952 resultados para Illinois. Dept. of Professional Regulation.
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To answer the question whether increased energy demand resulting from myocyte hypertrophy and enhanced $\beta$-myosin heavy chain mRNA, contractile protein synthesis and assembly leads to mitochondrial proliferation and differentiation, we set up an electrical stimulation model of cultured neonatal rat cardiac myocytes. We describe, as a result of increased contractile activity, increased mitochondrial profiles, cytochrome oxidase mRNA, and activity, as well as a switch in mitochondrial carnitine palmitoyltransferase-I (CPT-I) from the liver to muscle isoform. We investigate physiological pathways that lead to accumulation of gene transcripts for nuclear encoded mitochondrial proteins in the heart. Cardiomyocytes were stimulated for varying times up to 72 hr in serum-free culture. The mRNA contents for genes associated with transcriptional activation (c-fos, c-jun, junB, nuclear respiratory factor 1 (Nrf-1)), mitochondrial proliferation (cytochrome c (Cyt c), cytochrome oxidase), and mitochondrial differentiation (carnitine palmitonyltransferase I (CPT-I) isoforms) were measured. The results establish a temporal pattern of mRNA induction beginning with c-fos (0.25-3 hr) and followed by c-jun (0.5-3 hr), junB (0.5-6 hr), NRF-1 (1-12 hr), Cyt c (12-72 hr), cytochrome c oxidase (12-72 hr). Induction of the latter was accompanied by a marked decrease in the liver-specific CPT-I mRNA. Electrical stimulation increased c-fos, $\beta$-myosin heavy chain, and Cyt c promoter activities. These increases coincided with a rise in their respective endogenous gene transcripts. NRF-1, cAMP response element (CRE), and Sp-1 site mutations within the Cyt c promoter reduced luciferase expression in both stimulated and nonstimulated myocytes. Mutations in the Nrf-1 and CRE sites inhibited the induction by electrical stimulation or by transfection of c-jun into non-paced cardiac myocytes whereas mutation of the Sp-1 site maintained or increased the fold induction. This is consistent with the appearance of NRF-1 and fos/jun mRNAs prior to that of Cyt c. Overexpression of c-jun by transfection also activates the Nrf-1 and Cyt c mRNA sequentially. Electrical stimulation of cardiac myocytes activates the c-Jun-N-terminal kinase so that the fold-activation of the cyt c promoter is increased by pacing when either c-jun or c-fos/c-jun are cotransfected. We have identified physical association of Nrf-1 protein with the Nrf-1 enhancer element and of c-Jun with the CRE binding sites on the Cyt c promoter. This is the first demonstration that induction of Nrf-1 and c-Jun by pacing of cardiac myocytes directly mediates Cyt c gene expression and mitochondrial proliferation in response to hypertrophic stimuli in the heart.^ Subsequent to gene activation pathways that lead to mitochondrial proliferation, we observed an isoform switch in CPT-I from the liver to muscle mRNA. We have found that the half-life for the muscle CPT-I is not affected by electrical stimulation, but electrical decrease the T1/2 in the liver CPT-I by greater than 50%. This suggests that the liver CPT-I switch to muscle isoform is due to (1) a decrease in T1/2 of liver CPT-I and (2) activation of muscle CPT-Itranscripts by electrical stimulation. (Abstract shortened by UMI.) ^
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Professional guidance has developed in Argentina since 1940, influenced by the guidance which in that time came from Spain, France, Belgium and Switzerland. The paper analyzes the first experiences in professional guidance, developed both in the great industrial cities as Buenos Aires and Rosario, and little urban centres as Tucumán or San Luis. The institutional and political context of such experiences is also analyzed, as well as the State's full support of them.
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Professional guidance has developed in Argentina since 1940, influenced by the guidance which in that time came from Spain, France, Belgium and Switzerland. The paper analyzes the first experiences in professional guidance, developed both in the great industrial cities as Buenos Aires and Rosario, and little urban centres as Tucumán or San Luis. The institutional and political context of such experiences is also analyzed, as well as the State's full support of them.
