開発戦略としてのPro-Poor Growth －貧困層への雇用創出－

本稿は、後発開発途上国においてpro-poor growthを達成する開発戦略を、産業政策との関連から研究したものである。アジアとアフリカの経済発展プロセスにおいて、貧困層にどの産業が雇用機会を提供するか、を研究した。その結果明らかになったのは、1970-90年代の典型的な東アジア高成長経済であるタイと台湾において、貧困層に最も大きな雇用機会を与えたのが農業部門であったのに対して、貧困層の雇用機会増に最も貢献したのが製造業部門であったということである。同様の傾向は、アフリカの輸出指向成長国であるモーリシャスについても観察された。一方、グローバリゼーションの活力が十分に生かされなかったマラウィ、南アフリカ、インドにおいては、製造業が同様の雇用機会を貧困層に提供できなかった。結論として導かれるのは、典型的な後発発展途上国においては、雇用に関して農業のシェアが製造業のシェアを上回るのが一般的であるものの、もし比較優位がグローバリゼーションの中で顕在化できるのであれば、貧困層の雇用機会増に関して、農業よりも製造業の貢献度が大きくなりうる、ということである。一国全体の経済発展という観点のみならず、貧困層に利益をもたらすpro-poor growthという文脈においても、製造業の果たす役割が無視し得ないことが明らかになった。

Factores transcripcionales de la clase DOF en Brachypodium distachyon: caracterización molecular de BdDOF24 durante la germinación de las semillas

