in conjunction with other chaperone machines, including Hsp90. The Hsp110 chaperone Sse1 promotes Hsp90 activity in yeast, and functions as a nucleotide exchange factor (NEF) for cytosolic Hsp70, but the precise roles Sse1 plays in client maturation through the Hsp70-Hsp90 chaperone system are not fully understood. We find that upon pharmacological inhibition of Hsp90, a model protein kinase, Ste11DeltaN, is rapidly degraded, whereas heterologously expressed glucocorticoid receptor (GR) remains stable. Hsp70 binding and nucleotide exchange by Sse1 was required for GR maturation and signaling through endogenous Ste11, as well as to promote Ste11DeltaN degradation. Overexpression of another functional NEF partially compensated for loss of Sse1, whereas the paralog Sse2 fully restored GR maturation and Ste11DeltaN degradation. Sse1 was required for ubiquitinylation of Ste11DeltaN upon Hsp90 inhibition, providing a mechanistic explanation for its role in substrate degradation. Sse1/2 copurified with Hsp70 and other proteins comprising the "early-stage" Hsp90 complex, and was absent from "late-stage" Hsp90 complexes characterized by the presence of Sba1/p23. These findings support a model in which Hsp110 chaperones contribute significantly to the decision made by Hsp70 to fold or degrade a client protein.

Electrophysiological indices of response inhibition in a Go/NoGo task predict self-control in a social context.

Recent research demonstrates that response inhibition-a core executive function-may subserve self-regulation and self-control. However, it is unclear whether response inhibition also predicts self-control in the multifaceted, high-level phenomena of social decision-making. Here we examined whether electrophysiological indices of response inhibition would predict self-control in a social context. Electroencephalography was recorded as participants completed a widely used Go/NoGo task (the cued Continuous Performance Test). Participants then interacted with a partner in an economic exchange game that requires self-control. Results demonstrated that greater NoGo-Anteriorization and larger NoGo-P300 peak amplitudes-two established electrophysiological indices of response inhibition-both predicted more self-control in this social game. These findings support continued integration of executive function and self-regulation and help extend prior research into social decision-making processes.

Molecular control of cardiac sodium homeostasis in health and disease.

INTRODUCTION Cardiac myocytes utilize three high-capacity Na transport processes whose precise function can determine myocyte fate and the triggering of arrhythmias in pathological settings. We present recent results on the regulation of all three transporters that may be important for an understanding of cardiac function during ischemia/reperfusion episodes. METHODS AND RESULTS Refined ion selective electrode (ISE) techniques and giant patch methods were used to analyze the function of cardiac Na/K pumps, Na/Ca exchange (NCX1), and Na/H exchange (NHE1) in excised cardiac patches and intact myocytes. To consider results cohesively, simulations were developed that account for electroneutrality of the cytoplasm, ion homeostasis, water homeostasis (i.e., cell volume), and cytoplasmic pH. The Na/K pump determines the average life-time of Na ions (3-10 minutes) as well as K ions (>30 minutes) in the cytoplasm. The long time course of K homeostasis can determine the time course of myocyte volume changes after ion homeostasis is perturbed. In excised patches, cardiac Na/K pumps turn on slowly (-30 seconds) with millimolar ATP dependence, when activated for the first time. In steady state, however, pumps are fully active with <0.2 mM ATP and are nearly unaffected by high ADP (2 mM) and Pi (10 mM) concentrations as may occur in ischemia. NCX1s appear to operate with slippage that contributes to background Na influx and inward current in heart. Thus, myocyte Na levels may be regulated by the inactivation reactions of the exchanger which are both Na- and proton-dependent. NHE1 also undergo strong Na-dependent inactivation, whereby a brief rise of cytoplasmic Na can cause inactivation that persists for many minutes after cytoplasmic Na is removed. This mechanism is blocked by pertussis toxin, suggesting involvement of a Na-dependent G-protein. Given that maximal NCX1- and NHE1-mediated ion fluxes are much greater than maximal Na/K pump-mediated Na extrusion in myocytes, the Na-dependent inactivation mechanisms of NCX1 and NHE1 may be important determinants of cardiac Na homeostasis. CONCLUSIONS Na/K pumps appear to be optimized to continue operation when energy reserves are compromised. Both NCX1 and NHE1 activities are regulated by accumulation of cytoplasmic Na. These principles may importantly control cardiac cytoplasmic Na and promote myocyte survival during ischemia/reperfusion episodes by preventing Ca overload.

