1,720,983 research outputs found
Production, purification and structural characterisation of recombinant barley limit dextrinase and characterisation of its interaction with the endogenous limit dextrinase inhibitor
Get PDFHeterolog produktion af store multidomæne proteiner fra højere planter er ofte besværlig. Byg limit dextrinase (LD), som er et 98 kDa stort multidomæne stivelses og grænsedextrin afgreningsenzym, spiller en væsentlig rolle i stivelsesmobilisering under frøspiring og er muligvis også involveret i stivelse biosyntesen ved trimning af intermediære forgrenede α-glucan strukturer. Endvidere spiller LD en vigtig rolle ved nedbrydning af grænsedextriner i ølproduktion. LD isoleret fra spirende byg frø er fundet i en "bundet" inaktiv form, og en fri enzymatisk aktiv form. Den inaktive LD form blev foreslået til at bestå af et kompleks med den endogene limit dextrinase hæmmer (LDI) og isolering under reducerende forhold resulterede i enzymatisk fuldt aktivt LD. Tilstedeværelsen af LDI under ølproduktion menes at bidrage til en væsentlig reduktion i det teoretiske opnåelige ethanol indhold. Enzymatisk aktivet byg LD og inhibitorisk aktivt LDI blevet fremstillet ved sekretorisk ekspression ved en fermentering af Pichia pastoris med høj celledensitets. Generne som koder for henholdsvis LD og LDI (uden nativt signal peptid) blev subklonet in-frame med Saccharomyces cerevisiae -faktor sekretion signalet ind i P. pastoris ekspression vektor under kontrol af alkohol oxidase 1 promotoren. Optimering af en fed-batch fermentering procedure efterfulgt af affinitetskromatografi og gelfiltrering (LD) eller anionbytning (LDI) resulterede i gode udbytter af både LD og LDI. Kinetiske konstanter for LD katalyseret pullulan hydrolyse blev bestemt ved anvendelse af Michealis-Menten ligningen for ikke-konkurrencedygtige substrat hæmning, hvilket afspejler en væsentlig substrat-hæmning og/eller transglycosylation. Bindings kinetik og energetik af LD med LDI vildtype og N-terminal forlængede eller trunkerede varianter af LDI produceret i P. pastoris, blev bestemt ved hjælp af surface plasmon resonance (SPR) analyse. Anvendelse af en 1:1-bindingsmodel til de generede SPR data viste en høj bindingsaffinitet med KD i det subnanomolære område ved pH 6,0, 150 mM NaCl og 25 °C. Endvidere viste SPR dataene at KD var pH –afhængig primært pga. en forøgelse af dissociationshastigheden ved ændring af pH-værdien fra den optimale, pH=6,5. Undersøgelse af LD/LDI interaktionen ved forskellige ionstyrker viste at elektrostatiske kræfter ikke synes at bidrage væsentligt til LD/LDI stabiliteten. van't Hoff parametrene blev beregnet ud fra KD værdierne i temperaturintervallet 10-35 °C. Favorable enthalpi- og entropi-værdier indikerede at begge komponenter bidrog til kompleksdannelsen og resulterede i en bindings fri energi, ΔG° = - 57 kJ/mol. N-terminalt forlængede eller trunkerede LDI mutanter ledte kun til en marginal ændring af KD, hvilket indikerer at den N-terminale sekvens af vildtype LDI ikke er afgørende for binding til LD. Krystalstrukturerne af LD i kompleks med de kompetitive inhibitorer α- og β-CD er blevet forfinet til hhv. 2,5 Å og 2,1 Å. Strukturen af LD er den første krystalstruktur i verden af en plante limit dextrinase. Cyclodextrinerne er observeret ved det forventede bindingssted for den primære kulhydrat kæde og okkuperer aglycon subsite +1 og +2. Ny indsigt i substrat specifikke afgørende faktorer og den mulige rolle i stivelse biosyntesen er belyst ved hjælp af denne første struktur af en plante limit dextrinase. De unikke ekspression systemer som her er etableret for både byg LD og LDI åbner op for mulighederne med videre karakterisering af interaktionen mellem LD og LDI. Baseret på denne karakterisering såvel som på den første struktur af en plante limit dextrinase, kan der foretages ”protein engineering” af LD og/eller LDI, som resulterer i en ændret/nedsat inhibering af LD. Det vil give mulighed for en ølproduktion med bedre udnyttelse af byggen og resulterer i et højere ethanol indhold
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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