La semilla es el órgano que garantiza la propagación y continuidad evolutiva de las plantas espermatofitas y constituye un elemento indispensable en la alimentación humana y animal. La semilla de cereales acumula en el endospermo durante la maduración, mayoritariamente, almidón y proteínas de reserva. Estas reservas son hidrolizadas en la germinación por hidrolasas sintetizadas en la aleurona en respuesta a giberelinas (GA), siendo la principal fuente de energía hasta que la plántula emergente es fotosintéticamente activa. Ambas fases del desarrollo de la semilla, están reguladas por una red de factores de transcripción (TF) que unen motivos conservados en cis- en los promotores de sus genes diana. Los TFs son proteínas que han desempeñado un papel central en la evolución y en el proceso de domesticación, siendo uno de los principales mecanismos de regulación génica; en torno al 7% de los genes de plantas codifican TFs. Atendiendo al motivo de unión a DNA, éstos, se han clasificado en familias. La familia DOF (DNA binding with One Finger) participa en procesos vitales exclusivos de plantas superiores y sus ancestros cercanos (algas, musgos y helechos). En las semillas de las Triticeae (subfamilia Pooideae), se han identificado varias proteínas DOF que desempeñan un papel fundamental en la regulación de la expresión génica. Brachypodium distachyon es la primera especie de la subfamilia Pooideae cuyo genoma (272 Mbp) ha sido secuenciado. Su pequeño tamaño, ciclo de vida corto, y la posibilidad de ser transformado por Agrobacterium tumefaciens (plásmido Ti), hacen que sea el sistema modelo para el estudio de cereales de la tribu Triticeae con gran importancia agronómica mundial, como son el trigo y la cebada. En este trabajo, se han identificado 27 genes Dof en el genoma de B. distachyon y se han establecido las relaciones evolutivas entre estos genes Dof y los de cebada (subfamilia Pooideae) y de arroz (subfamilia Oryzoideae), construyendo un árbol filogenético en base al alineamiento múltiple del dominio DOF. La cebada contiene 26 genes Dof y en arroz se han anotado 30. El análisis filogenético establece cuatro grupos de genes ortólogos (MCOGs: Major Clusters of Orthologous Genes), que están validados por motivos conservados adicionales, además del dominio DOF, entre las secuencias de las proteínas de un mismo MCOG. El estudio global de expresión en diferentes órganos establece un grupo de nueve genes BdDof expresados abundantemente y/o preferencialmente en semillas. El estudio detallado de expresión de estos genes durante la maduración y germinación muestra que BdDof24, ortólogo putativo a BPBF-HvDOF24 de cebada, es el gen más abundante en las semillas en germinación de B. distachyon. La regulación transcripcional de los genes que codifican hidrolasas en la aleurona de las semillas de cereales durante la post‐germinación ha puesto de manifiesto la existencia en sus promotores de un motivo tripartito en cis- conservado GARC (GA-Responsive Complex), que unen TFs de la clase MYB-R2R3, DOF y MYBR1-SHAQKYF. En esta tesis, se ha caracterizado el gen BdCathB de Brachypodium que codifica una proteasa tipo catepsina B y es ortólogo a los genes Al21 de trigo y HvCathB de cebada, así como los TFs responsables de su regulación transcripcional BdDOF24 y BdGAMYB (ortólogo a HvGAMYB). El análisis in silico del promotor BdCathB ha identificado un motivo GARC conservado, en posición y secuencia, con sus ortólogos en trigo y cebada. La expresión de BdCathB se induce durante la germinación, así como la de los genes BdDof24 y BdGamyb. Además, los TFs BdDOF24 y BdGAMYB interaccionan en el sistema de dos híbridos de levadura e in planta en experimentos de complementación bimolecular fluorescente. En capas de aleurona de cebada, BdGAMYB activa el promotor BdCathB, mientras que BdDOF24 lo reprime; este resultado es similar al obtenido con los TFs ortólogos de cebada BPBF-HvDOF24 y HvGAMYB. Sin embargo, cuando las células de aleurona se transforman simultáneamente con los dos TFs, BdDOF24 tiene un efecto aditivo sobre la trans-activación mediada por BdGAMYB, mientras que su ortólogo BPBF-HvDOF24 produce el efecto contrario, revirtiendo el efecto de HvGAMYB sobre el promotor BdCathB. Las diferencias entre las secuencias deducidas de las proteínas BdDOF24 y BPBF-HvDOF24 podrían explicar las funciones opuestas que desempeñan en su interacción con GAMYB. Resultados preliminares con líneas de inserción de T-DNA y de sobre-expresión estable de BdGamyb, apoyan los resultados obtenidos en expresión transitoria. Además las líneas homocigotas knock-out para el gen BdGamyb presentan alteraciones en anteras y polen y no producen semillas viables. ABSTRACT The seed is the plant organ of the spermatophytes responsible for the dispersion and survival in the course of evolution. In addition, it constitutes one of the most importan elements of human food and animal feed. The main reserves accumulated in the endosperm of cereal seeds through the maturation phase of development are starch and proteins. Its degradation by hydrolases synthetized in aleurone cells in response to GA upon germination provides energy, carbon and nitrogen to the emerging seedling before it acquires complete photosynthetic capacity. Both phases of seed development are controlled by a network of transcription factors (TFs) that interact with specific cis- elements in the promoters of their target genes. TFs are proteins that have played a central role during evolution and domestication, being one of the most important regulatory mechanisms of gene expression. Around 7% of genes in plant genomes encode TFs. Based on the DNA binding motif, TFs are classified into families. The DOF (DNA binding with One Finger) family is involved in specific processes of plants and its ancestors (algae, mosses and ferns). Several DOF proteins have been described to play important roles in the regulation of genes in seeds of the Triticeae tribe (Pooideae subfamily). Brachypodium distachyon is the first member of the Pooideae subfamily to be sequenced. Its small size and compact structured genome (272 Mbp), the short life cycle, small plant size and the possibility of being transformed with Agrobacterium tumefaciens (Ti-plasmid) make Brachypodium the model system for comparative studies within cereals of the Triticeae tribe that have big economic value such as wheat and barley. In this study, 27 Dof genes have been identified in the genome of B. distachyon and the evolutionary relationships among these Dof genes and those frome barley (Pooideae subfamily) and those from rice (Oryzoideae subfamily) have been established by building a phylogenetic tree based on the multiple alignment of the DOF DNA binding domains. The barley genome (Hordeum vulgare) contains 26 Dof genes and in rice (Oryza sativa) 30 genes have been annotated. The phylogenetic analysis establishes four Major Clusters of Orthologous Genes (MCOGs) that are supported by additional conserved motives out of the DOF domain, between proteins of the same MCOG. The global expression study of BdDof genes in different organs and tissues classifies BdDof genes into two groups; nine of the 27 BdDof genes are abundantly or preferentially expressed in seeds. A more detailed expression analysis of these genes during seed maturation and germination shows that BdDof24, orholog to barley BPBF-HvDof24, is the most abundantly expressed gene in germinating seeds. Transcriptional regulation studies of genes that encode hydrolases in aleurone cells during post-germination of cereal seeds, have identified in their promoters a tripartite conserved cis- motif GARC (GA-Responsive Complex) that binds TFs of the MYB-R2R3, DOF and MYBR1-SHAQKYF families. In this thesis, the characterization of the BdCathB gene, encoding a Cathepsin B-like protease and that is ortholog to the wheat Al21 and the barley HvCathB genes, has been done and its transcriptional regulation by the TFs BdDOF24 and BdGAMYB (ortholog to HvGAMYB) studied. The in silico analysis of the BdCathB promoter sequence has identified a GARC motif. BdCathB expression is induced upon germination, as well as, those of BdDof24 and BdGamyb genes. Moreover, BdDOF24 and BdGAMYB interact in yeast (Yeast 2 Hybrid System, Y2HS) and in planta (Bimolecular Fluorecence Complementation, BiFC). In transient assays in aleurone cells, BdGAMYB activates the BdCathB promoter, whereas BdDOF24 is a transcriptional repressor, this result is similar to that obtained with the barley orthologous genes BPBF-HvDOF24 and HvGAMYB. However, when aleurone cells are simultaneously transformed with both TFs, BdDOF24 has an additive effect to the trans-activation mediated by BdGAMYB, while its ortholog BPBF-HvDOF24 produces an opposite effect by reducing the HvGAMYB activation of the BdCathB promoter. The differences among the deduced protein sequences between BdDOF24 and BPBF-HvDOF24 could explain their opposite functions in the interaction with GAMYB protein. Preliminary results of T-DNA insertion (K.O.) and stable over-expression lines of BdGamyb support the data obtained in transient expression assays. In addition, the BdGamyb homozygous T-DNA insertion (K.O.) lines have anther and pollen alterations and they do not produce viable seeds.