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Professional guidance has developed in Argentina since 1940, influenced by the guidance which in that time came from Spain, France, Belgium and Switzerland. The paper analyzes the first experiences in professional guidance, developed both in the great industrial cities as Buenos Aires and Rosario, and little urban centres as Tucumán or San Luis. The institutional and political context of such experiences is also analyzed, as well as the State's full support of them.
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Environmental constraints imposed on hydropoweroperation are usually given in the form of minimum environmental flows and maximum and minimum rates of change of flows, or ramp rates. One solution proposed to mitigate the environmental impact caused by the flows discharged by a hydropower plant while reducing the economic impact of the above-mentioned constraints consists in building a re-regulationreservoir, or afterbay, downstream of the power plant. Adding pumpingcapability between the re-regulationreservoir and the main one could contribute both to reducing the size of the re-regulationreservoir, with the consequent environmental improvement, and to improving the economic feasibility of the project, always fulfilling the environmental constraints imposed to hydropoweroperation. The objective of this paper is studying the contribution of a re-regulationreservoir to fulfilling the environmental constraints while reducing the economic impact of said constraints. For that purpose, a revenue-driven optimization model based on mixed integer linear programming is used. Additionally, the advantages of adding pumpingcapability are analysed. In order to illustrate the applicability of the methodology, a case study based on a real hydropower plant is presented
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Basic engineering skills are not the only key to professional development, particularly as engineering problems are everyday more and more complex and multifaceted, hence requiring the implementation of larger multidisciplinary teams, in many cases working in an international context and in a continuously evolving environment. Therefore other outcomes, sometimes referred to as professional skills, are also necessary for our students, as most universities are already aware. In this study we try to methodically analyze the main strategies for the promotion of professional skills, mainly linked to actuations which directly affect students or teachers (and teaching methodologies) and which take advantage of the environment and available resources. From an initial list of 51 strategies (in essence aimed at promotion of different drivers of change, linked to students, teachers, environment and resources), we focus on the 11 drivers of change considered more important after an initial evaluation. Subsequently, a systematic analysis of the typical problems linked to these main drivers of change, enables us to find and formulate 12 major and usually repeated and unsolved problems. After selecting these typical problems, we put forward 25 different solutions, for short-term actuation, and discuss their effects, while bearing in mind our team’s experience, together with the information from the studies carried out by numerous teaching staff from other universities.
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El análisis del rendimiento en deportes juega un papel esencial en el fútbol profesional. Aunque el estudio del análisis del juego en fútbol se ha utilizado desde diferentes ámbitos y situaciones, todavía existen diferentes aspectos y componentes del juego que siguen sin estar estudiados. En este sentido existen diferentes aspectos que deben de superar los estudios previos centrados en el componente descriptivo tales como el uso de variables/ indicadores de rendimiento que no se han definido ni estudiado, la validez de los métodos observaciones que no han sido testados con los softwares específicos en fútbol, la aplicación y utilidad de los resultados, así como las limitaciones del estudio de las variables