Outcomes after early administration of plasma exchange in pediatric central nervous system inflammatory demyelination.

The use of plasma exchange has been described in steroid-refractory central nervous system inflammatory demyelination in adults, but less has been published regarding its use in children and adolescents. We describe 12 children treated with plasma exchange for acute severe central nervous system inflammatory demyelination. The clinical attack leading to plasma exchange included symptomatic spinal cord lesions in 10 and symptomatic brainstem lesions in 2 children. Diagnosis was acute transverse myelitis in 6, relapsing-remitting multiple sclerosis in 5, and acute disseminated encephalomyelitis in 1 child. Adverse events related to plasma exchange necessitating intervention were observed in 3 children. Median Expanded Disability Status Scale score at plasma exchange start was 7.5 (range 4-9.5). At 3 months, 7 children were ambulatory without aid (Expanded Disability Status Scale score of ≤4). This retrospective study suggests that plasma exchange can be effective in ameliorating symptoms in severe pediatric central nervous system inflammatory demyelination, although lack of randomization or control group limits the ability to attribute this outcome entirely to plasma exchange.

Monetary and Exchange Rate Policy in Malaysia before the Asian Crisis

This paper provides a case study to characterize the monetary policy regime in Malaysia, from a medium- and long-term perspective. Specifically, we ask how the central bank of Malaysia, Bank Negara Malaysia (BNM), has structured its monetary policy regime, and how it has conducted monetary and exchange rate policy under the regime. By conducting three empirical analyses, we characterize the monetary and exchange rate policy regime in Malaysia by three intermediate solutions on three vectors: the degree of autonomy in monetary policy, the degree of variability of the exchange rate, and the degree of capital mobility.

Contributions to trajectory prediction theory and its application to arrival management for air traffic control