Metagenomic Anlaysis of microsymbiont selection by the legume plant host

Rhizobium leguminosarum bv.viciae is able to establish nitrogen-fixing symbioses with legumes of the genera Pisum, Lens, Lathyrus and Vicia. Classic studies using trap plants (Laguerre et al., Young et al.) provided evidence that different plant hosts are able to select different rhizobial genotypes among those available in a given soil. However, these studies were necessarily limited by the paucity of relevant biodiversity markers. We have now reappraised this problem with the help of genomic tools. A well-characterized agricultural soil (INRA Bretennieres) was used as source of rhizobia. Plants of Pisum sativum, Lens culinaris, Vicia sativa and V. faba were used as traps. Isolates from 100 nodules were pooled, and DNA from each pool was sequenced (BGI-Hong Kong; Illumina Hiseq 2000, 500 bp PE libraries, 100 bp reads, 12 Mreads). Reads were quality filtered (FastQC, Trimmomatic), mapped against reference R. leguminosarum genomes (Bowtie2, Samtools), and visualized (IGV). An important fraction of the filtered reads were not recruited by reference genomes, suggesting that plant isolates contain genes that are not present in the reference genomes. For this study, we focused on three conserved genomic regions: 16S-23S rDNA, atpD and nodDABC, and a Single Nucleotide Polymorphism (SNP) analysis was carried out with meta / multigenomes from each plant. Although the level of polymorphism varied (lowest in the rRNA region), polymorphic sites could be identified that define the specific soil population vs. reference genomes. More importantly, a plant-specific SNP distribution was observed. This could be confirmed with many other regions extracted from the reference genomes (data not shown). Our results confirm at the genomic level previous observations regarding plant selection of specific genotypes. We expect that further, ongoing comparative studies on differential meta / multigenomic sequences will identify specific gene components of the plant-selected genotypes

