Comparison of Airborne Laser Scanning Methods for Estimating Forest Structure Indicators Based on Lorenz Curves

The purpose of this study was to compare a number of state-of-the-art methods in airborne laser scan- ning (ALS) remote sensing with regards to their capacity to describe tree size inequality and other indi- cators related to forest structure. The indicators chosen were based on the analysis of the Lorenz curve: Gini coefficient ( GC ), Lorenz asymmetry ( LA ), the proportions of basal area ( BALM ) and stem density ( NSLM ) stocked above the mean quadratic diameter. Each method belonged to one of these estimation strategies: (A) estimating indicators directly; (B) estimating the whole Lorenz curve; or (C) estimating a complete tree list. Across these strategies, the most popular statistical methods for area-based approach (ABA) were used: regression, random forest (RF), and nearest neighbour imputation. The latter included distance metrics based on either RF (NN–RF) or most similar neighbour (MSN). In the case of tree list esti- mation, methods based on individual tree detection (ITD) and semi-ITD, both combined with MSN impu- tation, were also studied. The most accurate method was direct estimation by best subset regression, which obtained the lowest cross-validated coefficients of variation of their root mean squared error CV(RMSE) for most indicators: GC (16.80%), LA (8.76%), BALM (8.80%) and NSLM (14.60%). Similar figures [CV(RMSE) 16.09%, 10.49%, 10.93% and 14.07%, respectively] were obtained by MSN imputation of tree lists by ABA, a method that also showed a number of additional advantages, such as better distributing the residual variance along the predictive range. In light of our results, ITD approaches may be clearly inferior to ABA with regards to describing the structural properties related to tree size inequality in for- ested areas.

The TRANSPLANTA collection of Arabidopsis lines: a resource for functional analysis of transcription factors based on their conditional over-expression

Transcription factors (TFs) are key regulators of gene expression in all organisms. In eukaryotes, TFs are often represented by functionally redundant members of large gene families. Overexpression might prove a means to unveil the biological functions of redundant TFs; however, constitutive overexpression of TFs frequently causes severe developmental defects, preventing their functional characterization. Conditional overexpression strategies help to overcome this problem. Here, we report on the TRANSPLANTA collection of Arabidopsis lines, each expressing one of 949 TFs under the control of a β–estradiol-inducible promoter. Thus far, 1636 independent homozygous lines, representing an average of 2.6 lines for every TF, have been produced for the inducible expression of 634 TFs. Along with a GUS-GFP reporter, randomly selected TRANSPLANTA lines were tested and confirmed for conditional transgene expression upon β–estradiol treatment. As a proof of concept for the exploitation of this resource, β–estradiol-induced proliferation of root hairs, dark-induced senescence, anthocyanin accumulation and dwarfism were observed in lines conditionally expressing full-length cDNAs encoding RHD6, WRKY22, MYB123/TT2 and MYB26, respectively, in agreement with previously reported phenotypes conferred by these TFs. Further screening performed with other TRANSPLANTA lines allowed the identification of TFs involved in different plant biological processes, illustrating that the collection is a powerful resource for the functional characterization of TFs. For instance, ANAC058 and a TINY/AP2 TF were identified as modulators of ABA-mediated germination potential, and RAP2.10/DEAR4 was identified as a regulator of cell death in the hypocotyl–root transition zone. Seeds of TRANSPLANTA lines have been deposited at the Nottingham Arabidopsis Stock Centre for further distribution.

New insights into the regulation of NADPH oxidase dependent reactive oxygen species signaling during the plant immune response

