Extensive purification of a putative RNA polymerase I holoenzyme from plants that accurately initiates rRNA gene transcription in vitro

RNA polymerase I (pol I) is a nuclear enzyme whose function is to transcribe the duplicated genes encoding the precursor of the three largest ribosomal RNAs. We report a cell-free system from broccoli (Brassica oleracea) inflorescence that supports promoter-dependent RNA pol I transcription in vitro. The transcription system was purified extensively by DEAE-Sepharose, Biorex 70, Sephacryl S300, and Mono Q chromatography. Activities required for pre-rRNA transcription copurified with the polymerase on all four columns, suggesting their association as a complex. Purified fractions programmed transcription initiation from the in vivo start site and utilized the same core promoter sequences required in vivo. The complex was not dissociated in 800 mM KCl and had a molecular mass of nearly 2 MDa based on gel filtration chromatography. The most highly purified fractions contain ≈30 polypeptides, two of which were identified immunologically as RNA polymerase subunits. These data suggest that the occurrence of a holoenzyme complex is probably not unique to the pol II system but may be a general feature of eukaryotic nuclear polymerases.

Inosine stimulates extensive axon collateral growth in the rat corticospinal tract after injury

The purine nucleoside inosine has been shown to induce axon outgrowth from primary neurons in culture through a direct intracellular mechanism. For this study, we investigated the effects of inosine in vivo by examining whether it would stimulate axon growth after a unilateral transection of the corticospinal tract. Inosine applied with a minipump to the rat sensorimotor cortex stimulated intact pyramidal cells to undergo extensive sprouting of their axons into the denervated spinal cord white matter and adjacent neuropil. Axon growth was visualized by anterograde tracing with biotinylated dextran amine and by immunohistochemistry with antibodies to GAP-43. Thus, inosine, a naturally occurring metabolite without known side effects, might help to restore essential circuitry after injury to the central nervous system.

Spontaneous corticospinal axonal plasticity and functional recovery after adult central nervous system injury

Although it is believed that little recovery occurs after adult mammalian spinal cord injury, in fact significant spontaneous functional improvement commonly occurs after spinal cord injury in humans. To investigate potential mechanisms underlying spontaneous recovery, lesions of defined components of the corticospinal motor pathway were made in adult rats in the rostral cervical spinal cord or caudal medulla. Following complete lesions of the dorsal corticospinal motor pathway, which contains more than 95% of all corticospinal axons, spontaneous sprouting from the ventral corticospinal tract occurred onto medial motoneuron pools in the cervical spinal cord; this sprouting was paralleled by functional recovery. Combined lesions of both dorsal and ventral corticospinal tract components eliminated sprouting and functional recovery. In addition, functional recovery was also abolished if dorsal corticospinal tract lesions were followed 5 weeks later by ventral corticospinal tract lesions. We found extensive spontaneous structural plasticity as a mechanism correlating with functional recovery in motor systems in the adult central nervous system. Experimental enhancement of spontaneous plasticity may be useful to promote further recovery after adult central nervous system injury.

Functional complementation of xeroderma pigmentosum complementation group E by replication protein A in an in vitro system.

Xeroderma pigmentosum (XP) is caused by a defect in nucleotide excision repair. Patients in the complementation group E (XP-E) have the mildest form of the disease and the highest level of residual repair activity. About 20% of the cell strains derived from XP-E patients lack a damaged DNA-binding protein (DDB) activity that binds to ultraviolet-induced (6-4) photoproducts with high affinity. We report here that cell-free extracts prepared from XP-E cell strains that either lacked or contained DDB activity were severely defective in excising DNA damage including (6-4) photoproducts. However, this excision activity defect was not restored by addition of purified DDB that, in fact, inhibited removal of (6-4) photoproducts by the human excision nuclease reconstituted from purified proteins. Extensive purification of correcting activity from HeLa cells revealed that the correcting activity is inseparable from the human replication/repair protein A [RPA (also known as human single stranded DNA binding protein, HSSB)]. Indeed, supplementing XP-E extracts with recombinant human RPA purified from Escherichia coli restored excision activity. However, no mutation was found in the genes encoding the three subunits of RPA in an XP-E (DDB-) cell line. It is concluded that RPA functionally complements XP-E extracts in vitro, but it is not genetically altered in XP-E patients.

