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.

Interaction of cyclooxygenases with an apoptosis- and autoimmunity-associated protein.

Cyclooxygenases (COXs) 1 and 2 are 72-kDa, intralumenal residents of the endoplasmic reticulum (ER) and nuclear envelope, where they catalyze the rate-limiting steps in the conversion of arachidonate to the physiologically dynamic prostanoids. Recent studies, including the generation of knockout mice, show COX-1 and COX-2 to have biologically distinct roles within cells and organisms. Also apparent is that arachidonate substrate is selectably metabolized by COX-2 after mitogen stimulation in many cells that contain both isoforms. Because COX-1 and COX-2 are highly conserved in all residues needed for catalysis and in their purified forms have almost identical kinetic properties, we have searched for COX-interacting ER proteins that might mediate these unique isoenzymic properties. Using COXs as bait in the yeast two-hybrid system, we identified autoimmunity- and apoptosis-associated nucleobindin (Nuc) as a protein that specifically interacts with both isoenzymes. COX-Nuc binding was substantiated by immunoprecipitation experiments, which showed that COX-1 and, to a lesser extent, COX-2 form complexes with Nuc in vitro. When overexpressed in COS-1 cells, Nuc was found to be extracellularly released. However, when Nuc was co-overexpressed with COX-1 or COX-2, its release was reduced by >80%. This finding suggests that COX isoenzymes participate in the retention of Nuc within the lumen of the ER, where COX may regulate the release of Nuc from the cell. It also identifies Nuc as a potential regulator of COXs through this interaction.

Embryonic stem cells express multiple Eph-subfamily receptor tyrosine kinases.

Eph and its homologues form the largest subfamily of receptor tyrosine kinases. Normal expression patterns of this subfamily indicate roles in differentiation and development, whereas their overexpression has been linked to oncogenesis. This study investigated the potential role of Eph-related molecules during very early embryonic development by examining their expression in embryonic stem (ES) cells and embryoid bodies differentiated from ES cells in vitro. By use of a strategy based on reverse transcriptase-mediated PCR, nine clones containing Eph-subfamily sequence were isolated from ES cells. Of these, eight were almost identical to one of four previously identified molecules (Sek, Nuk, Eck, and Mek4). However, one clone contained sequence from a novel Eph-subfamily member, which was termed embryonic stem-cell kinase or Esk. Northern analysis showed expression of Esk in ES cells, embryoid bodies, day 12 mouse embryos, and some tissues of the adult animal. Levels of expression were similar in ES cells and embryoid bodies. By comparison, Mek4 showed no significant transcription in the ES cell cultures by Northern analysis, whereas Eck displayed stronger signals in ES cells than in the embryoid bodies. These results suggest that Eph-subfamily molecules may play roles during the earliest phases of embryogenesis. Furthermore, the relative importance of different members of this subfamily appears to change as development proceeds.

The specificity of the transforming growth factor beta receptor kinases determined by a spatially addressable peptide library.

Type I and II receptors for the transforming growth factor beta (TGF-beta) are transmembrane serine/threonine kinases that are essential for TGF-beta signaling. However, little is known about their in vivo substrates or signal transduction pathways. To determine the substrate specificity of these kinases, we developed combinatorial peptide libraries synthesized on a hydrophilic matrix that is easily accessible to proteins in aqueous solutions. When we subjected these libraries to phosphorylation by the cAMP-dependent protein kinase, we obtained the optimal peptide sequence RRXS (I/L/V), in perfect agreement with the substrate sequence deduced from mutagenesis and crystal structure analyses. By using the same libraries, we showed that the optimal substrate peptide for both the type I and II TGF-beta receptors was KKKKKK(S/T)XXX. Since the two kinases are thought to play different roles in intracellular signal transduction, it was a surprise to find that they have almost identical substrate specificity. Our method is direct, sensitive, and simple and provides information about the kinase specificity for all the amino acid residues at each position.

Modes of expression and common structural features of the complete phenylalanine ammonia-lyase gene family in parsley.