Las teorías del gótico y su representación gráfica en España el último tercio del siglo XIX. Un estudio sobre: Some Account of Gothic Architecture in Spain, de George Edmund Street

La tesis se centra en el estudio, descripción y análisis del libro publicado por el arquitecto británico George Edmund Street en 1865, bajo el título Some Account of Gothic Architecture in Spain. El libro dio a conocer una de la colecciones más importantes de dibujos realizada en el siglo XIX sobre la arquitectura gótica española, y por lo tanto fue una primera referencia para su estudio, hasta bien entrado el siglo veinte. El volumen incluyó 107 grabados de diversos tipos de edificios con perspectivas y detalles, y 25 láminas con 45 planos de plantas de iglesias y claustros, muchos de ellos publicados por primera vez. Gracias a varias campañas de verano por la mitad norte del país, la casi inexplorada -desde un punto de vista académico- arquitectura española medieval fue finalmente descubierta. Este hecho conduce a una pregunta intrigante que está en el origen de esta investigación ¿cómo pudo Street en pocos viajes sentar las bases de la historia del gótico español que hasta entonces los estudiosos españoles no habían sido capaces de definir? Esta tesis comienza describiendo la obra de Street en su contexto cultural con un breve repaso a su biografía y a su posición profesional y teórica. También su relación con las personas más representativas que participaron en el estudio de la arquitectura gótica, como Robert Willis, William Whewell, Augustus Pugin, o George G. Scott. Se ha prestado especial atención, en explicar su papel relevante en el Gothic Revival, para entender el significado de su interés en la arquitectura gótica continental. Estos capítulos preliminares son seguidos por una revisión del papel del dibujo como herramienta para la arquitectura de los viajeros en sus rutas en busca de la arquitectura gótica. También se trata la influencia de la Royal Academy y sus académicos, (entre los cuales estuvo Street) y su formación académica. Finalmente la tesis entra en el estudio de los planos arquitectónicos que Street hizo durante sus viajes continentales de arquitectura, seguido por una descripción detallada de sus dibujos de España, analizando su método, su técnica, y las nuevas características aportadas, que fueron una novedad en el contexto español. También se lleva a cabo algunos estudios comparativos de los dibujos de España, gracias a una recopilación exhaustiva de bocetos y dibujos originales de Street, que en su gran mayoría se conservan en los archivos del RIBA, cotejándolos con sus versiones finales, con dibujos de la época de otros autores sobre los mismos edificios, y con fotos recientes. La tesis deja claro por qué y cómo Street, gracias a su soporte teórico y habilidades para el dibujo, pudo realizar algo que había pasado desapercibido para los estudiosos españoles de la época (construcción, historia de los estilos, señalamiento de períodos constructivos), lo que le permitió encontrar el lugar adecuado de la arquitectura gótica española en la historia y en el mapa de la arquitectura gótica europea. ABSTRACT This thesis focuses on the study, description and analysis of the book published by the British architect George Edmund Street in 1865, under the title Some Account of Gothic Architecture in Spain. The book displayed one of the most significant collections of drawings on Spanish Gothic Architecture made in the nineteenth century, and therefore was a first reference for its study, until well into the twentieth century. The book included 107 engravings, the surveying of various types of buildings with perspectives and details, and 25 sheets containing 45 ground plans of churches and cloisters, many of them new and published for the first time. Thanks to several summer campaigns in the north half of the country, the almost unexplored -from a scholar point of view- medieval Spanish architecture was eventually revealed. This fact lead to an intriguing question that is at the origin of this research: how could Street in a few trips lay the foundations of the history of Spanish Gothic that until then Spanish scholars had not been able to define? This thesis begins inscribing this Street's work in his cultural context. A brief review of his biography and professional and theoretical positions has been seen as necessary. Also his debts and relationship with the most representative people involved in the study of Gothic architecture, like Robert Willis, William Whewell, Augustus Pugin, or George G. Scott are discussed. Special attention has been paid, taken into account his relevant role in the Gothic Revival, to understand the significance of his interest in continental Gothic architecture. These preliminary chapters are followed by a review of role of drawing as a tool for Architectural travellers in their Tours in search of the Gothic architecture. The influence of the Royal Academy and its academicians, (among which was Street) and his educational background are here tackled. Eventually this thesis enters into the study of the architectural drawings Street made during his continental architectural journeys, which is followed by a detailed description and analysis of the Spanish ones: his methods, his technique, and the new features which were a novelty in the Spanish context are explored. Also in this thesis is carried out some comparative studies thanks to a previous exhaustive gathering of Street's sketches and original drawings, most of which are preserved in the RIBA archives. Their final versions, drawings of the same buildings from other contemporary draughtsman and pictures of their current state are compared with them. This thesis makes clear why and how Street thanks to his theoretical back-ground and portraying skills could realize what have passed unnoticed by contemporary Spanish scholars (construction, genealogy of forms, dating of periods) allowing him to find the proper place of the Spanish architecture in the history and the map of European Gothic architecture.