situacionales/contextuales. Con el objetivo de cubrir las citadas limitaciones se han diseñado 6 estudios independientes e inter-relacionados que tratan de estudiar los aspectos anteriormente referidos. El primer estudio evalua la fiabilidad inter-observadores de las estadísticas de juego de la empresa privada OPTA Sportsdata, estos datos son la muestra de estudio de la presente tesis doctoral. Dos grupos de observadores experimentados se requieren para analizar un partido de la liga española de manera independiente. Los resultados muestran que los eventos de equipos y porteros codificados por los inter-operadores alcanzan un acuerdo muy bueno (valores kappa entre 0.86 y 0.94). La validez inter-observadores de las acciones de juego y los datos de jugadores individuales se evaluó con elevados niveles de acuerdo (valores del coeficiente de correlación intraclase entre 0.88 hasta 1.00, el error típico estandarizado variaba entre 0.00 hasta 0.37). Los resultados sugieren que las estadísticas de juego registradas por los operadores de la empresa OPTA Sportsdata están bien entrenados y son fiables. El segundo, tercer y cuarto estudio se centran en resaltar la aplicabilidad del análisis de rendimiento en el fútbol así como para explicar en profundidad las influencias de las variables situacionales. Utilizando la técnica de los perfiles de rendimiento de jugadores y equipos de fútbol se puede evaluar y comparar de manera gráfica, fácil y visual. Así mismo, mediante esta técnica se puede controlar el efecto de las variables situacionales (localización del partido, nivel del equipo y del oponente, y el resultado final del partido). Los perfiles de rendimiento de porteros (n = 46 porteros, 744 observaciones) y jugadores de campo (n = 409 jugadores, 5288 observaciones) de la primera division professional de fútbol Española (La Liga, temporada 2012-13), los equipos (n = 496 partidos, 992 observaciones) de la UEFA Champions League (temporadas 2009-10 a 2012-13) fueron analizados registrando la media, desviación típica, mediana, cuartiles superior e inferior y el recuento de valores de cada indicador de rendimiento y evento, los cuales se presentaron en su forma tipificada y normalizada. Los valores medios de los porteros de los equipos de diferentes niveles de La Liga y de los equipos de diferente nivel de la UEFA Champions League cuando jugaban en diferentes contextos de juego y situaciones (variables situacionales) fueron comparados utilizando el ANOVA de un factor y la prueba t para muestras independientes (localización del partido, diferencias entre casa y fuera), y fueron establecidos en los perfiles de red después de unificar todos los registros en la misma escala derivada con valores estandarizados. Mientras que las diferencias de rendimiento entre los jugadores de los mejores equipos (Top3) y los peores (Bottom3) fueron comparados mediante el uso de diferencias en la magnitud del tamaño del efecto. El quinto y el sexto estudio analizaban el rendimiento del fútbol desde un punto de vista de predicción del rendimiento. El modelo linear general y el modelo lineal general mixto fue empleado para analizar la magnitud de las relaciones de los indicadores y estadísticas de juego con el resultado final del partido en función del tipo de partido (partidos ajustados o todos los partidos) en la fase de grupos de la Copa del Mundo 2014 de Brasil (n = 48 partidos, 38 partidos ajustados) y La Liga 2012-13 (n = 320 partidos ajustados). Las relaciones fueron evaluadas mediante las inferencias en la magnitud de las diferencias y se expresaron como partidos extra ganados o perdidos por cada 10 partidos mediante la variable calculada en 2 desviaciones típicas. Los resultados mostraron que, para los 48 partidos de la fase de grupos de la Copa del Mundo 2014, nueve variables tuvieron un efecto positive en la probabilidad de ganar (tiros, tiros a puerta, tiros de contraataque, tiros dentro del área, posesión de balón, pases en corto, media de secuencia de pases, duelos aéreos y entradas), cuatro tuvieron efectos negativos (tiros bloqueados, centros, regates y tarjetas amarillas), y otras 12 variables