Las NADPH oxidasas de plantas, denominadas “respiratory burst oxidase homologues” (RBOHs), producen especies reactivas del oxígeno (ROS) que median un amplio rango de funciones. En la célula vegetal, el ajuste preciso de la producción de ROS aporta la especificidad de señal para generar una respuesta apropiada ante las amenazas ambientales. RbohD y RbohF, dos de los diez genes Rboh de Arabidopsis, son pleiotrópicos y median diversos procesos fisiológicos en respuesta a patógenos. El control espacio-temporal de la expresión de los genes RbohD y RbohF podría ser un aspecto crítico para determinar la multiplicidad de funciones de estas oxidasas. Por ello, generamos líneas transgénicas de Arabidopsis con fusiones de los promoters de RbohD y RbohF a los genes delatores de la B-glucuronidasa y la luciferasa. Estas líneas fueron empleadas para revelar el patrón de expresión diferencial de RbohD y RbohF durante la respuesta inmune de Arabidopsis a la bacteria patógena Pseudomonas syringae pv. tomato DC3000, el hongo necrótrofo Plectosphaerella cucumerina y en respuesta a señales relacionadas con la respuesta inmune. Nuestros experimentos revelan un patrón de expresión diferencial de los promotores de RbohD y RbohF durante el desarrollo de la planta y en la respuesta inmune de Arabidopsis. Además hemos puesto de manifiesto que existe una correlación entre el nivel de actividad de los promotores de RbohD y RbohF con la acumulación de ROS y el nivel de muerte celular en respuesta a patógenos. La expression de RbohD y RbohF también es modulada de manera diferencial en respuesta a patrones moleculares asociados a patógenos (PAMPs) y por ácido abscísico (ABA). Cabe destacar que, mediante una estrategia de intercambio de promotores, hemos revelado que la región promotora de RbohD, es necesaria para dirigir la producción de ROS en respuesta a P. cucumerina. Adicionalmente, la activación del promotor de RbohD en respuesta al aislado de P. cucumerina no adaptado a Arabidopsis 2127, nos llevó a realizar ensayos de susceptibilidad con el doble mutante rbohD rbohF que han revelado un papel desconocido de estas oxidasas en resistencia no-huesped. La interacción entre la señalización dependiente de las RBOHs y otros componentes de la respuesta inmune de plantas podría explicar también las distintas funciones que median estas oxidasas en relación con la respuesta inmune. Entre la gran cantidad de señales coordinadas con la actividad de las RBOHs, existen evidencias genéticas y farmacológicas que indican que las proteínas G heterotriméricas están implicadas en algunas de las rutas de señalización mediadas por ROS derivadas de los RBOHs en respuesta a señales ambientales. Por ello hemos estudiado la relación entre estas RBOH-NADPH oxidasas y AGB1, la subunidad β de las proteínas G heterotriméricas en la respuesta inmune de Arabidopsis. Análisis de epistasis indican que las proteínas G heterotriméricas están implicadas en distintas rutas de señalización en defensa mediadas por las RBOHs. Nuestros resultados ilustran la relación compleja entre la señalización mediada por las RBOHs y las proteínas G heterotriméricas, que varía en función de la interacción planta-patógeno analizada. Además, hemos explorado la posible asociación entre AGB1 con RBOHD y RBOHF en eventos tempranos de la respuesta immune. Cabe señalar que experimentos de coímmunoprecipitación apuntan a una posible asociación entre AGB1 y la kinasa citoplasmática reguladora de RBOHD, BIK1. Esto indica un posible mecanismo de control de la función de esta NADPH oxidase por AGB1. En conjunto, estos datos aportan nuevas perspectivas sobre cómo, a través del control transcripcional o mediante la interacción con las proteínas G heterotriméricas, las NADPH oxidases de plantas median la producción de ROS y