Os impactos ambientais no meio físico: erosão e assoreamento na bacia hidrográfica do rio Taquari, MS, em decorrência da pecuária

O aumento da inundação em áreas do baixo curso do rio Taquari, no Pantanal do estado do Mato Grosso do Sul, tem transformado a pecuária desta região numa atividade com baixa rentabilidade, à medida que extensas áreas de campo passaram a ser inundadas vários meses durante o ano a partir da década de 70. A pecuária realizada em campos naturais de regiões úmidas do Pantanal indica que há necessidade de se investigar metodologias apropriadas para avaliação de impacto ambiental, que abordem impactos diretos, indiretos, cumulativos e processos do meio físico que alteram, de maneira prejudicial, o meio ambiente. Supõe-se que a inundação na planície do rio Taquari esteja relacionada com a ocupação antrópica nas áreas de planalto da bacia do rio Taquari. O presente trabalho tem por objetivo avaliar os impactos ambientais na planície de inundação do baixo curso do Taquari, decorrentes da ocupação antrópica da bacia hidrográfica do rio Taquari em sua totalidade, considerando os impactos ambientais causados pela pecuária à medida que se configura como principal atividade econômica da bacia bem como os processos erosivos e de assoreamento no quadro atual do regime de inundações. As etapas de caracterização da área, de análise dos impactos e as propostas de ações mitigadoras, previstas num Estudo de Impacto Ambiental, foram aqui analisadas. Foram utilizadas informações sobre as características do meio físico, biótico e socioeconômico, selecionadas a partir do levantamento dos dados existentes com recorte efetuado para a bacia hidrográfica do rio Taquari. Na maior parte dos temas, este foi um processo de levantamento, ordenamento e recuperação de informações, na escala original de 1:250.000, do Plano de Conservação da Bacia do Alto Paraguai-PCBAP, gerenciado no SPRING. Foram também realizadas viagens de campo para a complementação dos dados e para o levantamento de atividades antrópicas com verificações \"in loco\" da ocorrência de impacto ambiental. A maioria dos dados socioeconômicos compilados para o presente trabalho teve por base os censos agropecuários e demográficos realizados pelo IBGE. Os resultados obtidos demonstram que os impactos ambientais decorrentes da pecuária no planalto interferem no regime de inundação na planície da bacia, o que só foi possível de ser identificado a partir de análises integradas em toda a bacia hidrográfica do rio Taquari. Verificou-se que os métodos de EIA são adequados para identificar os impactos diretos decorrentes da pecuária, mas não são adequados para identificar os processos e seus efeitos cumulativos na extensão da bacia hidrográfica do rio Taquari. Além disto, a abordagem da avaliação ambiental estratégica, como procedimento para análise ambiental em políticas, planos e programas, mostra-se adequada para as análises na BHRT à medida que está centralizada nos efeitos do ambiente sobre as necessidades e oportunidades de desenvolvimento. Contudo, somente a recuperação de danos ambientais, o controle das origens dos impactos no ambiente e um sistema de gestão consciente de seus compromissos podem levar, juntamente com a melhora dos procedimentos técnicos e administrativos para análises ambientais, à uma maior proximidade da sustentabilidade ambiental na BHRT.

Description of atmospheric conditions at the Pierre Auger Observatory using the Global Data Assimilation System (GDAS)

Atmospheric conditions at the site of a cosmic ray observatory must be known for reconstructing observed extensive air showers. The Global Data Assimilation System (GDAS) is a global atmospheric model predicated on meteorological measurements and numerical weather predictions. GDAS provides altitude-dependent profiles of the main state variables of the atmosphere like temperature, pressure, and humidity. The original data and their application to the air shower reconstruction of the Pierre Auger Observatory are described. By comparisons with radiosonde and weather station measurements obtained on-site in Malargue and averaged monthly models, the utility of the GDAS data is shown. (C) 2012 Elsevier B.V. All rights reserved.