La gestión del tráfico aéreo (Air Traffic Management, ATM) está experimentando un cambio de paradigma hacia las denominadas operaciones basadas trayectoria. Bajo dicho paradigma se modifica el papel de los controladores de tráfico aéreo desde una operativa basada su intervención táctica continuada hacia una labor de supervisión a más largo plazo. Esto se apoya en la creciente confianza en las soluciones aportadas por las herramientas automatizadas de soporte a la decisión más modernas. Para dar soporte a este concepto, se precisa una importante inversión para el desarrollo, junto con la adquisición de nuevos equipos en tierra y embarcados, que permitan la sincronización precisa de la visión de la trayectoria, basada en el intercambio de información entre ambos actores. Durante los últimos 30 a 40 años las aerolíneas han generado uno de los menores retornos de la inversión de entre todas las industrias. Sin beneficios tangibles, la industria aérea tiene dificultades para atraer el capital requerido para su modernización, lo que retrasa la implantación de dichas mejoras. Esta tesis tiene como objetivo responder a la pregunta de si las capacidades actualmente instaladas en las aeronaves comerciales se pueden aplicar para lograr la sincronización de la trayectoria con el nivel de calidad requerido. Además, se analiza en ella si, conjuntamente con mejoras en las herramientas de predicción trayectorias instaladas en tierra en para facilitar la gestión de las arribadas, dichas capacidades permiten obtener los beneficios esperados en el marco de las operaciones basadas en trayectoria. Esto podría proporcionar un incentivo para futuras actualizaciones de la aviónica que podrían llevar a mejoras adicionales. El concepto operacional propuesto en esta tesis tiene como objetivo permitir que los aviones sean pilotados de una manera consistente con las técnicas actuales de vuelo optimizado. Se permite a las aeronaves que desciendan en el denominado “modo de ángulo de descenso gestionado” (path-managed mode), que es el preferido por la mayoría de las compañías aéreas, debido a que conlleva un reducido consumo de combustible. El problema de este modo es que en él no se controla de forma activa el tiempo de llegada al punto de interés. En nuestro concepto operacional, la incertidumbre temporal se gestiona en mediante de la medición del tiempo en puntos estratégicamente escogidos a lo largo de la trayectoria de la aeronave, y permitiendo la modificación por el control de tierra de la velocidad de la aeronave. Aunque la base del concepto es la gestión de las ordenes de velocidad que se proporcionan al piloto, para ser capaces de operar con los niveles de equipamiento típicos actualmente, dicho concepto también constituye un marco en el que la aviónica más avanzada (por ejemplo, que permita el control por el FMS del tiempo de llegada) puede integrarse de forma natural, una vez que esta tecnología este instalada. Además de gestionar la incertidumbre temporal a través de la medición en múltiples puntos, se intenta reducir dicha incertidumbre al mínimo mediante la mejora de las herramienta de predicción de la trayectoria en tierra. En esta tesis se presenta una novedosa descomposición del proceso de predicción de trayectorias en dos etapas. Dicha descomposición permite integrar adecuadamente los datos de la trayectoria de referencia calculada por el Flight Management System (FMS), disponibles usando Futuro Sistema de Navegación Aérea (FANS), en el sistema de predicción de trayectorias en tierra. FANS es un equipo presente en los aviones comerciales de fuselaje ancho actualmente en la producción, e incluso algunos aviones de fuselaje estrecho pueden tener instalada avionica FANS. Además de informar automáticamente de la posición de la aeronave, FANS permite proporcionar (parte de) la trayectoria de referencia en poder de los FMS, pero la explotación de esta capacidad para la mejora de la predicción de trayectorias no se ha estudiado en profundidad en el pasado. La predicción en dos etapas proporciona una solución adecuada al problema de sincronización de trayectorias aire-tierra dado que permite la sincronización de las dimensiones controladas por el sistema de guiado utilizando la información de la trayectoria de referencia proporcionada mediante FANS, y también facilita la mejora en la predicción de las dimensiones abiertas restantes usado un modelo del guiado que explota los modelos meteorológicos mejorados disponibles en tierra. Este proceso de predicción de la trayectoria de dos etapas se aplicó a una muestra de 438 vuelos reales que realizaron un descenso continuo (sin intervención del controlador) con destino Melbourne. Dichos vuelos son de aeronaves del modelo Boeing 737-800, si bien la metodología descrita es extrapolable a otros tipos de aeronave. El método propuesto de predicción de trayectorias permite una mejora en la desviación estándar del error de la estimación del tiempo de llegada al punto de interés, que es un 30% menor que la que obtiene el FMS. Dicha trayectoria prevista mejorada se puede utilizar para establecer la secuencia de arribadas y para la asignación de las franjas horarias para cada aterrizaje (slots). Sobre la base del slot asignado, se determina un perfil de velocidades que permita cumplir con dicho slot con un impacto mínimo en la eficiencia del vuelo. En la tesis se propone un nuevo algoritmo que determina las velocidades requeridas sin necesidad de un proceso iterativo de búsqueda sobre el sistema de predicción de trayectorias. El algoritmo se basa en una parametrización inteligente del proceso de predicción de la