Isolation and characterization of proteins that bind to yeast origins of DNA replication

Yeast chromosomes contain sequences called ARSs which function as origins of replication in vitro and in vivo. We have carried out a systematic deletion analysis of ARS1, allowing us to define three functionally distinct domains, designated A, B, and C. Domain A is a sequence of 11 to 19bp, containing the core consensus element that is required for replication. The core consensus sequence, A/TTTTATPuTTTA/T, is conserved at all ARSs sequenced to date. A fragment containing only element A and 8 flanking nucleotides enables autonomous replication of centromeric plasmids. These plasmids replicate very inefficiently, suggesting that flanking sequences must be important for ARS function. Domain B also provides important sequences needed for efficient replication. Deletion of domain B drastically increases the doubling times of transformants and reduces plasmid stability. Domain B contains a potential consensus sequence conserved at some ARSs which overlaps a region of bent DNA. Mutational analysis suggests this bent DNA may be important for ARS function. Deletion of domain C has only a slight effect on replication of plasmids carrying those deletions.

We have identified a protein called ARS binding factor I (ABF-I) that binds to the HMR-E ARS and ARS1. We have purified this protein to homogeneity using conventional and oligonucleotide affinity chromatography. The protein has an apparent molecular weight of 135kDa and is present at about 700 molecules per diploid cell, based on the yield of purified protein and in situ antibody staining. DNaseI footprinting reveals that ABF-I binds sequence-specifically to an approximately 24bp sequence that overlaps element Bat ARSl. This same protein binds to and protects a similar size region at the HMR-E ARS.

We also find evidence for another ARS binding protein, ABF-III, based on DN asei footprint analysis and gel retardation assays. The protein protects approximately 22bp adjacent to the ABF-I site. There appears to be no interaction between ABF-I and ABF-III despite the proximity of their binding sites.

To address the function of ABF-I in DNA replication, we have cloned the ABF-I gene using rabbit polyclonal anti-sera and murine monoclonal antibodies against ABF-I to screen a λgt11 expression library. Four EcoRI restriction fragments were isolated which encoded proteins that were recognized by both polyclonal and monoclonal antibodies. A gene disruption can now be constructed to determine the in vivo function of ABF-I.

Donati Artis Grammaticae Index et Concordantia

615 p.

Retórica y derecho en la Alta Edad Media: una defensa judicial del siglo IX

[ES] El objetivo de este artículo es Mostrar un aspecto particular de la Retórica en la Alta Edad Media, a saber, la vinculación de esta "Ars" con la práctica judicial y los estudios de Derecho. Para ello, hemoss analizado un documento relativamente "raro" en tanto que es casi el único de sus características en la Hispania del siglo IX: una defensa judicial incluída en el «Epistolario» de Álvaro de Córdoba (n.c. 800), uno de los mejores escritores en lengua latina de su época. La elección del citado documento tiene su razón de ser en la oscuridad e incertidumbre que rodean nuestro conocimiento de las fuentes y de los procedimientos jurídicos de dicha época.

Fish assemblages associated with three types of artificial reefs: density of assemblages and possible impacts on adjacent fish abundance

We evaluated the effectiveness of wooden artificial reefs (ARs) as fish habitat. Three types of ARs, made of cedar logs, broadleaf tree logs, and PVC pipes, respectively, were deployed in triplicate at 8-m depth off Maizuru, Kyoto Prefecture, Sea of Japan, in May 2004. Fish assemblages associated with each of the nine ARs were observed by using SCUBA twice a month for four years. Fish assemblages in the adjacent habitat were also monitored for two years before and four years after reef deployment. In the surveyed areas (ca. 10 m2) associated with each of the cedar, broadleaf, and PVC ARs, the average number of fish species was 4.14, 3.49, and 3.00, and the average number of individuals was 40.7, 27.9, and 20.3, respectively. The estimated biomass was also more greater when associated with the cedar ARs than with other ARs. Visual censuses of the habitat adjacent to the ARs revealed that the number of fish species and the density of individuals were not affected by the deployment of the ARs. Our results support the superiority of cedar as an AR material and indicate that deployment of wooden ARs causes no reduction of fish abundance in adjacent natural reefs.