Evidence-Based Practice in the Juvenile Justice System: Balancing Clinical Expertise with Best Available Research and Client Variables

Each year, thousands of adolescents are processed through the juvenile justice system -- a system that is complicated, expensive, and inadequately addressing the needs of the youth in its care. While there is extensive literature available in support of interventions for youthful offenders that are clinically superior to current care and more cost-effective than the existing structure, there is a gap between research and practice that is preventing their implementation. The use of Evidence-Based Practice in Psychology (EBPP) as defined by the American Psychological Association is presented as one method to bridge this gap. This paper identifies and discusses each of five barriers to effective use of EBPP: cost, fragmentation of the mental health system, historical and systemic variables, research methodology, and clinician variables. These barriers are first defined and then illustrated using examples from the author's experience working in the juvenile justice field. Finally, recommendations for the field are presented.

The Second Phase of the Common European Asylum System: A Step Forward in the Protection of Asylum Seekers? IES WORKING PAPER 7/2013

The aim of this paper is to analyse what is the impact of the second phase of the creation of the Common European Asylum System (CEAS) in the protection of rights of Asylum Seekers in the European Union. The establishment of a CEAS has been always a part of the development of the Area of Freedom, Security and Justice. Its implementation was planned in two phases: the first one, focused on the harmonisation of internal legislation on minimum common standards; the second, based on the result of an evaluation of the effectiveness of the agreed legal instruments, should improve the effectiveness of the protection granted. The five instruments adopted between 2002 and 2005, three Directives, on Qualification, Reception Conditions and Asylum Procedures, and two Regulations, the so-called “Dublin System”, were subjected to an extensive evaluation and modification, which led to the end of the recasting in 2013. The paper discusses briefly the international obligations concerning the rights of asylum seekers and continues with the presentation of the legal basis of the CEAS and its development, together with the role of the Charter of Fundamental Rights of the European Union in asylum matters. The research will then focus on the development in the protection of asylum seekers after the recasting of the legislative instruments mentioned above. The paper will note that the European standards result now improved, especially concerning the treatment of vulnerable people, the quality of the application procedure, the effectiveness of the appeal, the treatment of gender issues in decision concerning procedures and reception. However, it will be also highlighted that Member States maintained a wide margin of appreciation in many fields, which can lead to the compression of important guarantees. This margin concerns, for example, the access to free legal assistance, the definition of the material support to be granted to each applicant for international protection, the access to labour market, the application of the presumptions of the “safety” of a third country. The paper will therefore stress that the long negotiations that characterised the second phase of the CEAS undoubtedly led to some progress in the protection of Asylum Seekers in the EU. However, some provisions are still in open contrast with the international obligations concerning rights of asylum seekers, while others require to the Member State consider carefully its obligation in the choice of internal policies concerning asylum matters.

Clinicopathological Phenotype of Autosomal Recessive Cholesterol Deficiency in Holstein Cattle.

BACKGROUND Cholesterol deficiency (CD), a newly identified autosomal recessive genetic defect in Holstein cattle, is associated with clinical signs of diarrhea, failure to thrive, and hypocholesterolemia. HYPOTHESIS/OBJECTIVES The objective is to describe the clinicopathological phenotype of affected Holstein cattle homozygous for the causative apolipoprotein B gene (APOB) mutation. ANIMALS Six Holstein cattle, 5 calves with a clinical history of chronic diarrhea, and 1 heifer with erosions in the buccal cavity and neurologic symptoms were admitted to the Clinic for Ruminants. METHODS This case review included a full clinical examination, a complete blood count, blood chemistry, and measurements of cholesterol and triglycerides. The animals were euthanized and necropsied. A PCR-based direct gene test was applied to determine the APOB genotype. RESULTS All 6 animals were inbred, could be traced back to the sire Maughlin Storm, and were confirmed homozygous for the APOB mutation. The clinical phenotype included poor development, underweight, and intermittent diarrhea in the calves, and neurologic signs in the heifer included hypermetria and pacing. Hypocholesterolemia and low triglycerides concentrations were present in all animals. The pathological phenotype of all animals was steatorrhea with enterocytes of the small intestine containing intracytoplasmic lipid vacuoles. The peripheral nervous system of the heifer displayed degenerative changes. CONCLUSIONS AND CLINICAL IMPORTANCE Suspicion of CD in Holstein cattle is based on the presence of chronic diarrhea with no evidence of primary infections. Confirmation of the associated APOB gene mutation is needed. Additionally, the heifer demonstrated primarily signs of neurologic disease providing an unexpected phenotype of CD.