trayectoria, que permite relacionar el tiempo estimado de llegada con una función polinómica. Resolviendo dicho polinomio para el tiempo de llegada deseado, se obtiene de forma natural el perfil de velocidades optimo para cumplir con dicho tiempo de llegada sin comprometer la eficiencia. El diseño de los sistemas de gestión de arribadas propuesto en esta tesis aprovecha la aviónica y los sistemas de comunicación instalados de un modo mucho más eficiente, proporcionando valor añadido para la industria. Por tanto, la solución es compatible con la transición hacia los sistemas de aviónica avanzados que están desarrollándose actualmente. Los beneficios que se obtengan a lo largo de dicha transición son un incentivo para inversiones subsiguientes en la aviónica y en los sistemas de control de tráfico en tierra. ABSTRACT Air traffic management (ATM) is undergoing a paradigm shift towards trajectory based operations where the role of an air traffic controller evolves from that of continuous intervention towards supervision, as decision making is improved based on increased confidence in the solutions provided by advanced automation. To support this concept, significant investment for the development and acquisition of new equipment is required on the ground as well as in the air, to facilitate the high degree of trajectory synchronisation and information exchange required. Over the past 30-40 years the airline industry has generated one of the lowest returns on invested capital among all industries. Without tangible benefits realised, the airline industry may find it difficult to attract the required investment capital and delay acquiring equipment needed to realise the concept of trajectory based operations. In response to these challenges facing the modernisation of ATM, this thesis aims to answer the question whether existing aircraft capabilities can be applied to achieve sufficient trajectory synchronisation and improvements to ground-based trajectory prediction in support of the arrival management process, to realise some of the benefits envisioned under trajectory based operations, and to provide an incentive for further avionics upgrades. The proposed operational concept aims to permit aircraft to operate in a manner consistent with current optimal aircraft operating techniques. It allows aircraft to descend in the fuel efficient path managed mode as preferred by a majority of airlines, with arrival time not actively controlled by the airborne automation. The temporal uncertainty is managed through metering at strategically chosen points along the aircraft’s trajectory with primary use of speed advisories. While the focus is on speed advisories to support all aircraft and different levels of equipage, the concept also constitutes a framework in which advanced avionics as airborne time-of-arrival control can be integrated once this technology is widely available. In addition to managing temporal uncertainty through metering at multiple points, this temporal uncertainty is minimised by improving the supporting trajectory prediction capability. A novel two-stage trajectory prediction process is presented to adequately integrate aircraft trajectory data available through Future Air Navigation Systems (FANS) into the ground-based trajectory predictor. FANS is standard equipment on any wide-body aircraft in production today, and some single-aisle aircraft are easily capable of being fitted with FANS. In addition to automatic position reporting, FANS provides the ability to provide (part of) the reference trajectory held by the aircraft’s Flight Management System (FMS), but this capability has yet been widely overlooked. The two-stage process provides a ‘best of both world’s’ solution to the air-ground synchronisation problem by synchronising with the FMS reference trajectory those dimensions controlled by the guidance mode, and improving on the prediction of the remaining open dimensions by exploiting the high resolution meteorological forecast available to a ground-based system. The two-stage trajectory prediction process was applied to a sample of 438 FANS-equipped Boeing 737-800 flights into Melbourne conducting a continuous descent free from ATC intervention, and can be extrapolated to other types of aircraft. Trajectories predicted through the two-stage approach provided estimated time of arrivals with a 30% reduction in standard deviation of the error compared to estimated time of arrival calculated by the FMS. This improved predicted trajectory can subsequently be used to set the sequence and allocate landing slots. Based on the allocated landing slot, the proposed system calculates a speed schedule for the aircraft to meet this landing slot at minimal flight efficiency impact. A novel algorithm is presented that determines this speed schedule without requiring an iterative process in which multiple calls to a trajectory predictor need to be made. The algorithm is based on parameterisation of the trajectory prediction process, allowing the estimate time of arrival to be represented by a polynomial function of the speed schedule, providing an analytical solution to the speed schedule required to meet a set arrival time. The arrival management solution proposed in this thesis leverages the use of existing avionics and communications systems resulting in new value for industry for current investment. The solution therefore supports a transition concept from mixed equipage towards advanced avionics currently under development. Benefits realised under this transition may provide an incentive for ongoing investment in avionics.