We describe a complete gene family encoding phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) in one particular plant species. In parsley (Petroselinum crispum), the PAL gene family comprises two closely related members, PAL1 and PAL2, whose TATA-proximal promoter and coding regions are almost identical, and two additional members, PAL3 and PAL4, with less similarity to one another and to the PAL1 and PAL2 genes. Using gene-specific probes derived from the 5' untranslated regions of PAL1/2, PAL3, and PAL4, we determined the respective mRNA levels in parsley leaves and cell cultures treated with UV light or fungal elicitor and in wounded leaves and roots. For comparison, the functionally closely related cinnamate 4-hydroxylase (C4H) and 4-coumarate:CoA ligase (4CL) mRNAs were measured in parallel. The results indicate various degrees of differential responsiveness of PAL4 relative to the other PAL gene family members, in contrast to a high degree of coordination in the overall expression of the PAL, C4H, and 4CL genes. The only significant sequence similarities shared by all four PAL gene promoters are a TATA-proximal set of three putative cis-acting elements (boxes P, A, and L). None of these elements alone, or the promoter region containing all of them together, conferred elicitor or light responsiveness on a reporter gene in transient expression assays. The elements appear to be necessary but not sufficient for elicitor- or light-mediated PAL gene activation, similar to the situation previously reported for 4CL.

Complex flexibility of the transforming growth factor beta superfamily.

The transforming growth factors beta (TGF-beta s) are important modulators of growth and differentiation. They are intermolecular disulfide-bonded homodimeric molecules. The monomer fold has a conserved cystine knot and lacks a hydrophobic core. The biological specificity of a given member of the family is believed to be determined by the conformational flexibility of the variable loop regions of the monomer. The monomer subunit assembly in the dimer is stabilized mainly by hydrophobic contacts and a few hydrogen bonds. Since these interactions are nondirectional, we examined subunit assemblies of TGF-beta by using conformational analysis. The different subunit assemblies in TGF-beta 2 dimer were characterized in terms of the intersubunit disulfide torsion. Our analyses show that the subunit assemblies fall into two states: the crystallographically observed gauche+conformation and the previously not reported gauche--conformation, both having almost identical interaction energies. Furthermore, there is significant flexibility in the subunit assembly within the gauche+ and the gauche- states of the disulfide bond. The monomer subunit assembly is independent of the variations about the loop regions. The variations in the loop regions, coupled with flexibility in the monomer assembly, lead to a complex flexibility in the dimer of the TGF-beta superfamily. For the TGF-beta superfamily, the cystine knot acts as a scaffold and complex flexibility provides for biological selectivity. Complex flexibility might provide an explanation for the diverse range of biological activities that these important molecules display.

Aplicação de modelos de defeitos na geração de conjuntos de teste completos a partir de Sistemas de Transição com Entrada/Saída

O Teste Baseado em Modelos (TBM) emergiu como uma estratégia promissora para minimizar problemas relacionados à falta de tempo e recursos em teste de software e visa verificar se a implementação sob teste está em conformidade com sua especificação. Casos de teste são gerados automaticamente a partir de modelos comportamentais produzidos durante o ciclo de desenvolvimento de software. Entre as técnicas de modelagem existentes, Sistemas de Transição com Entrada/Saída (do inglês, Input/Output Transition Systems - IOTSs), são modelos amplamente utilizados no TBM por serem mais expressivos do que Máquinas de Estado Finito (MEFs). Apesar dos métodos existentes para geração de testes a partir de IOTSs, o problema da seleção de casos de testes é um tópico difícil e importante. Os métodos existentes para IOTS são não-determinísticos, ao contrário da teoria existente para MEFs, que fornece garantia de cobertura completa com base em um modelo de defeitos. Esta tese investiga a aplicação de modelos de defeitos em métodos determinísticos de geração de testes a partir de IOTSs. Foi proposto um método para geração de conjuntos de teste com base no método W para MEFs. O método gera conjuntos de teste de forma determinística além de satisfazer condições de suficiência de cobertura da especificação e de todos os defeitos do domínio de defeitos definido. Estudos empíricos avaliaram a aplicabilidade e eficácia do método proposto: resultados experimentais para analisar o custo de geração de conjuntos de teste utilizando IOTSs gerados aleatoriamente e um estudo de caso com especificações da indústria mostram a efetividade dos conjuntos gerados em relação ao método tradicional de Tretmans.

Lists of books donated by individuals and organizations in Scotland, ca. 1766 (3 manuscript copies)

These lists include books donated by Anthony Ferguson, an Aberdeen merchant; the Society for Promoting Religious Knowledge; William Gray, bookseller; Mr. Hog, merchant; Messrs. Gray and Alston, printers; Mr. Miller, bookseller; John Erskine, who donated his own work, Theological Dissertations; the Reverend Mr. Randal; and the Reverend Walter Scot Baxter. The lists are almost identical, though there are several variations and discrepancies.