tenían efectos triviales o poco claros. Mientras que los 38 partidos ajustados, el efecto de duelos aéreos y tarjetas amarillas fueron triviales y claramente negativos respectivamente. En la La Liga, existió un efecto moderado positive para cada equipo para los tiros a puerta (3.4 victorias extras por cada 10 partidos; 99% IC ±1.0), y un efecto positivo reducido para tiros totales (1.7 victorias extrsa; ±1.0). Los efectos de la mayoría de los eventos se han relacionado con la posesión del balón, la cual obtuvo efectos negativos entre equipos (1.2 derrotas extras; ±1.0) pero un efecto positivo pequeño entra equipos (1.7 victorias extras; ±1.4). La localización del partido mostró un efecto positive reducido dentro de los equipos (1.9 victorias extras; ±0.9). Los resultados obtenidos en los perfiles y el modelado del rendimiento permiten ofrecer una información detallada y avanzada para el entrenamiento, la preparación previa a los partidos, el control de la competición y el análisis post-partido, así como la evaluación e identificación del talento de los jugadores. ABSTRACT Match performance analysis plays an important role in the modern professional football. Although the research in football match analysis is well-developed, there are still some issues and problems remaining in this field, which mainly include the lack of operational definitions of variables, reliability issues, applicability of the findings, the lack of contextual/situational variables, and focusing too much on descriptive and comparative analysis. In order to address these issues, six independent but related studies were conducted in the current thesis. The first study evaluated the inter-operator reliability of football match statistics from OPTA Sportsdata Company which is the data resourse of the thesis. Two groups of experienced operators were required to analyse a Spanish league match independently in the experiment. Results showed that team events and goalkeeper actions coded by independent operators reached a very good agreement (kappa values between 0.86 and 0.94). The inter-operator reliability of match actions and events of individual outfield players was also tested to be at a high level (intra-class correlation coefficients ranged from 0.88 to 1.00, standardised typical error varied from 0.00 to 0.37). These results suggest that the football match statistics collected by well-trained operators from OPTA Sportsdata Company are reliable. The second, third and fourth study aims to enhance the applicability of football match performance analysis and to explore deeply the influences of situational variables. By using a profiling technique, technical and tactical performances of football players and teams can be interpreted, evaluated and compared more easily and straightforwardly, meanwhile, influences and effects from situational variables (match location, strength of team and opposition, and match outcome) on the performances can be properly incorporated. Performance profiles of goalkeepers (n = 46 goalkeepers, 744 full match observations) and outfield players (n = 409 players, 5288 full match observations) from the Spanish First Division Professional Football League (La Liga, season 2012-13), teams (n = 496 matches, 992 observations) from UEFA Champions League (seasons 2009-10 to 2012-13) were set up by presenting the mean, standard deviation, median, lower and upper quartiles of the count values of each performance-related match action and event to represent their typical performances and spreads. Means of goalkeeper from different levels of team in La Liga and teams of different strength in UEFA Champions League when playing under different situational conditions were compared by using one-way ANOVA and independent sample t test (for match location, home and away differences), and were plotted into the same radar charts after unifying all the event counts by standardised score. While differences between the performances of outfield players from Top3 and from Bottom3 teams were compared by magnitude-based inferences. The fifth and sixth study aims to move from the descriptive and comparative