Control of phosphatidylserine biosynthesis through phosphatidylserine-mediated inhibition of phosphatidylserine synthase I in Chinese hamster ovary cells

Phosphatidylserine (PtdSer) synthesis in Chinese hamster ovary (CHO) cells occurs through the exchange of l-serine with the base moiety of phosphatidylcholine or phosphatidylethanolamine. The synthesis is depressed on the addition of PtdSer to the culture medium. A CHO cell mutant named mutant 29, whose PtdSer biosynthesis is highly resistant to this depression by exogenous PtdSer, has been isolated from CHO-K1 cells. In the present study, the PtdSer-resistant PtdSer biosynthesis in the mutant was traced to a point mutation in the PtdSer synthase I gene, pssA, resulting in the replacement of Arg-95 of the synthase by lysine. Introduction of the mutant pssA cDNA, but not the wild-type pssA cDNA, into CHO-K1 cells induced the PtdSer-resistant PtdSer biosynthesis. In a cell-free system, the serine base-exchange activity of the wild-type pssA-transfected cells was inhibited by PtdSer, but that of the mutant pssA-transfected cells was resistant to the inhibition. Like the mutant 29 cells, the mutant pssA-transfected cells grown without exogenous PtdSer exhibited an ≈2-fold increase in the cellular PtdSer level compared with that in CHO-K1 cells, although the wild-type pssA-transfected cells did not exhibit such a significant increase. These results indicated that the inhibition of PtdSer synthase I by PtdSer is essential for the maintenance of a normal PtdSer level in CHO-K1 cells and that Arg-95 of the synthase is a crucial residue for the inhibition.

A Rap guanine nucleotide exchange factor enriched highly in the basal ganglia

Ras proteins, key regulators of growth, differentiation, and malignant transformation, recently have been implicated in synaptic function and region-specific learning and memory functions in the brain. Rap proteins, members of the Ras small G protein superfamily, can inhibit Ras signaling through the Ras/Raf-1/mitogen-activated protein (MAP) kinase pathway or, through B-Raf, can activate MAP kinase. Rap and Ras proteins both can be activated through guanine nucleotide exchange factors (GEFs). Many Ras GEFs, but to date only one Rap GEF, have been identified. We now report the cloning of a brain-enriched gene, CalDAG-GEFI, which has substrate specificity for Rap1A, dual binding domains for calcium (Ca2+) and diacylglycerol (DAG), and enriched expression in brain basal ganglia pathways and their axon-terminal regions. Expression of CalDAG-GEFI activates Rap1A and inhibits Ras-dependent activation of the Erk/MAP kinase cascade in 293T cells. Ca2+ ionophore and phorbol ester strongly and additively enhance this Rap1A activation. By contrast, CalDAG-GEFII, a second CalDAG-GEF family member that we cloned and found identical to RasGRP [Ebinu, J. O., Bottorff, D. A., Chan, E. Y. W., Stang, S. L., Dunn, R. J. & Stone, J. C. (1998) Science 280, 1082–1088], exhibits a different brain expression pattern and fails to activate Rap1A, but activates H-Ras, R-Ras, and the Erk/MAP kinase cascade under Ca2+ and DAG modulation. We propose that CalDAG-GEF proteins have a critical neuronal function in determining the relative activation of Ras and Rap1 signaling induced by Ca2+ and DAG mobilization. The expression of CalDAG-GEFI and CalDAG-GEFII in hematopoietic organs suggests that such control may have broad significance in Ras/Rap regulation of normal and malignant states.

Controlling small guanine–nucleotide-exchange factor function through cytoplasmic RNA intramers

ADP-ribosylation factor (ARF) GTPases and their regulatory proteins have been implicated in the control of diverse biological functions. Two main classes of positive regulatory elements for ARF have been discovered so far: the large Sec7/Gea and the small cytohesin/ARNO families, respectively. These proteins harbor guanine–nucleotide-exchange factor (GEF) activity exerted by the common Sec7 domain. The availability of a specific inhibitor, the fungal metabolite brefeldin A, has enabled documentation of the involvement of the large GEFs in vesicle transport. However, because of the lack of such tools, the biological roles of the small GEFs have remained controversial. Here, we have selected a series of RNA aptamers that specifically recognize the Sec7 domain of cytohesin 1. Some aptamers inhibit guanine–nucleotide exchange on ARF1, thereby preventing ARF activation in vitro. Among them, aptamer M69 exhibited unexpected specificity for the small GEFs, because it does not interact with or inhibit the GEF activity of the related Gea2-Sec7 domain, a member of the class of large GEFs. The inhibitory effect demonstrated in vitro clearly is observed as well in vivo, based on the finding that M69 produces similar results as a dominant-negative, GEF-deficient mutant of cytohesin 1: when expressed in the cytoplasm of T-cells, M69 reduces stimulated adhesion to intercellular adhesion molecule-1 and results in a dramatic reorganization of F-actin distribution. These highly specific cellular effects suggest that the ARF-GEF activity of cytohesin 1 plays an important role in cytoskeletal remodeling events of lymphoid cells.