An Investigation of Career Tenure in the European Parliament: Electoral, Party and Individual Considerations. ACES Working Papers, 2012

What explains the length of a Member of the European Parliament’s career? Little evidence of careerism has been uncovered in the European Parliament, particularly when compared to studies of legislator tenure in the U.S. Congress. Due to the different historical contexts in which these two legislatures developed, it seems reasonable to rule out many of the explanations used to account for increasing careerism in Congress in searching for the influences on legislator tenure in the European Parliament. This paper therefore proposes three potential models of careerism in the European Parliament: an electoral systems model, a party model, and an individual model. While the data necessary to test these models has not been fully compiled, this paper outlines the major hypotheses of each model and details plans for the operationalization of all independent and control variables. These models are not intended to be mutually exclusive alternatives, but rather each explanation is expected to influence each MEP in varying degrees.

Geochemistry of basalts from ODP Leg 49, North Atlantic Ocean

The geochemistry of basalts recovered from seven sites in the North Atlantic is described with particular reference to minor elements. Three sites (407, 408, and 409) along the same mantle flow line, transverse to the Reykjanes Ridge at about 63°N, provide information on the composition of basalts erupted over a 34-m.y. interval between 2.3 and 36 m.y. ago. At Site 410, at 45°N, penetration into 10 m.y.-old crust west of the ridge axis permits comparisons with young basalts dredged from the median valley at 45°N. Three sites in the FAMOUS area at about 36°N provided material from very young (1 m.y.) basaltic crust (Site 411), and material to test the geochemical coherence of basalts of different ages (1.5 and 3.5 m.y.) on either side of a fracture zone (Sites 412 and 413). These sites complement earlier data from dredged and drilled sites (Leg 37) in the FAMOUS area. At Site 407, four geochemically distinct basalt units occur, with different normative and rare-earth element (REE) characteristics, and there is a clear correlation with magnetic stratigraphy. Yet there is a remarkable consistency in incompatible element ratios between these units, indicating derivation from an essentially similar mantle source. The basalts from the younger sites, 408 and 409, show a similar range of normative and REE variation, but incompatible element ratios are identical to those at Site 407, indicating that basalts at all three sites were produced from a mantle source which was geochemically relatively uniform. Rare-earth differences between the basalts can be interpreted in terms of variations in the degree and depth of partial melting causing HREE (+Y) retention in the source, although there may be some inter-site differences with respect to REE. A similar picture is presented at 45°N. Apparently a range of tholeiitic, transitional, and alkalic basalts were being erupted 10 m.y. ago, which have almost identical geochemical characteristics to those recently erupted in the median valley at 45°N. Incompatible element ratios are markedly different from those recorded at the Reykjanes Ridge. Basalts recovered from the FAMOUS sites are geochemically similar to previous samples recovered from the FAMOUS area, and their incompatible element ratios are similar, but not identical, to those at 45°N. However, total trace element levels are consistently lower than in 45°N basalts, which might imply smaller degrees of partial melting and/or greater depths of magma generation at 45°N, or higher trace element levels in the mantle source at 45°N. Few of the basalts recovered on Leg 49 have the geochemical characteristics of typical "MORB" (e.g., Nazca Plate, Leg 34). The data strongly support models invoking geochemical inhomogeneity in the source regions of basalts produced at the Mid-Atlantic Ridge. However, the data also introduce an additional time factor into such models and demonstrate the uniformity of the mantle source at a particular ridge sector (over periods in excess of 30 m.y.), while emphasizing the marked differences along the ridge. Mixing models invoking "depleted" and "enriched" mantle sources would seem to be inadequate to account for the observed variations.

Local records; or, Historical register of remarkable events which have occurred in Northumberland and Durham, Newcastle-upon-Tyne, and Berwick-upon-Tweed, with biographical notices of deceased persons of talent, eccentricity, and longevity;

A compilation of similar character, bearing a title almost identical, was made by J. Latimer, covering the period from Oct. 8, 1832, to Aug. 10, 1857.

Influence of phytase and xylanase supplementation on growth performance and nutrient utilisation of broilers offered wheat-based diets