The Extensive (but Fragile) Authority of the WTO Appellate Body

The authority of an international court (IC) is not necessarily evolutionary and its development unidirectional. This article addresses the authority of the Appellate Body (AB) of the World Trade Organization (WTO) and shows how it rapidly and almost immediately became extensive, but has since exhibited signs of becoming more fragile. The article applies a typology of IC authority developed by Alter, Helfer and Madsen (2014) and explains the transformation from narrow authority (a dispute resolution venue under the GATT based on political negotiations) to extensive authority (a judicialized WTO dispute settlement system with a sophisticated case law) and presents empirical indicators of the rise of the AB’s authority. Such rapid development of extensive authority is arguably a unique case in international politics at the multilateral level. That authority nonetheless remains fragile, and shows signs that it could decline significantly for reasons we explain.

Experimental study of phase equilibria in the Al-Fe-Zn-O system in air

The phase equilibria in the Al-Fe-Zn-O system in the range 1250 °C to 1695 °C in air have been experimentally studied using equilibration and quenching techniques followed by electron probe X-ray microanalysis. The phase diagram of the binary Al2O3-ZnO system and isothermal sections of the Al2O3-“Fe2O3”-ZnO system at 1250 °C, 1400 °C, and 1550 °C have been constructed and reported for the first time. The extents of solid solutions in the corundum (Al,Fe)2O3, hematite (Fe,Al)2O3, Al2O3*Fe2O3 phase (Al,Fe)2O3, spinel (Al,Fe,Zn)O4, and zincite (Al,Zn,Fe)O primary phase fields have been measured. Corundum, hematite, and Al2O3*Fe2O3 phases dissolve less than 1 mol pct zinc oxide. The limiting compositions of Al2O3*Fe2O3 phase measured in this study at 1400 °C are slightly nonstoichiometric, containing more Al2O3 then previously reported. Spinel forms an extensive solid solution in the Al2O3-“Fe2O3”-ZnO system in air with increasing temperature. Zincite was found to dissolve up to 7 mole pct of aluminum in the presence of iron at 1550 °C in air. A meta-stable Al2O3-rich phase of the approximate composition Al8FeZnO14+x was observed at all of the conditions investigated. Aluminum dissolved in the zincite in the presence of iron appears to suppress the transformation from a round to platelike morphology.

Phase equilibria at sub-solidus conditions in the Fe-Mg-Zn-O system in air

The phase equilibria in the Fe-Mg-Zn-O system in the temperature range 1100-1550degreesC in air have been experimentally studied using equilibration and quenching followed by electron probe X-ray microanalysis. The compositions of condensed phases in equilibrium in the binary MgO-ZnO system and the ternary Fe-Mg-O system have been reported at sub-solidus in air. Pseudo-ternary sections of the quaternary Fe-Mg-Zn-O system at 1100, 1250 and 1400degreesC in air were constructed using the experimental data. The solid solution of iron oxide, MgO and ZnO in the periclase (Mg, Zn, Fe)O, spinel (Mg2+, Fe2+, Zn2+)(x)Fe(2+y)3+O4 and zincite (Zn, Mg, Fe)O phases were found to be extensive under the conditions investigated. A continuous spinel solid solution is formed between the magnesioferrite (Mg2+, Fe2+)(x)Fe(2+y)3+O4 and franklinite (Zn2+, Fe2+)(x)Fe(2+y)3+O4 end-members at 1100 and 1250degreesC, extending to magnetite (Fe2+)(x)Fe(2+y)3+O4 at 1400degreesC in air. The compositions along the spinel boundaries were found to be non-stoichiometric, the magnitude of the non-stoichiometry being a function of composition and temperature in air. It was found that hematite dissolves neither MgO nor ZnO in air.