A Análise de redes sociais e a perspectiva geoespacial: um estudo de caso para avaliar a capilaridade do fluxo de informações em redes socioespaciais.

A noção de rede social e os métodos em análise em redes sociais (ARS) tem atraído considerável interesse e curiosidade para a comunidade científica nestas últimas décadas, assim como a crescente fascinação pública sobre as relações mais complexas da sociedade moderna. A ARS está baseada na importância de relações entre a interação de unidades. Numa análise de rede social a observação dos atributos sociais de um ator pode ser entendida como padrão ou como uma estrutura de relação entre as unidades. Desta forma, quando empregados a uma perspectiva de rede, é possível estudar os padrões de estruturas relacionais diretamente, sem referência a atributos dos indivíduos envolvidos. Na análise convencional de redes sociais a distância geográfica (ou geoespacial) não tem efeito mapeado na rede. Quando utilizada, esta informação normalmente adquire caráter de um atributo complementar associado individualmente a cada ator (ou aglomerados de atores) da própria rede. O objetivo deste trabalho é demonstrar, numa escala local (de maneira mais granular) como o posicionamento geoespacial dos atores, considerado como elementos da rede, pode desvendar aspectos significativos do comportamento destes que não seriam revelados em análises convencionais. No estudo de caso utilizou-se ainda conceitos de outras disciplinas, como a Matemática e a Física, para verificar a capilaridade do fluxo da informação da rede em três perspectivas distintas: a social, a espacial e a socioespacial. O trabalho descreve o campo emergente de pesquisa em ARS, abordando também questões fundamentais sobre como estas diferentes perspectivas podem trazer resultados mais assertivos para orientar uma tomada de decisão no mercado.

Empatia e trajetórias de desenvolvimento em famílias de jovens do Rio de Janeiro

A empatia, definida como a habilidade de compartilhar, compreender e de expressar o entendimento dos pensamentos e sentimentos dos outros, tem sido amplamente estudada. As trajetórias de desenvolvimento, que representam os diversos caminhos que o desenvolvimento do self pode tomar, também têm sido amplamente estudadas por pesquisadores transculturais, geralmente em contextos prototípicos e envolvendo três modelos de desenvolvimento do self: autonomia, interdependência e autonomia relacionada. Estudos anteriores mostraram que os participantes do Rio de Janeiro tendem para a autonomia relacionada, equlibrando características autônomas e relacionais, que são parte do nosso ambiente sociocultural, no qual habilidades sociais, como a empatia, são requeridas. O desenvolvimento da empatia pode ser relacionado à demanda ambiental por comportamentos, de acordo com aspectos sociodemográficos e valores compartilhados. Esta tese visa apresentar dados de 106 famílias do Rio de Janeiro: pai (idade média M = 51,6 anos), mãe (idade média M = 48,3 anos) e seus filhos jovens (17-25 anos, idade média M = 20,9 anos, 67,9% mulheres). A maioria dos pais, mães e dos filhos tinham Ensino Superior incompleto ou acima. Este estudo objetivou investigar a relação entre empatia e os modelos de desenvolvimento de self e a transmissão transgeracional em contexto não prototípico. O Inventário de Empatia de Falcone e colaboradores (IE) foi utilizado para avaliar a empatia. As Escalas de Desenvolvimento do Self de Kağitçibaşi (EDS), (Self Autônomo (AutS), Self Interdependente (IntS) de Self Autônomo-Relacionado (ARS)) foram utilizadas para avaliar o modelo de desenvolvimento do self. Filhos também responderam quantas horas semanais conviviam com pais e mães. Testes t pareados mostraram, como esperado, que os escores de ARS foram os mais altos para todos os participantes. Correlações de Pearson foram feitas entre os escores de IE e EDS de ambos os pais e filhos. Os escores de ARS de pais, mães e filhos correlacionaram-se positivamente, assim como seus escores de IE. Examinando entre instrumentos, para pais e filhos, altos escores em ARS foram correlacionados a altos escores de IE. O tempo de convívio relatado entre pais e filhos correlacionou-se com IntS e IE dos pais. Para testar o modelo teórico proposto de ARS de pais e mães e dos filhos e a empatia de ambos os pais como preditores da empatia dos filhos, análises de regressão foram utilizadas. A empatia dos pais e das mães previu, separadamente, a empatia dos filhos, mas não em conjunto. Nenhum dos escores ARS dos participantes previu o IE dos filhos. Estes resultados favorecem a hipótese da prevalência de selves autônomo-relacionados no Rio de Janeiro. Pais, mães e filhos mostraram similaridades e tiveram relações importantes entre seus escores de EDS, assim como para o IE. Apesar de o modelo com todos juntos não ter sido significativo, separadamente o IE de pais e de mães predisse o IE dos filhos. Este resultado sugere que a empatia parental pode ter algum efeito sobre a dos filhos jovens, mas não o modelo parental de desenvolvimento do self, inesperadamente. De modo interessante, os pais apresentaram um papel importante no desenvolvimento de seus filhos