Energy efficiency evaluation of zero energy houses

Given the global energy and environmental situation, the European Union has been issuing directives with increasingly demanding requirements in term of the energy efficiency in buildings. The international competition of sustainable houses, Solar Decathlon Europe (SDE), is aligned with these European objectives. SDE houses are low energy solar buildings that must reach the near to zero energy houses’ goal. In the 2012 edition, in order to emphasize its significance, the Energy Efficiency Contest was added. SDE houses’ interior comfort, functioning and energy performance is monitored. The monitoring data can give an idea about the efficiency of the houses. However, a jury comprised by international experts is responsible for carrying out the houses energy efficiency evaluation. Passive strategies and houses services are analyzed. Additionally, the jury's assessment has been compared with the behavior of the houses during the monitoring period. Comparative studies make emphasis on the energy aspects, houses functioning and their interior comfort. Conclusions include thoughts related with the evaluation process, the results of the comparative studies and suggestions for the next competitions.

JUVENTUDE, RELIGIÃO E PODER: UM ESTUDO DOS CONFLITOS GERACIONAIS NA IGREJA PENTECOSTAL DEUS É AMOR NA PERIFERIA DE GOIÂNIA

A presente pesquisa, situada no âmbito teórico das Ciências Sociais e Religião, linha de pesquisa em Instituições e Movimentos Religiosos, analisa os conflitos geracionais na Igreja Pentecostal Deus é Amor (IPDA), em regiões periféricas de Goiânia. O objeto da pesquisa é o jovem e sua possível influência nos processos internos de conflitos geracionais dentro desta instituição. A IPDA é um importante ramo do pentecostalismo clássico, com ampla atuação em diferentes capitais e cidades brasileiras, e com significativo exercício em regiões periféricas de grandes centros urbanos. Além da inserção em território nacional, a Igreja Pentecostal Deus é Amor (IPDA), tem presença notória em diferentes países da América Latina e representantes em todos os continentes. Considerando sua existência além do território brasileiro, estima-se que a Igreja Pentecostal Deus é Amor, atinja ao número de 136 países, totalizando 11 mil templos. O método utilizado na investigação cientifica é a Análise Sociológica, para isso privilegia metodologias de entrevistas semi estruturadas e pesquisa bibliográfica. A relevância da pesquisa esta na sua intencionalidade de apontar fatores novos na compreensão do complexo fenômeno religioso bem como suas implicações no cenário religioso da sociedade brasileira. Também é contribuir para o debate acadêmico de estudiosos da área da sociologia da religião com o interesse em realizar estudos comparativos de conflitos geracionais existentes no segmento pentecostal e outras instituições religiosas, bem como do tema pentecostalismo e periferia.(AU)

JUVENTUDE, RELIGIÃO E PODER: UM ESTUDO DOS CONFLITOS GERACIONAIS NA IGREJA PENTECOSTAL DEUS É AMOR NA PERIFERIA DE GOIÂNIA