The importance of mating system in translocation programs: reproductive success of released male bridled nailtail wallabies

Translocation is an important tool for the conservation of species that have suffered severe range reductions. The success of a translocation should be measured not only by the survival of released animals, but by the reproductive output of individuals and hence the establishment of a self-sustaining population. The bridled nailtail wallaby is an endangered Australian macropod that suffered an extensive range contraction to a single remaining wild population. A translocated population was established and subsequently monitored over a four year period. The aim of this study was to measure the reproductive success of released males using genetic tools and to determine the factors that predicted reproductive success. Captive-bred and wild-caught animals were released and we found significant variation in male reproductive success among release groups. Variation in reproductive success was best explained by individual male weight, survival and release location rather than origin. Only 26% of candidate males were observed to sire an offspring during the study. The bridled nailtail wallaby is a sexually dimorphic, polygynous macropod and reproductive success is skewed toward large males. Males over 5800 g were six times more likely to sire an offspring than males below this weight. This study highlights the importance of considering mating system when choosing animals for translocation. Translocation programs for polygynous species should release a greater proportion of females, and only release males of high breeding potential. By maximizing the reproductive output of released animals, conservation managers will reduce the costs of translocation and increase the chance of successfully establishing a self-sustaining population. (C) 2004 Elsevier Ltd. All rights reserved.

Phase equilibria in the Fe-Zn-O system at conditions relevant to zinc sintering and smelting

Zincite and spinel phases are present in the complex slag systems encountered in zinc/lead sintering and zinc smelting processes. These phases form extensive solid solutions and are stable over a wide range of compositions, temperatures and oxygen partial pressures. Accurate information on the stability of these phases is required in order to develop thermodynamic models of these slag systems. Phase equilibria in the Fe–Zn–O system have been experimentally studied for a range of conditions, between 900°C and 1580°C and oxygen partial pressures (pO2) between air and metallic iron saturation, using equilibration and quenching techniques. The compositions of the phases were measured using Electron probe X-ray microanalysis (EPMA). The ferrous and ferric bulk iron concentrations were determined using a specially developed wet-chemical analysis procedure based on the use of ammonium metavanadate. XRD was used to confirm phase identification. A procedure was developed to overcome the problems associated with evaporation of zinc at low pO2 values and to ensure the achievement of equilibria. An isothermal section of the system FeO–Fe2O3–ZnO at high ZnO concentrations at 1200°C was constructed. The maximum solubilities of iron and zinc in zincite and spinel phases in equilibrium were determined at pO2 = 1 × 10-6 atm at 1200°C and 1300°C. The morphology of the zincite crystals sharply changes in air between 1200–1300°C from rounded to plate-like. This is shown to be associated with significant increase in total iron concentration, the additional iron being principally in the form of ferric iron. Calculations performed by FactSage with a thermodynamically optimised database have been compared with the experimental results.

MULTIPRED: A computational system for prediction of promiscuous HLA binding peptides

MULTIPRED is a web-based computational system for the prediction of peptide binding to multiple molecules ( proteins) belonging to human leukocyte antigens (HLA) class I A2, A3 and class II DR supertypes. It uses hidden Markov models and artificial neural network methods as predictive engines. A novel data representation method enables MULTIPRED to predict peptides that promiscuously bind multiple HLA alleles within one HLA supertype. Extensive testing was performed for validation of the prediction models. Testing results show that MULTIPRED is both sensitive and specific and it has good predictive ability ( area under the receiver operating characteristic curve A(ROC) > 0.80). MULTIPRED can be used for the mapping of promiscuous T-cell epitopes as well as the regions of high concentration of these targets termed T-cell epitope hotspots. MULTIPRED is available at http:// antigen.i2r.a-star.edu.sg/ multipred/.