A baseline assessment of the ecological resources of Jobos Bay, Puerto Rico

This baseline assessment of Jobos Bay and surrounding marine ecosystems consists of a two part series. The first report (Zitello et al., 2008) described the characteristics of the Bay and its watershed, including modeling work related to nutrients and sediment fluxes, based on existing data. The second portion of this assessment, presented in this document, presents the results of new field studies conducted to fill data gaps identified in previous studies, to provide a more complete characterization of Jobos Bay and the surrounding coral reef ecosystems. Specifically, the objective was to establish baseline values for the distribution of habitats, nutrients, contaminants, fi sh, and benthic communities. This baseline assessment is the first step in evaluating the effectiveness in changes in best management practices in the watershed. This baseline assessment is part of the Conservation Effects Assessment Project (CEAP), which is a multi-agency effort to quantify the environmental benefits of conservation practices used by agricultural producers participating in selected U.S. Department of Agriculture (USDA) conservation programs. Partners in the CEAP Jobos Bay Special Emphasis Watershed (SEW) included USDA’s Agricultural Research Service (ARS) and the Natural Resources Conservation Service (NRCS), National Oceanic and Atmospheric Administration (NOAA) and the Government of Puerto Rico. The project originated from an on-going collaboration between USDA and NOAA on the U.S. Coral Reef Task Force. The Jobos Bay watershed was chosen because the predominant land use is agriculture, including agricultural lands adjacent to the Jobos Bay National Estuarine Research Reserve (JBNERR or Reserve), one of NOAA’s 26 National Estuarine Research Reserves (NERR). This report is organized into six chapters that represent a suite of interrelated studies. Chapter 1 provides a short introduction to Jobos Bay, including the land use and hydrology of the watershed. Chapter 2 is focused on benthic mapping and provides the methods and results of newly created benthic maps for Jobos Bay and the surrounding coral reef ecosystem. Chapter 3 presents the results of new surveys of fish, marine debris, and reef communities of the system. Chapter 4 is focused on the distribution of chemical contaminants in sediments within the Bay and corals outside of the Bay. Chapter 5 focuses on quantifying nutrient and pesticide concentrations in the surface waters at the Reserve’s System-Wide Monitoring Program (SWMP) sites. Chapter 6 is a brief summary discussion that highlights key findings of the entire suite of studies.

Technology for the community: SEAFDEC-designed artificial reefs

An account is given of the fabrication and deployment of artificial reefs used in the SEAFDEC/AQD's Community Fishery Resource Management project, which focused on Malalison Island located in west Central Philippines. The project aimed to apply community-based techniques of fishery resource management through the collaboration of community organizations, biologists and social scientists. The 3 types of reefs (building blocks, concrete pipe culvert, and modified concrete pipe culvert) were deployed at Gui-ob reef covering an area of less than 1 ha.