A presente pesquisa, situada no âmbito teórico das Ciências Sociais e Religião, linha de pesquisa em Instituições e Movimentos Religiosos, analisa os conflitos geracionais na Igreja Pentecostal Deus é Amor (IPDA), em regiões periféricas de Goiânia. O objeto da pesquisa é o jovem e sua possível influência nos processos internos de conflitos geracionais dentro desta instituição. A IPDA é um importante ramo do pentecostalismo clássico, com ampla atuação em diferentes capitais e cidades brasileiras, e com significativo exercício em regiões periféricas de grandes centros urbanos. Além da inserção em território nacional, a Igreja Pentecostal Deus é Amor (IPDA), tem presença notória em diferentes países da América Latina e representantes em todos os continentes. Considerando sua existência além do território brasileiro, estima-se que a Igreja Pentecostal Deus é Amor, atinja ao número de 136 países, totalizando 11 mil templos. O método utilizado na investigação cientifica é a Análise Sociológica, para isso privilegia metodologias de entrevistas semi estruturadas e pesquisa bibliográfica. A relevância da pesquisa esta na sua intencionalidade de apontar fatores novos na compreensão do complexo fenômeno religioso bem como suas implicações no cenário religioso da sociedade brasileira. Também é contribuir para o debate acadêmico de estudiosos da área da sociologia da religião com o interesse em realizar estudos comparativos de conflitos geracionais existentes no segmento pentecostal e outras instituições religiosas, bem como do tema pentecostalismo e periferia.(AU)

A structural view of evolutionary divergence

Two directed evolution experiments on p-nitrobenzyl esterase yielded one enzyme with a 100-fold increased activity in aqueous-organic solvents and another with a 17°C increase in thermostability. Structures of the wild type and its organophilic and thermophilic counterparts are presented at resolutions of 1.5 Å, 1.6 Å, and 2.0 Å, respectively. These structures identify groups of interacting mutations and demonstrate how directed evolution can traverse complex fitness landscapes. Early-generation mutations stabilize flexible loops not visible in the wild-type structure and set the stage for further beneficial mutations in later generations. The mutations exert their influence on the esterase structure over large distances, in a manner that would be difficult to predict. The loops with the largest structural changes generally are not the sites of mutations. Similarly, none of the seven amino acid substitutions in the organophile are in the active site, even though the enzyme experiences significant changes in the organization of this site. In addition to reduction of surface loop flexibility, thermostability in the evolved esterase results from altered core packing, helix stabilization, and the acquisition of surface salt bridges, in agreement with other comparative studies of mesophilic and thermophilic enzymes. Crystallographic analysis of the wild type and its evolved counterparts reveals networks of mutations that collectively reorganize the active site. Interestingly, the changes that led to diversity within the α/β hydrolase enzyme family and the reorganization seen in this study result from main-chain movements.

Statistical methods for characterizing similarities and differences between semantic structures

This paper describes a variety of statistical methods for obtaining precise quantitative estimates of the similarities and differences in the structures of semantic domains in different languages. The methods include comparing mean correlations within and between groups, principal components analysis of interspeaker correlations, and analysis of variance of speaker by question data. Methods for graphical displays of the results are also presented. The methods give convergent results that are mutually supportive and equivalent under suitable interpretation. The methods are illustrated on the semantic domain of emotion terms in a comparison of the semantic structures of native English and native Japanese speaking subjects. We suggest that, in comparative studies concerning the extent to which semantic structures are universally shared or culture-specific, both similarities and differences should be measured and compared rather than placing total emphasis on one or the other polar position.

Cochlear mechanisms from a phylogenetic viewpoint

The hearing organ of the inner ear was the last of the paired sense organs of amniotes to undergo formative evolution. As a mechanical sensory organ, the inner-ear hearing organ's function depends highly on its physical structure. Comparative studies suggest that the hearing organ of the earliest amniote vertebrates was small and simple, but possessed hair cells with a cochlear amplifier mechanism, electrical frequency tuning, and incipient micromechanical tuning. The separation of the different groups of amniotes from the stem reptiles occurred relatively early, with the ancestors of the mammals branching off first, approximately 320 million years ago. The evolution of the hearing organ in the three major lines of the descendents of the stem reptiles (e.g., mammals, birds-crocodiles, and lizards-snakes) thus occurred independently over long periods of time. Dramatic and parallel improvements in the middle ear initiated papillar elongation in all lineages, accompanied by increased numbers of sensory cells with enhanced micromechanical tuning and group-specific hair-cell specializations that resulted in unique morphological configurations. This review aims not only to compare structure and function across classification boundaries (the comparative approach), but also to assess how and to what extent fundamental mechanisms were influenced by selection pressures in times past (the phylogenetic viewpoint).