la señalización por ROS en la respuesta inmune. Nuestro trabajo ejemplifica cómo la regulación diferencial de dos miembros de una familia multigénica, les permite realizar distintas funciones fisiológicas especializadas usando un mismo mecanismo enzimático. ABSTRACT The plant NADPH oxidases, termed respiratory burst oxidase homologues (RBOHs), produce reactive oxygen species (ROS) which mediate a wide range of functions. Fine tuning this ROS production provides the signaling specificity to the plant cell to produce the appropriate response to environmental threats. RbohD and RbohF, two of the ten Rboh genes present in Arabidopsis, are pleiotropic and mediate diverse physiological processes in response to pathogens. One aspect that may prove critical to determine the multiplicity of functions of RbohD and RbohF is the spatio-temporal control of their gene expression. Thus, we generated Arabidopsis transgenic lines with RbohD- and RbohF-promoter fusions to the β-glucuronidase and the luciferase reporter genes. These transgenics were employed to reveal RbohD and RbohF promoter activity during Arabidopsis immune response to the pathogenic bacterium Pseudomonas syringae pv tomato DC3000, the necrotrophic fungus Plectosphaerella cucumerina and in response to immunity-related cues. Our experiments revealed a differential expression pattern of RbohD and RbohF throughout plant development and during Arabidopsis immune response. Moreover, we observed a correlation between the level of RbohD and RbohF promoter activity, the accumulation of ROS and the amount of cell death in response to pathogens. RbohD and RbohF gene expression was also differentially modulated by pathogen associated molecular patterns and abscisic acid. Interestingly, a promoter-swap strategy revealed the requirement for the promoter region of RbohD to drive the production of ROS in response to P. cucumerina. Additionally, since the RbohD promoter was activated during Arabidopsis interaction with a non-adapted P. cucumerina isolate 2127, we performed susceptibility tests to this fungal isolate that uncovered a new role of these oxidases on non-host resistance. The interplay between RBOH-dependent signaling with other components of the plant immune response might also explain the different immunity-related functions mediated by these oxidases. Among the plethora of signals coordinated with RBOH activity, pharmacological and genetic evidence indicates that heterotrimeric G proteins are involved in some of the signaling pathways mediated by RBOH–derived ROS in response to environmental cues. Therefore, we analysed the interplay between these RBOH-NADPH oxidases and AGB1, the Arabidopsis β-subunit of heterotrimeric G proteins during Arabidopsis immune response. We carried out epistasis studies that allowed us to test the implication of AGB1 in different RBOH-mediated defense signaling pathways. Our results illustrate the complex relationship between RBOH and heterotrimeric G proteins signaling, that varies depending on the type of plant-pathogen interaction. Furthermore, we tested the potential association between AGB1 with RBOHD and RBOHF during early immunity. Interestingly, our co-immunoprecipitation experiments point towards an association of AGB1 and the RBOHD regulatory kinase BIK1, thus providing a putative mechanism in the control of the NADPH oxidase function by AGB1. Taken all together, these studies provide further insights into the role that transcriptional control or the interaction with heterotrimeric G-proteins have on RBOH-NADPH oxidase-dependent ROS production and signaling in immunity. Our work exemplifies how, through a differential regulation, two members of a multigenic family achieve specialized physiological functions using a common enzymatic mechanism.