Design and modeling of an integrated device for acoustic resonance spectroscopy

This paper investigates the design and modelling of an integrated device for acoustic resonance spectroscopy (ARS). Miniaturisation of such platforms can be achieved using MEMS technology thereby enabling scaling of device dimensions to investigate smaller specimens while simultaneously operating at higher frequencies. We propose an integrated device where the transducers are mounted in close proximity with the specimen to be analysed (e.g. by integrating ultrasound transducers within a microfluidic channel). A finite element (FE) model and a simplified analytical model have been constructed to predict the acoustic response of a sample embedded in such a device configuration. A FE simulation is performed in COMSOL by embedding the piezoelectric transducers in representative fluid media. Resonant frequencies associated with the measurement can be extracted from this data. The response of various media modelled through FEA matches with analytical predictions for a range of biological media. A variety of biological media may be identified by using the measured resonant frequencies as a signature of relevant physical characteristics. The paper establishes the modelling basis of an integrated acoustic resonant spectrometer that is then applied to examine the impact of geometrical scaling on system resolution. © 2013 IEEE.

肿瘤耐药与心肌肥厚两种生物医学体系的比较蛋白质组学研究

随着人类基因组计划的完成，蛋白质组学研究已成为生命科学乃至自然科学中最活跃的学科领域之一。蛋白质组学将对我们理解后基因组学时代的基因功能有极大的帮助。差异表达蛋白质组学通过对不同样品蛋白质表达水平的比较，已经被广泛地应用在疾病的研究领域。P糖蛋白已经被证实参与介导了在急性粒白血病中发生的多药耐药，然而越来越多的研究结果表明，在急性粒白血病中多药耐药的机制是多因素的。为了寻找白血病肿瘤细胞中与耐药相关的靶蛋白，我们采用蛋白质组学研究技术，对多药耐药细胞系K562／A02的蛋白质表达谱进行了分析。 本研究采用蛋白质组学研究技术，对多药耐药细胞系K562／A02和药物敏感细胞系K562／S的总蛋白质进行双向凝胶电泳（2 DE）的差异分析，结果发现在K562／A02细胞中有41个蛋白质点差异显著，并用基质辅助激光解离飞行时间质谱（MALDI-MS）成功鉴定了大多数的蛋自点。在被鉴定出的差异蛋白质中，有一些是首次发现与耐药相关，包括chloride intracellular charmel，chaperonin，proteindisulfide-isomerase ER6o precursor and constitutive heat shock protein 70等。所得结果不但能够使我们更加深入的理解肿瘤耐药的病理机制，更重要的是通过找到肿瘤细胞发生耐药的标志性蛋白，可以为开发肿瘤耐药预测方法和寻找抗耐药肿瘤靶标新药的线索，同时也为计算机虚拟筛选设计抗耐药肿瘤新药奠定基础。除了详细的研究了K562／A02细胞系差异蛋白质组，我们还采用蛋白质组学的方法，以乳鼠心肌细胞为实验模型，研究在ARs激动的不同清况下心肌细胞蛋白质表达谱的变化。心肌肥厚是心脏工作超负荷的一种代偿反应。目前，越来越多的证据表明ARs在心肌肥厚的发生中起着非常重要的作用，但人们对其机制了解尚不全面。通过检测心肌肥厚时全蛋白表达的变化，将有助于我们全面、深入了解ARs在心肌肥厚中的作用机制。通过严格的2D胶图象分析，蛋白取点后胶内水解，高灵敏度的质谱鉴定及数据库检索，我们成功地进行了乳鼠心肌细胞肥厚为模型的蛋白质组学的研究。本实验通过比较研究不同肾上腺素受体激动剂norePinephrine伽E），phenylephxine（PE）and isoprenaline（ISO）诱导组与未诱导对照组的蛋白质组学变化，分析确定蛋白丰度差异在2倍以上的蛋白质点，并通过MALDI-MS进行蛋白鉴定。20个具有显著差异的蛋白质被鉴定，包括caspase-11，vimeniin，4-hydroxyphenylpyruvic acid dioxygenase，Modl protein，Txndcs protein等，其中一些蛋白在此之前并没发现与心肌肥厚相关，对它们在心肌肥厚中的意义和机制我们将做进一步的探讨。本研究结果将为我们更好理解肾上腺素受体之间以及肾上腺素受体与心肌肥厚之间的关系提供有益的线索，并将为最终发现可应用于临床的新的诊断和治疗心血管疾病的标志性蛋白打下基础。