On the origin of and phylogenetic relationships among living amphibians

The phylogenetic relationships among the three orders of modern amphibians (Caudata, Gymnophiona, and Anura) have been estimated based on both morphological and molecular evidence. Most morphological and paleontological studies of living and fossil amphibians support the hypothesis that salamanders and frogs are sister lineages (the Batrachia hypothesis) and that caecilians are more distantly related. Previous interpretations of molecular data based on nuclear and mitochondrial rRNA sequences suggested that salamanders and caecilians are sister groups to the exclusion of frogs. In an attempt to resolve this apparent conflict, the complete mitochondrial genomes of a salamander (Mertensiella luschani) and a caecilian (Typhlonectes natans) were determined (16,656 and 17,005 bp, respectively) and compared with previously published sequences from a frog (Xenopus laevis) and several other groups of vertebrates. Phylogenetic analyses of the mitochondrial data supported with high bootstrap values the monophyly of living amphibians with respect to other living groups of tetrapods, and a sister group relationship of salamanders and frogs. The lack of phylogenetically informative sites in the previous rRNA data sets (because of its shorter size and higher among-site rate variation) likely explains the discrepancy between our results and those based on previous molecular data. Strong support of the Batrachia hypothesis from both molecule- and morphology-based studies provides a robust phylogenetic framework that will be helpful to comparative studies among the three living orders of amphibians and will permit better understanding of the considerably divergent vertebral, brain, and digit developmental patterns found in frogs and salamanders.

The Kinetics of Zeaxanthin Formation Is Retarded by Dicyclohexylcarbodiimide1

The de-epoxidation of violaxanthin to antheraxanthin (Anth) and zeaxanthin (Zeax) in the xanthophyll cycle of higher plants and the generation of nonphotochemical fluorescence quenching in the antenna of photosystem II (PSII) are induced by acidification of the thylakoid lumen. Dicyclohexylcarbodiimide (DCCD) has been shown (a) to bind to lumen-exposed carboxy groups of antenna proteins and (b) to inhibit the pH-dependent fluorescence quenching. The possible influence of DCCD on the de-epoxidation reactions has been investigated in isolated pea (Pisum sativum L.) thylakoids. The Zeax formation was found to be slowed down in the presence of DCCD. The second step (Anth → Zeax) of the reaction sequence seemed to be more affected than the violaxanthin → Anth conversion. Comparative studies with antenna-depleted thylakoids from plants grown under intermittent light and with unstacked thylakoids were in agreement with the assumption that binding of DCCD to antenna proteins is probably responsible for the retarded kinetics. Analyses of the DCCD-induced alterations in different antenna subcomplexes showed that Zeax formation in the PSII antenna proteins was predominantly influenced by DCCD, whereas Zeax formation in photosystem I was nearly unaffected. Our data support the suggestion that DCCD binding to PSII antenna proteins is responsible for the observed alterations in xanthophyll conversion.

Characterization of SKT1, an Inwardly Rectifying Potassium Channel from Potato, by Heterologous Expression in Insect Cells