Evolución de parámetros de condición física a lo largo de la temporada en un equipo de fútbol profesional

Se han realizado muchos estudios relacionados con el fútbol, pero pocos con jugadores profesionales evaluándolos en un periodo de dos temporadas consecutivas. El objetivo de este trabajo fue investigar las variaciones que sufren parámetros relacionados con la composición corporal, la velocidad, la fuerza y la resistencia de futbolistas profesionales de alto y medio nivel en el periodo de una temporada. Material y métodos: 20 jugadores de categoría alta (CA) y 21 jugadores de categoría media (CM) fueron evaluados a lo largo de una temporada en tres ocasiones, la primera a mitad de pretemporada (M1), la segunda a mitad del periodo competitivo (M2) y la última al final de la temporada (M3). Algunos de ellos, 16 de CA y 8 de CM participaron durante dos temporadas seguidas realizando un total de seis evaluaciones. En cada evaluación se midió la talla (TA), peso corporal (PC), sumatorio de pliegues grasos (ZP), el índice de masa corporal (IMC), el tiempo en 5 (T5) y 20 (T20) metros, el salto en contra movimiento (CMJ), Abalakov (ABA), la velocidad del umbral anaeróbico (VUAN), el peso de potencia media (PPM) y la potencia media (PMW) en el ejercicio de media sentadilla. Resultados: Hubo diferencias significativas en el ZP en el grupo de CA entre la M1 (45,28±8,98) y la M2 (40,57±5,89) al igual que en la VUAN entre la M1 (12,49±0,67) con respecto a la M2 (12,93±0,62) y la M3 (12,93±0,44). Además, se encontraron diferencias significativas en el grupo CA en la (T5) entre la M1 (1,04±0,06) y la M2 (0,99±0,04) y la M3 (1,00±0,04) y en el grupo CM sólo entre la M1 (1,05±0,05) y la M3 (1,01±0,04). El PPM fue superior en la M2 (94±13,74) con respecto a la M1 (82±13,64) de forma significativa, así como la PMW, M1 (1642,87±207,05) frente a M2 (1735,07±168,01) en el grupo de CA y también hubo diferencias significativas en el PPM entre la M1 (70,67±10,74) y la M2 (80,50±13,20) en el grupo de CM, así como en la PMW en la M1 (1574,90±205,66) con la M2 (1642,87±207,05). Conclusiones: Hubo parámetros condicionales que sufrieron variaciones a lo largo de la temporada. Los cambios más importantes se produjeron desde la mitad del periodo preparatorio hasta la mitad del periodo competitivo, no apreciándose cambios desde la mitad del periodo competitivo hasta el final del mismo. ABSTRACT They have been many studies on soccer, but few with professional players evaluating them during two consecutive seasons. The purpose of this study was to investigate seasonal variations in fitness parameters as body composition, speed, strength and stamina in top and medium class professional soccer players during a hole season. Methods: 20 Top Class male (CA) and 21 Medium Class male (CM) soccer players were evaluated during a season in three different periods. The first one at half preseason (M1), second one at the middle of the competitive period (M2) and last one at the end of the season (M3). 16 players from group CA and 8 from group CM were registered from two seasons in a row, doing a total of 6 evaluation periods. In each evaluation period was measured body height (TA), body mass (PC), skinfolds sum (ZP), body mass index (IMC), acceleration on 5 (T5) y 20 (T20) meters, countermovement jump (CMJ), free arms countermovement jump (ABA), anaerobic threshold speed (VUAN), average power strength weight (PPM) and average power (PMW) on half squat. Results: significant decrease on ZP were found in group CA from M1 (45,28±8,98) to M2 (40,57±5,89) and a significant increase on VUAN from M1 (12,49±0,67) to M2 (12,93±0,62) and M3 (12,93±0,44). Also a significant decrease was found in group CA on (T5) from M1 (1,04±0,06) to M2 (0,99±0,04) and M3 (1,00±0,04). PPM was significantly higher from M2 (94±13,74) to M1 (82±13,64) as PMW from M1 (1642,87±207,05) to M2 (1735,07±168,01) in CA group and also was found a significant increase in group CM on PPM from M1 (70,67±10,74) to M2 (80,50±13,20) and on PMW from M1 (1574,90±205,66) to M2 (1642,87±207,05). Conclusions: Some of the fitness parameters did suffer seasonal variations. The most important changes took place from the middle of the preseason period until middle of the competitive period, remaining unchanged from middle of the competitive period until the end of the season.

Diseño e implementación de un demostrador web de mejoras de prestaciones wi-fi

Las características de Wi-Fi en combinación con el protocolo TCP hacen que se produzca una reducción del caudal resultante, causada por el diseño del propio protocolo TCP, desaprovechando en gran medida la capacidad de la red. El objetivo del proyecto ABA-WIFI era elaborar una solución que permitiera corregir el comportamiento anómalo del protocolo de transporte TCP que sufren las redes de acceso de banda ancha a través de Wi-Fi. Para ello se presentaron una serie de mejoras destinadas a los puntos de acceso Wi-Fi basadas en técnicas de TCP Split y TCP Spoof que solucionan el problema identificado de manera notable. Este trabajo proporciona una herramienta web para probar y medir cuantitativamente en tiempo real el efecto de dichas mejoras sobre las prestaciones de la red para distintas condiciones de la misma, atendiendo a la posición del cliente respecto del punto de acceso, el tipo de tráfico cursado, las condiciones de carga de la red y la situación geográfica.