football match analysis to a more predictive one. Generalised linear modelling and generalised mixed linear modelling were undertaken to quantify relationships of the performance-related match events, actions and variables with the match outcome in different types of games (close games and all games) in the group stage of 2014 Brazil FIFA World Cup (n = 48 games, 38 close games) and La Liga 2012-13 (n = 320 close games). Relationships were evaluated with magnitude-based inferences and were expressed as extra matches won or lost per 10 matches for an increase of two standard deviations of a variable. Results showed that, for all the 48 games in the group stage of 2014 FIFA World Cup, nine variables had clearly positive effects on the probability of winning (shot, shot on target, shot from counter attack, shot from inside area, ball possession, short pass, average pass streak, aerial advantage, and tackle), four had clearly negative effects (shot blocked, cross, dribble and red card), other 12 variabless had either trivial or unclear effects. While for the 38 close games, the effects of aerial advantage and yellow card turned to trivial and clearly negative, respectively. In the La Liga, there was a moderate positive within-team effect from shots on target (3.4 extra wins per 10 matches; 99% confidence limits ±1.0), and a small positive within-team effect from total shots (1.7 extra wins; ±1.0). Effects of most other match events were related to ball possession, which had a small negative within-team effect (1.2 extra losses; ±1.0) but a small positive between-team effect (1.7 extra wins; ±1.4). Game location showed a small positive within-team effect (1.9 extra wins; ±0.9). Results from the established performance profiles and modelling can provide detailed and straightforward information for training, pre-match preparations, in-match tactical approaches and post-match evaluations, as well as for player identification and development. 摘要 比赛表现分析在现代足球中起着举足轻重的作用。尽管如今对足球比赛表现分析的研究已经相对完善,但仍有很多不足之处。这些不足主要体现在:研究中缺乏对研究变量的清晰定义、数据信效度缺失、研究结果的实用性受限、比赛情境因素缺失以及过于集中在描述性和对比性分析等。针对这些问题,本论文通过六个独立而又相互联系的研究,进一步对足球比赛表现分析进行完善。 第一个研究对本论文的数据源--OPTA Sportsdata公司的足球比赛数据的信效度进行了实验检验。实验中,两组数据收集人员被要求对同一场西班牙足球甲级联赛的比赛进行分析。研究结果显示,两组收集人员记录下的球队比赛事件和守门员比赛行为具有高度的一致性(卡帕系数介于0.86和0.94)。收集人员输出的外场球员的比赛行为和比赛事件也具有很高的组间一致性(ICC相关系数介于0.88和1.00,标准化典型误差介于0.00和0.37)。实验结果证明了OPTA Sportsdata公司收集的足球比赛数据具有足够高的信效度。 第二、三、四个研究旨在提升足球比赛表现分析研究结果的实用性以及深度探讨比赛情境因素对足球比赛表现的影响。通过对足球运动员和运动队的比赛技战术表现进行档案创建,可以对运动员和运动队的比赛表现进行简直接而直观的呈现、评价和对比,同时,情境变量(比赛场地、球队和对手实力、比赛结果)对比赛表现的影响也可以被整合到表现档案中。本部分对2012-13赛季西班牙足球甲级联赛的参赛守门员(n = 46球员人次,744比赛场次)和外场球员(n = 409球员人次, 5288比赛场次)以及2009-10至2012-13赛季欧洲足球冠军联赛的参赛球队(n = 496比赛场次)的比赛技战术表现进行了档案创建。在表现档案中,各项比赛技战术指标的均值、标准差、中位数和大小四分位数被用来展现守门员、外场球员和球队的普遍表现和表现浮动性。方差分析(ANOVA)被用来对西甲不同水平球队的守门员、欧冠中不同水平球队在不同比赛情境下的普遍表现(各项指标的均值)进行对比,独立样本t检验被用来对比主客场比赛普遍表现的差异。数据量级推断(magnitude-based inferences)的方法则被用来对西甲前三名和最后三名球队外场球员的普遍表现进行对比分析。所有来自不同水平球队的运动员和不同水平运动队的各项比赛指标皆被转换成了标准分数,从而能把他们在各种不同比赛情境下的普遍表现(各项比赛指标的均值)投到相同的雷达图中进行直观的对比。 第五和第六个研究目的在于进行预测性足球比赛表现分析,从而跨越之前固有的描述性和对比性分析。广义线性模型和广义混合线性模型被用来对2014年巴西世界杯小组赛(n = 48 比赛场次,38小分差场次)和2012-13赛季西甲联赛(n = 320小分差场次)的比赛中各表现相关比赛事件、行为和变量与比赛结果(胜、平、负)的关系进行建模。模型中的关系通过数据量级推断(magnitude-based inferences)的方法来界定,具体表现为某个变量增加两个标准差对比赛结果的影响(每10场比赛中额外取胜或失利的场数)。研究结果显示,在2014年巴西世界杯小组赛的所有48场比赛中,9个变量(射门、射正、反击中射门、禁区内射门、控球、短传、连续传球平均次数、高空球争抢成功率和抢断)与赢球概率有清晰的正相关关系,4个变量(射门被封堵、传中、过人和红牌)与赢球概率有清晰的负相关关系,其他12个被分析的变量与赢球概率的相关关系微小或不清晰。而在38场小分差比赛中,高空球争抢成功率由正相关变为微小关系,黄牌则由微小关系变为清晰的负相关。在西甲联赛中,每一支球队增加两个标准差的“射正球门”可以给每10场比赛带来3.4场额外胜利(99%置信区间±1.0场),而所有球队作为一个整体,每增加两个标准差的“射正球门”可以给每10场比赛带来1.7场额外胜利(99%置信区间±1.0场)。其他大多数比赛相关事件与比赛结果的相关关系与“控球”相关联。每一支球队增加两个标准差的“控球”将会给每10场比赛带来1.2场额外失利(99%置信区间±1.0场),而所有球队作为一个整体,每增加两个标准差的“控球”可以给每10场比赛带来1.7场额外胜利(99%置信区间±1.4场)。与客场比赛相对,主场能给球队带来1.9 /10场额外胜利(99%置信区间±0.9场)。 比赛表现档案和模型中得出的研究结果可以为俱乐部、足球队、教练组、表现分析师和运动员提供详细而直接的参考信息。这些信息可用于训练指导、赛前备战、赛中技战术调整和赛后技战术表现分析,也可运用于足球运动员选材、培养和发展。
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Peer reviewed