Individual and combined supplementation of phosphorus-adequate, wheat-based broiler diets with exogenous phytase and xylanase was evaluated in three experiments. The effects of the enzyme combination in lysine-deficient diets containing wheat and sorghum were more pronounced than those of the individual feed enzymes. The inclusion of phytase plus xylanase improved (p<0.05) weight gains (7.3%) and feed efficiency (7.0%) of broilers (7-28 days post-hatch) and apparent metabolisable energy (AME) by 0.76 MJ/kg DM. Phytase plus xylanase increased (p<0.05) the overall, apparent ileal digestibility of amino acids by 4.5% (0.781 to 0.816); this was greater than the responses to either phytase (3.6%; 0.781 to 0.809) or xylanase (0.7%; 0.781 to 0.784). Absolute increases in amino acid digestibility with the combination exceeded the sum of the individual increases generated by phytase and xylanase for alanine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, phenylalanine, threonine, tyrosine and valine. These synergistic responses may have resulted from phytase and xylanase having complementary modes of action for enhancing amino acid digestibilities and/or facilitating substrate access. The two remaining experiments were almost identical except wheat used in Experiment 2 had a higher phytate concentration and a lower estimated AME content than wheat used in Experiment 3. Individually, phytase and xylanase were generally more effective in Experiment 2, which probably reflects the higher dietary substrate levels present. Phytase plus xylanase increased (p<0.05) gains (15.4%) and feed efficiency (7.0%) of broiler chicks from 4-24 days post-hatch in Experiment 2; whereas, in Experiment 3, the combination increased (p<0.05) growth to a lesser extent (5.6%) and had no effect on feed efficiency. This difference in performance responses appeared to be 'protein driven' as the combination increased (p<0.05) nitrogen retention in Experiment 2 but not in Experiment 3; whereas phytase plus xylanase significantly increased AME in both experiments. In Experiments 2 and 3 the combined inclusion levels of phytase and xylanase were lower that the individual additions, which demonstrates the benefits of simultaneously including phytase and xylanase in wheat-based poultry diets.

Characteristics of banana fruit from nearby organic versus conventional plantations: A case study

This case study reports the post-harvest qualities of conventionally versus organically grown banana fruit from nearby plantations in the Dominican Republic. The comparison involved six repeated harvests over the transition from cooler to hotter seasons. Green mature Cavendish 'Grande Naine' banana fruit were shipped to the UK. They were triggered to ripen with ethylene gas and kept under simulated retail conditions. Fruit mass, colour, firmness and flavour parameters were measured every second day over 12 d of shelf life. Sensory comparisons were conducted on four of the six harvest times. Significant differences (P<0.05) in measured quality attributes between conventionally and organically grown fruit were few and marginal. Moreover, any differences were inconsistent across harvest-times and during shelf life. Thus, organically and conventionally grown product had almost identical qualities. Sensory comparison confirmed that there was no flavour difference. This case study provides data that challenge a general perception that organic bananas have better flavour than conventional bananas.

Classification and analysis of integrating frameworks in multiscale modelling

Chemical engineers are turning to multiscale modelling to extend traditional modelling approaches into new application areas and to achieve higher levels of detail and accuracy. There is, however, little advice available on the best strategy to use in constructing a multiscale model. This paper presents a starting point for the systematic analysis of multiscale models by defining several integrating frameworks for linking models at different scales. It briefly explores how the nature of the information flow between the models at the different scales is influenced by the choice of framework, and presents some restrictions on model-framework compatibility. The concepts are illustrated with reference to the modelling of a catalytic packed bed reactor. (C) 2004 Elsevier Ltd. All rights reserved.

A PCR method for the identification of methicillin-resistant Staphylococcus aureus (MRSA) from screening swabs

Aim: The aim of this study was to assess the discriminatory power and potential turn around time ( TAT) of a PCR-based method for the detection of methicillin-resistant Staphylococcus aureus (MRSA) from screening swabs. Methods: Screening swabs were examined using the current laboratory protocol of direct culture on mannitol salt agar supplemented with oxacillin (MSAO-direct). The PCR method involved pre-incubation in broth for 4 hours followed by a multiplex PCR with primers directed to mecA and nuc genes of MRSA. The reference standard was determined by pre-incubation in broth for 4 hours followed by culture on MSAO (MSAO-broth). Results: A total of 256 swabs was analysed. The rates of detection of MRSA using MSAO-direct, MSAO-broth and PCR were 10.2, 13.3 and 10.2%, respectively. For PCR, the sensitivity, specificity, positive predictive value and negative predictive values were 66.7% (95% CI 51.9 - 83.3%), 98.6% ( 95% CI 97.1 - 100%), 84.6% ( 95% CI 76.2 - 100%) and 95.2% ( 95% CI 92.4 - 98.0%), respectively, and these results were almost identical to those obtained from MSAO-direct. The agreement between MSAO-direct and PCR was 61.5% ( 95% CI 42.8 - 80.2%) for positive results, 95.6% ( 95% CI 93.0 - 98.2%) for negative results and overall was 92.2% ( 95% CI 88.9 - 95.5%). Conclusions: ( 1) The discriminatory power of PCR and MSAO-direct is similar but the level of agreement, especially for true positive results, is low. ( 2) The potential TAT for the PCR method provides a marked advantage over conventional methods. ( 3) Further modifications to the PCR method such as increased broth incubation time, use of selective broth and adaptation to real-time PCR may lead to improvement in sensitivity and TAT.