INTERVENÇÕES FISIOTERAPÊUTICAS NUM CONTEXTO LÚDICO JUNTO A CRIANÇAS COM DOENÇAS RESPIRATÓRIAS CRÔNICAS

Verifica-se hoje em dia o aumento da incidência de problemas pulmonares que ocasiona internações hospitalares infantis com maior freqüência. Este projeto baseia-se na visão biopsicossocial da Psicologia da Saúde e leva em conta a valorização de atividades do cotidiano da criança, como o brincar, para sua recuperação. Tem por objetivo principal investigar se a utilização de técnicas interventivas lúdicas em fisioterapia contribui para a melhora dos níveis de oxigenação de crianças asmáticas e por objetivos específicos traçar um breve perfil clínico da criança que procura o Ambulatório de Alergia e Imunologia da Faculdade de Medicina do ABC e verificar a adesão da criança com problemas respiratórios a uma intervenção fisioterapêutica lúdica da criança. A análise qualitativa da intervenção lúdica registra grande envolvimento e participação prazerosa das crianças e ampla aceitação dos pais. Estes resultados sugerem que novas pesquisas sejam realizadas a respeito de intervenções lúdicas em fisioterapia. Utiliza-se de método avaliativo-interventivo quase-experimental. Seus participantes são 58 crianças de ambos os sexos com idade de quatro a 11 anos com as patologias de asma brônquica, bronquite e rinite alérgica, sem a presença da crise. Desenvolve-se por meio de sessão única com cada paciente, com avaliação antes e após da intervenção fisioterápica lúdica por meio das aferições dos sinais vitais, (PA, FC, FR,T°C, incluindo oximetria de pulso e PFE) técnicas de aquecimento, alongamento torácico, fortalecimento abdominal e expansão torácica. Realiza análise qualitativa da responsividade das crianças às técnicas interventivas empregadas no contexto lúdico. Os resultados a respeito do perfil clinico indicam maior incidência de Asma, com 44 casos (75,8620 %), para 11 casos de Rinite (18,9655 %) e apenas três de Bronquite (5,1724 %). Os dados relativos a PA, FC, FR,T°C e ao PFE mostram-se dentro da normalidade. Este estudo não constata diferença significativa em relação ao nível de saturação de O2 após a intervenção lúdica. Quanto à adesão, 55,2 % dos participantes aderiram, sendo que a não adesão deu-se por motivos particulares alegados pelos responsáveis ou indisposição da criança, de dor de cabeça, sonolência e/ou vômito. A análise qualitativa da intervenção lúdica registra grande envolvimento e participação prazerosa das crianças e ampla aceitação dos pais. Estes resultados sugerem que novas pesquisas sejam realizadas a respeito de intervenções lúdicas em fisioterapia.(AU)

INTERVENÇÕES FISIOTERAPÊUTICAS NUM CONTEXTO LÚDICO JUNTO A CRIANÇAS COM DOENÇAS RESPIRATÓRIAS CRÔNICAS

Verifica-se hoje em dia o aumento da incidência de problemas pulmonares que ocasiona internações hospitalares infantis com maior freqüência. Este projeto baseia-se na visão biopsicossocial da Psicologia da Saúde e leva em conta a valorização de atividades do cotidiano da criança, como o brincar, para sua recuperação. Tem por objetivo principal investigar se a utilização de técnicas interventivas lúdicas em fisioterapia contribui para a melhora dos níveis de oxigenação de crianças asmáticas e por objetivos específicos traçar um breve perfil clínico da criança que procura o Ambulatório de Alergia e Imunologia da Faculdade de Medicina do ABC e verificar a adesão da criança com problemas respiratórios a uma intervenção fisioterapêutica lúdica da criança. A análise qualitativa da intervenção lúdica registra grande envolvimento e participação prazerosa das crianças e ampla aceitação dos pais. Estes resultados sugerem que novas pesquisas sejam realizadas a respeito de intervenções lúdicas em fisioterapia. Utiliza-se de método avaliativo-interventivo quase-experimental. Seus participantes são 58 crianças de ambos os sexos com idade de quatro a 11 anos com as patologias de asma brônquica, bronquite e rinite alérgica, sem a presença da crise. Desenvolve-se por meio de sessão única com cada paciente, com avaliação antes e após da intervenção fisioterápica lúdica por meio das aferições dos sinais vitais, (PA, FC, FR,T°C, incluindo oximetria de pulso e PFE) técnicas de aquecimento, alongamento torácico, fortalecimento abdominal e expansão torácica. Realiza análise qualitativa da responsividade das crianças às técnicas interventivas empregadas no contexto lúdico. Os resultados a respeito do perfil clinico indicam maior incidência de Asma, com 44 casos (75,8620 %), para 11 casos de Rinite (18,9655 %) e apenas três de Bronquite (5,1724 %). Os dados relativos a PA, FC, FR,T°C e ao PFE mostram-se dentro da normalidade. Este estudo não constata diferença significativa em relação ao nível de saturação de O2 após a intervenção lúdica. Quanto à adesão, 55,2 % dos participantes aderiram, sendo que a não adesão deu-se por motivos particulares alegados pelos responsáveis ou indisposição da criança, de dor de cabeça, sonolência e/ou vômito. A análise qualitativa da intervenção lúdica registra grande envolvimento e participação prazerosa das crianças e ampla aceitação dos pais. Estes resultados sugerem que novas pesquisas sejam realizadas a respeito de intervenções lúdicas em fisioterapia.(AU)