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CP12 is a small nuclear encoded chloroplast protein of higher plants, which was recently shown to interact with NAD(P)H–glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.13), one of the key enzymes of the reductive pentosephosphate cycle (Calvin cycle). Screening of a pea cDNA library in the yeast two-hybrid system for proteins that interact with CP12, led to the identification of a second member of the Calvin cycle, phosphoribulokinase (PRK; EC 2.7.1.19), as a further specific binding partner for CP12. The exchange of cysteines for serines in CP12 demonstrate that interaction with PRK occurs at the N-terminal peptide loop of CP12. Size exclusion chromatography and immunoprecipitation assays reveal the existence of a stable 600-kDa PRK/CP12/GAPDH complex in the stroma of higher plant chloroplasts. Its stoichiometry is proposed to be of two N-terminally dimerized CP12 molecules, each carrying one PRK dimer on its N terminus and one A2B2 complex of GAPDH subunits on the C-terminal peptide loop. Incubation of the complex with NADP or NADPH, in contrast to NAD or NADH, causes its dissociation. Assays with the stromal 600-kDa fractions in the presence of the four different nicotinamide-adenine dinucleotides indicate that PRK activity depends on complex dissociation and might be further regulated by the accessible ratio of NADP/NADPH. From these results, we conclude that light regulation of the Calvin cycle in higher plants is not only via reductive activation of different proteins by the well-established ferredoxin/thioredoxin system, but in addition, by reversible dissociation of the PRK/CP12/GAPDH complex, mediated by NADP(H).
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The protein Sex-lethal (SXL) controls pre-mRNA splicing of two genes involved in Drosophila sex determination: transformer (tra) and the Sxl gene itself. Previous in vitro results indicated that SXL antagonizes the general splicing factor U2AF65 to regulate splicing of tra. In this report, we have used transgenic flies expressing chimeric proteins between SXL and the effector domain of U2AF65 to study the mechanisms of splicing regulation by SXL in vivo. Conferring U2AF activity to SXL relieves its inhibitory activity on tra splicing but not on Sxl splicing. Therefore, antagonizing U2AF65 can explain tra splicing regulation both in vitro and in vivo, but this mechanism cannot explain splicing regulation of Sxl pre-mRNA. These results are a direct proof that Sxl, the master regulatory gene in sex determination, has multiple and separable activities in the regulation of pre-mRNA splicing.
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The circadian clock in the suprachiasmatic nucleus (SCN) of the hypothalamus organizes behavioral rhythms, such as the sleep–wake cycle, on a near 24-h time base and synchronizes them to environmental day and night. Light information is transmitted to the SCN by direct retinal projections via the retinohypothalamic tract (RHT). Both glutamate (Glu) and pituitary adenylyl cyclase-activating peptide (PACAP) are localized within the RHT. Whereas Glu is an established mediator of light entrainment, the role of PACAP is unknown. To understand the functional significance of this colocalization, we assessed the effects of nocturnal Glu and PACAP on phasing of the circadian rhythm of neuronal firing in slices of rat SCN. When coadministered, PACAP blocked the phase advance normally induced by Glu during late night. Surprisingly, blocking PACAP neurotransmission, with either PACAP6–38, a specific PACAP receptor antagonist, or anti-PACAP antibodies, augmented the Glu-induced phase advance. Blocking PACAP in vivo also potentiated the light-induced phase advance of the rhythm of hamster wheel-running activity. Conversely, PACAP enhanced the Glu-induced delay in the early night, whereas PACAP6–38 inhibited it. These results reveal that PACAP is a significant component of the Glu-mediated light-entrainment pathway. When Glu activates the system, PACAP receptor-mediated processes can provide gain control that generates graded phase shifts. The relative strengths of the Glu and PACAP signals together may encode the amplitude of adaptive circadian behavioral responses to the natural range of intensities of nocturnal light.