A cDNA encoding a novel, inwardly rectifying K+ (K+in) channel protein, SKT1, was cloned from potato (Solanum tuberosum L.). SKT1 is related to members of the AKT family of K+in channels previously identified in Arabidopsis thaliana and potato. Skt1 mRNA is most strongly expressed in leaf epidermal fragments and in roots. In electrophysiological, whole-cell, patch-clamp measurements performed on baculovirus-infected insect (Spodoptera frugiperda) cells, SKT1 was identified as a K+in channel that activates with slow kinetics by hyperpolarizing voltage pulses to more negative potentials than −60 mV. The pharmacological inhibitor Cs+, when applied externally, inhibited SKT1-mediated K+in currents half-maximally with an inhibitor concentration (IC50) of 105 μm. An almost identical high Cs+ sensitivity (IC50 = 90 μm) was found for the potato guard-cell K+in channel KST1 after expression in insect cells. SKT1 currents were reversibly activated by a shift in external pH from 6.6 to 5.5, which indicates a physiological role for pH-dependent regulation of AKT-type K+in channels. Comparative studies revealed generally higher current amplitudes for KST1-expressing cells than for SKT1-expressing insect cells, which correlated with a higher targeting efficiency of the KST1 protein to the insect cell's plasma membrane, as demonstrated by fusions to green fluorescence protein.

Up-regulation of pressure-activated Ca(2+)-permeable cation channel in intact vascular endothelium of hypertensive rats.

In endothelial cells, stretch-activated cation channels have been proposed to act as mechanosensors for changes in hemodynamic forces. We have identified a novel mechanosensitive pressure-activated channel in intact endothelium from rat aorta and mesenteric artery. The 18-pS cation channel responded with a multifold increase in channel activity when positive pressure was applied to the luminal cell surface with the patch pipette and inactivated at negative pipette pressure. Channel permeability ratio for K+, Na+, and Ca2+ ions was 1:0.98:0.23. Ca2+ influx through the channel was sufficient to activate a neighboring Ca2(+)-dependent K+ channel. Hemodynamic forces are chronically disturbed in arterial hypertension. Endothelial cell dysfunction has been implicated in the pathogenesis of arterial hypertension. In two comparative studies, density of the pressure-activated channel was found to be significantly higher in spontaneously hypertensive rats and renovascular hypertensive rats compared with their respective normotensive controls. Channel activity presumably leads to mechanosensitive Ca2+ influx and induces cell hyperpolarization by K+ channel activity. Both Ca2+ influx and hyperpolarization are known to induce a vasodilatory endothelial response by stimulating endothelial nitric oxide (NO) production. Up-regulation of channel density in hypertension could, therefore, represent a counterregulatory mechanism of vascular endothelium.

Transgenic mice carrying a human mutant superoxide dismutase transgene develop neuronal cytoskeletal pathology resembling human amyotrophic lateral sclerosis lesions.

Mutations in the human Cu,Zn superoxide dismutase gene (SOD1) are found in 20% of kindreds with familial amyotrophic lateral sclerosis. Transgenic mice (line G1H) expressing a human SOD1 containing a mutation of Gly-93 --> Ala (G93A) develop a motor neuron disease similar to familial amyotrophic lateral sclerosis, but transgenic mice (line N1029) expressing a wild-type human SOD1 transgene do not. Because neurofilament (NF)-rich inclusions in spinal motor neurons are characteristic of amyotrophic lateral sclerosis, we asked whether mutant G1H and/or N1029 mice develop similar NF lesions. NF inclusions (i.e., spheroids, Lewy body-like inclusions) were first detected in spinal cord motor neurons of the G1H mice at 82 days of age about the time these mice first showed clinical evidence of disease. Other neuronal intermediate filament proteins (alpha-internexin, peripherin) also accumulated in these spheroids. The onset of accumulations of ubiquitin immunoreactivity in the G1H mice paralleled the emergence of vacuoles and NF-rich spheroids in neurons, but they did not colocalize exclusively with spheroids. In contrast, NF inclusions were not seen in the N1029 mice until they were 132 days old, and ubiquitin immunoreactivity was not increased in the N1029 mice even at 199 days of age. Astrocytosis in spinal cord was associated with a marked increase in glial fibrillary acidic protein immunoreactivity in the G1H mice, but not in the N1029 mice. Finally, comparative studies revealed a striking similarity between the cytoskeletal pathology in the G1H transgenic mice and in patients with amyotrophic lateral sclerosis. These findings link a specific SOD1 mutation with alterations in the neuronal cytoskeleton of patients with amyotrophic lateral sclerosis. Thus, neuronal cytoskeletal abnormalities may be implicated in the pathogenesis of human familial amyotrophic lateral sclerosis.