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.

Membrane voltage initiates Ca2+ waves and potentiates Ca2+ increases with abscisic acid in stomatal guard cells

In higher plants changes and oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) are central to hormonal physiology, including that of abscisic acid (ABA), which signals conditions of water stress and alters ion channel activities in guard cells of higher-plant leaves. Such changes in [Ca2+]i are thought to encode for cellular responses to different stimuli, but their origins and functions are poorly understood. Because transients and oscillations in membrane voltage also occur in guard cells and are elicited by hormones, including ABA, we suspected a coupling of [Ca2+]i to voltage and its interaction with ABA. We recorded [Ca2+]i by Fura2 fluorescence ratio imaging and photometry while bringing membrane voltage under experimental control with a two-electrode voltage clamp in intact Vicia guard cells. Free-running oscillations between voltages near −50 mV and −200 mV were associated with oscillations in [Ca2+]i, and, under voltage clamp, equivalent membrane hyperpolarizations caused [Ca2+]i to increase, often in excess of 1 μM, from resting values near 100 nM. Image analysis showed that the voltage stimulus evoked a wave of high [Ca2+]i that spread centripetally from the peripheral cytoplasm within 5–10 s and relaxed over 40–60 s thereafter. The [Ca2+]i increases showed a voltage threshold near −120 mV and were sensitive to external Ca2+ concentration. Substituting Mn2+ for Ca2+ to quench Fura2 fluorescence showed that membrane hyperpolarization triggered a divalent influx. ABA affected the voltage threshold for the [Ca2+]i rise, its amplitude, and its duration. In turn, membrane voltage determined the ability of ABA to raise [Ca2+]i. These results demonstrate a capacity for voltage to evoke [Ca2+]i increases, they point to a dual interaction with ABA in triggering and propagating [Ca2+]i increases, and they implicate a role for voltage in “conditioning” [Ca2+]i signals that regulate ion channels for stomatal function.

The yeast halotolerance determinant Hal3p is an inhibitory subunit of the Ppz1p Ser/Thr protein phosphatase

Components of cellular stress responses can be identified by correlating changes in stress tolerance with gain or loss of function of defined genes. Previous work has shown that yeast cells deficient in Ppz1 protein phosphatase or overexpressing Hal3p, a novel regulatory protein of unknown function, exhibit increased resistance to sodium and lithium, whereas cells lacking Hal3p display increased sensitivity. These effects are largely a result of changes in expression of ENA1, encoding the major cation extrusion pump of yeast cells. Disruption or overexpression of HAL3 (also known as SIS2) has no effect on salt tolerance in the absence of PPZ1, suggesting that Hal3p might function upstream of Ppz1p in a novel signal transduction pathway. Hal3p is recovered from crude yeast homogenates by using immobilized, bacterially expressed Ppz1p fused to glutathione S-transferase, and it also copurifies with affinity-purified glutathione S-transferase-Ppz1p from yeast extracts. In both cases, the interaction is stronger when only the carboxyl-terminal catalytic phosphatase domain of Ppz1p is expressed. In vitro experiments reveal that the protein phosphatase activity of Ppz1p is inhibited by Hal3p. Overexpression of Hal3p suppresses the reduced growth rate because of the overexpression of Ppz1p and aggravates the lytic phenotype of a slt2/mpk1 mitogen-activated protein kinase mutant (thus mimicking the deletion of PPZ1). Therefore, Hal3p might modulate diverse physiological functions of the Ppz1 phosphatase, such as salt stress tolerance and cell cycle progression, by acting as a inhibitory subunit.