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Rad is the prototypic member of a new class of Ras-related GTPases. Purification of the GTPase-activating protein (GAP) for Rad revealed nm23, a putative tumor metastasis suppressor and a development gene in Drosophila. Antibodies against nm23 depleted Rad-GAP activity from human skeletal muscle cytosol, and bacterially expressed nm23 reconstituted the activity. The GAP activity of nm23 was specific for Rad, was absent with the S105N putative dominant negative mutant of Rad, and was reduced with mutations of nm23. In the presence of ATP, GDP⋅Rad was also reconverted to GTP⋅Rad by the nucleoside diphosphate (NDP) kinase activity of nm23. Simultaneously, Rad regulated nm23 by enhancing its NDP kinase activity and decreasing its autophosphorylation. Melanoma cells transfected with wild-type Rad, but not the S105N-Rad, showed enhanced DNA synthesis in response to serum; this effect was lost with coexpression of nm23. Thus, the interaction of nm23 and Rad provides a potential novel mechanism for bidirectional, bimolecular regulation in which nm23 stimulates both GTP hydrolysis and GTP loading of Rad whereas Rad regulates activity of nm23. This interaction may play important roles in the effects of Rad on glucose metabolism and the effects of nm23 on tumor metastasis and developmental regulation.
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The focal adhesion kinase (FAK) is discretely localized to focal adhesions via its C-terminal focal adhesion–targeting (FAT) sequence. FAK is regulated by integrin-dependent cell adhesion and can regulate tyrosine phosphorylation of downstream substrates, like paxillin. By the use of a mutational strategy, the regions of FAK that are required for cell adhesion–dependent regulation and for inducing tyrosine phosphorylation of paxillin were determined. The results show that the FAT sequence was the single region of FAK that was required for each function. Furthermore, the FAT sequence of FAK was replaced with a focal adhesion–targeting sequence from vinculin, and the resulting chimera exhibited cell adhesion–dependent tyrosine phosphorylation and could induce paxillin phosphorylation like wild-type FAK. These results suggest that subcellular localization is the major determinant of FAK function.
Resumo:
Sugar transport by some permeases in Escherichia coli is allosterically regulated by the phosphorylation state of the intracellular regulatory protein, enzyme IIAglc of the phosphoenolpyruvate:sugar phosphotransferase system. A sensitive radiochemical assay for the interaction of enzyme IIAglc with membrane-associated lactose permease was used to characterize the binding reaction. The binding is stimulated by transportable substrates such as lactose, melibiose, and raffinose, but not by sugars that are not transported (maltose and sucrose). Treatment of lactose permease with N-ethylmaleimide, which blocks ligand binding and transport by alkylating Cys-148, also blocks enzyme IIAglc binding. Preincubation with the substrate analog β-d-galactopyranosyl 1-thio-β-d-galactopyranoside protects both lactose transport and enzyme IIAglc binding against inhibition by N-ethylmaleimide. A collection of lactose permease replacement mutants at Cys-148 showed, with the exception of C148V, a good correlation of relative transport activity and enzyme IIAglc binding. The nature of the interaction of enzyme IIAglc with the cytoplasmic face of lactose permease was explored. The N- and C-termini, as well as five hydrophilic loops in the permease, are exposed on the cytoplasmic surface of the membrane and it has been proposed that the central cytoplasmic loop of lactose permease is the major determinant for interaction with enzyme IIAglc. Lactose permease mutants with polyhistidine insertions in cytoplasmic loops IV/V and VI/VII and periplasmic loop VII/VIII retain transport activity and therefore substrate binding, but do not bind enzyme IIAglc, indicating that these regions of lactose permease may be involved in recognition of enzyme IIAglc. Taken together, these results suggest that interaction of lactose permease with substrate promotes a conformational change that brings several cytoplasmic loops into an arrangement optimal for interaction with the regulatory protein, enzyme IIAglc. A topological map of the proposed interaction is presented.