Rapid activation of Na+/H+ exchange by aldosterone in renal epithelial cells requires Ca2+ and stimulation of a plasma membrane proton conductance.

There is increasing evidence for an additional acute, nongenomic action of the mineralocorticoid hormone aldosterone on renal epithelial cells, leading to a two-step model of mineralocorticoid action on electrolyte excretion. We investigated the acute effect of aldosterone on intracellular free Ca2+ and on intracellular pH in an aldosterone-sensitive Madin-Darby canine kidney cell clone. Within seconds of application of aldosterone, but not of the glucocorticoid hydrocortisone, there was a 3-fold sustained increase of intracellular Ca2+ at a half-maximal concentration of 10(-10) mol/liter. Omission of extracellular Ca2+ prevented this hormone response. In the presence of extracellular Ca2+ aldosterone led to intracellular alkalinization. The Na+/H+ exchange inhibitor ethyl-isopropanol-amiloride (EIPA) prevented the aldosterone-induced alkalinization but not the aldosterone-induced increase of intracellular Ca2+. Omission of extracellular Ca2+ also prevented aldosterone-induced alkalinization. Instead, aldosterone led to a Zn(2+)-dependent intracellular acidification in the presence of EIPA, indicative of an increase of plasma membrane proton conductance. Under control conditions, Zn2+ prevented the aldosterone-induced alkalinization completely. We conclude that aldosterone stimulated net-entry of Ca2+ from the extracellular compartment and a plasma membrane H+ conductance as prerequisites for the stimulation of plasma membrane Na+/H+ exchange which in turn modulates K+ channel acitivity. It is probable that the aldosterone-sensitive H+ conductance maintains Na+/H+ exchange activity by providing an acidic environment in the vicinity of the exchanger. Thus, genomic action of aldosterone determines cellular transport equipment, whereas the nongenomic action regulates transporter activity that requires responses within seconds or minutes, which explains the rapid effects on electrolyte excretion.

31P NMR magnetization transfer study of the control of ATP turnover in Saccharomyces cerevisiae.

31P NMR magnetization transfer measurements have been used to measure the steady state flux between Pi and ATP in yeast cells genetically modified to overexpress an adenine nucleotide translocase isoform. An increase in Pi -> ATP flux and apparent ratio of moles of ATP synthesized/atoms of oxygen consumed (P:O ratio), when these cells were incubated with glucose, demonstrated that the reactions catalyzed by the translocase and F1F0 ATP synthase were readily reversible in vivo. However, when the same cells were incubated with ethanol alone, translocase overexpression had no effect on the measured Pi -> ATP flux or apparent P:O ratio, suggesting that the synthase was now operating irreversibly. This change was accompanied by an increase in the intracellular ADP concentration. These observations are consistent with a model proposed for the kinetic control of mitochondrial ATP synthesis, which was based on isotope exchange measurements with isolated mammalian mitochondria [LaNoue, K. F., Jeffries, F. M. H. & Radda, G. K. (1986) Biochemistry 25, 7667-7675].

A human RNase E-like activity that cleaves RNA sequences involved in mRNA stability control.

We have detected an endoribonucleolytic activity in human cell extracts that processes the Escherichia coli 9S RNA and outer membrane protein A (ompA) mRNA with the same specificity as RNase E from E. coli. The human enzyme was partially purified by ion-exchange chromatography, and the active fractions contained a protein that was detected with antibodies shown to recognize E. coli RNase E. RNA containing four repeats of the destabilizing motif AUUUA and RNA from the 3' untranslated region of human c-myc mRNA were also found to be cleaved by E. coli RNase E and its human counterpart in a fashion that may suggest a role of this activity in mammalian mRNA decay. It was also found that RNA containing more than one AUUUA motif was cleaved more efficiently than RNA with only one or a mutated motif. This finding of a eukaryotic endoribonucleolytic activity corresponding to RNase E indicates an evolutionary conservation of the components of mRNA degradation systems.