Nuclear-cytoplasmic shuttling of C-ABL tyrosine kinase

The ubiquitously expressed nonreceptor tyrosine kinase c-Abl contains three nuclear localization signals, however, it is found in both the nucleus and the cytoplasm of proliferating fibroblasts. A rapid and transient loss of c-Abl from the nucleus is observed upon the initial adhesion of fibroblasts onto a fibronectin matrix, suggesting the possibility of nuclear export [Lewis, J., Baskaran, R., Taagepera, S., Schwartz, M. & Wang, J. (1996) Proc. Natl. Acad. Sci. USA 93, 15174–15179]. Here we show that the C terminus of c-Abl does indeed contain a functional nuclear export signal (NES) with the characteristic leucine-rich motif. The c-Abl NES can functionally complement an NES-defective HIV Rev protein (RevΔ3NI) and can mediate the nuclear export of glutathione-S-transferase. The c-Abl NES function is sensitive to the nuclear export inhibitor leptomycin B. Mutation of a single leucine (L1064A) in the c-Abl NES abrogates export function. The NES-mutated c-Abl, termed c-Abl NES(−), is localized exclusively to the nucleus. Treatment of cells with leptomycin B also leads to the nuclear accumulation of wild-type c-Abl protein. The c-Abl NES(−) is not lost from the nucleus when detached fibroblasts are replated onto fibronectin matrix. Taken together, these results demonstrate that c-Abl shuttles continuously between the nucleus and the cytoplasm and that the rate of nuclear import and export can be modulated by the adherence status of fibroblastic cells.

Cross-lineage expression of Ig-β (B29) in thymocytes: Positive and negative gene regulation to establish T cell identity

Developmental commitment involves activation of lineage-specific genes, stabilization of a lineage-specific gene expression program, and permanent inhibition of inappropriate characteristics. To determine how these processes are coordinated in early T cell development, the expression of T and B lineage-specific genes was assessed in staged subsets of immature thymocytes. T lineage characteristics are acquired sequentially, with germ-line T cell antigen receptor-β transcripts detected very early, followed by CD3ɛ and terminal deoxynucleotidyl transferase, then pTα, and finally RAG1. Only RAG1 expression coincides with commitment. Thus, much T lineage gene expression precedes commitment and does not depend on it. Early in the course of commitment to the T lineage, thymocytes lose the ability to develop into B cells. To understand how this occurs, we also examined expression of well defined B lineage-specific genes. Although λ5 and Ig-α are not expressed, the μ0 and Iμ transcripts from the unrearranged IgH locus are expressed early, in distinct patterns, then repressed just before RAG1 expression. By contrast, RNA encoding the B cell receptor component Ig-β was found to be transcribed in all immature thymocyte subpopulations and throughout most thymocyte differentiation. Ig-β expression is down-regulated only during positive selection of CD4+CD8– cells. Thus several key participants in the B cell developmental program are expressed in non-B lineage-committed cells, and one is maintained even through commitment to an alternative lineage, and repressed only after extensive T lineage differentiation. The results show that transcriptional activation of “lymphocyte-specific” genes can occur in uncommitted precursors, and that T lineage commitment is a composite of distinct positive and negative regulatory events.

Histidine kinase activity of the ETR1 ethylene receptor from Arabidopsis

ETR1 represents a prototypical ethylene receptor. Homologues of ETR1 have been identified in Arabidopsis as well as in other plant species, indicating that ethylene perception involves a family of receptors and that the mechanism of ethylene perception is conserved in plants. The amino-terminal half of ETR1 contains a hydrophobic domain responsible for ethylene binding and membrane localization. The carboxyl-terminal half of the polypeptide contains domains with homology to histidine kinases and response regulators, signaling motifs originally identified in bacteria. The putative histidine kinase domain of ETR1 was expressed in yeast as a fusion protein with glutathione S-transferase and affinity purified. Autophosphorylation of the purified fusion protein was observed on incubation with radiolabeled ATP. The incorporated phosphate was resistant to treatment with 3 M NaOH, but was sensitive to 1 M HCl, consistent with phosphorylation of histidine. Autophosphorylation was abolished by mutations that eliminated either the presumptive site of phosphorylation (His-353) or putative catalytic residues within the kinase domain. Truncations were used to delineate the region required for histidine kinase activity. An examination of cation requirements indicated that ETR1 requires Mn2+ for autophosphorylation. These results demonstrate that higher plants contain proteins with histidine kinase activity. Furthermore, these results indicate that aspects of ethylene signaling may be regulated by changes in histidine kinase activity of the receptor.

A gene cluster for macrolide antibiotic biosynthesis in Streptomyces venezuelae: Architecture of metabolic diversity

In a survey of microbial systems capable of generating unusual metabolite structural variability, Streptomyces venezuelae ATCC 15439 is notable in its ability to produce two distinct groups of macrolide antibiotics. Methymycin and neomethymycin are derived from the 12-membered ring macrolactone 10-deoxymethynolide, whereas narbomycin and pikromycin are derived from the 14-membered ring macrolactone, narbonolide. This report describes the cloning and characterization of the biosynthetic gene cluster for these antibiotics. Central to the cluster is a polyketide synthase locus (pikA) that encodes a six-module system comprised of four multifunctional proteins, in addition to a type II thioesterase (TEII). Immediately downstream is a set of genes for desosamine biosynthesis (des) and macrolide ring hydroxylation. The study suggests that Pik TEII plays a role in forming a metabolic branch through which polyketides of different chain length are generated, and the glycosyl transferase (encoded by desVII) has the ability to catalyze glycosylation of both the 12- and 14-membered ring macrolactones. Moreover, the pikC-encoded P450 hydroxylase provides yet another layer of structural variability by introducing regiochemical diversity into the macrolide ring systems. The data support the notion that the architecture of the pik gene cluster as well as the unusual substrate specificity of particular enzymes contributes to its ability to generate four macrolide antibiotics.

Single prenyl-binding site on protein prenyl transferases

Three distinct protein prenyl transferases, one protein farnesyl transferase (FTase) and two protein geranylgeranyl transferases (GGTase), catalyze prenylation of many cellular proteins. One group of protein substrates contains a C-terminal CAAX motif (C is Cys, A is aliphatic, and X is a variety of amino acids) in which the single cysteine residue is modified with either farnesyl or geranylgeranyl (GG) by FTase or GGTase type-I (GGTase-I), respectively. Rab proteins constitute a second group of substrates that contain a C-terminal double-cysteine motif (such as XXCC in Rab1a) in which both cysteines are geranylgeranylated by Rab GG transferase (RabGGTase). Previous characterization of CAAX prenyl transferases showed that the enzymes form stable complexes with their prenyl pyrophosphate substrates, acting as prenyl carriers. We developed a prenyl-binding assay and show that RabGGTase has a prenyl carrier function similar to the CAAX prenyl transferases. Stable RabGGTase:GG pyrophosphate (GGPP), FTase:GGPP, and GGTase-I:GGPP complexes show 1:1 (enzyme:GGPP) stoichiometry. Chromatographic analysis of prenylated products after single turnover reactions by using isolated RabGGTase:GGPP complex revealed that Rab is mono-geranylgeranylated. This study establishes that all three protein prenyl transferases contain a single prenyl-binding site and suggests that RabGGTase transfers two GG groups to Rabs in independent and consecutive reactions.