Charakterisierung der Vinorin-Synthase aus Rauvolfia serpentina durch Reinigung, Expression, Mutation und Kristallisation

ABSTRACTDie vorliegende Arbeit befasste sich mit der Reinigung,heterologen Expression, Charakterisierung, molekularenAnalyse, Mutation und Kristallisation des EnzymsVinorin-Synthase. Das Enzym spielt eine wichtige Rolle inder Ajmalin-Biosynthese, da es in einerAcetyl-CoA-abhängigen Reaktion die Umwandlung desSarpagan-Alkaloids 16-epi-Vellosimin zu Vinorin unterBildung des Ajmalan-Grundgerüstes katalysiert. Nach der Reinigung der Vinorin-Synthase ausHybrid-Zellkulturen von Rauvolfia serpentina/Rhazya strictamit den fünf chromatographischen TrennmethodenAnionenaustauschchromatographie an SOURCE 30Q, HydrophobeInteraktionen Chromatographie an SOURCE 15PHE,Chromatographie an MacroPrep Ceramic Hydroxyapatit,Anionenaustauschchromatographie an Mono Q undGrößenausschlußchromatographie an Superdex 75 konnte dieVinorin-Synthase aus 2 kg Zellkulturgewebe 991fachangereichert werden.Das nach der Reinigung angefertigte SDS-Gel ermöglichte eineklare Zuordnung der Protein-Bande als Vinorin-Synthase.Der Verdau der Enzymbande mit der Endoproteinase LysC unddie darauffolgende Sequenzierung der Spaltpeptide führte zuvier Peptidsequenzen. Der Datenbankvergleich (SwissProt)zeigte keinerlei Homologien zu Sequenzen bekannterPflanzenenzyme. Mit degenerierten Primern, abgeleitet voneinem der erhaltenen Peptidfragmente und einer konserviertenRegion bekannter Acetyltransferasen gelang es, ein erstescDNA-Fragment der Vinorin-Synthase zu amplifizieren. Mit derMethode der RACE-PCR wurde die Nukleoidsequenzvervollständigt, was zu einem cDNA-Vollängenklon mit einerGröße von 1263 bp führte, der für ein Protein mit 421Aminosäuren (46 kDa) codiert.Das Vinorin-Synthase-Gen wurde in den pQE2-Expressionsvektorligiert, der für einen N-terminalen 6-fachen His-tagcodiert. Anschließend wurde sie erstmals erfolgreich in E.coli im mg-Maßstab exprimiert und bis zur Homogenitätgereinigt. Durch die erfolgreiche Überexpression konnte dieVinorin-Synthase eingehend charakterisiert werden. DerKM-Wert für das Substrat Gardneral wurde mit 20 µM, bzw.41.2 µM bestimmt und Vmax betrug 1 pkat, bzw. 1.71 pkat.Nach erfolgreicher Abspaltung des His-tags wurden diekinetischen Parameter erneut bestimmt (KM- Wert 7.5 µM, bzw.27.52 µM, Vmax 0.7 pkat, bzw. 1.21 pkat). Das Co-Substratzeigt einen KM- Wert von 60.5 µM (Vmax 0.6 pkat). DieVinorin-Synthase besitzt ein Temperatur-Optimum von 35 °Cund ein pH-Optimum bei 7.8.Homologievergleiche mit anderen Enzymen zeigten, dass dieVinorin-Synthase zu einer noch kleinen Familie von bisher 10Acetyltransferasen gehört. Alle Enzyme der Familie haben einHxxxD und ein DFGWG-Motiv zu 100 % konserviert. Basierendauf diesen Homologievergleichen und Inhibitorstudien wurden11 in dieser Proteinfamilie konservierte Aminosäuren gegenAlanin ausgetauscht, um so die Aminosäuren einer in derLiteratur postulierten katalytischen Triade(Ser/Cys-His-Asp) zu identifizieren.Die Mutation aller vorhandenen konservierten Serine undCysteine resultierte in keiner Mutante, die zumvollständigen Aktivitätsverlust des Enzyms führte. Nur dieMutationen H160A und D164A resultierten in einemvollständigen Aktivitätsverlust des Enzyms. Dieses Ergebniswiderlegt die Theorie einer katalytischen Triade und zeigte,dass die Aminosäuren H160A und D164A exklusiv an derkatalytischen Reaktion beteiligt sind.Zur Überprüfung dieser Ergebnisse und zur vollständigenAufklärung des Reaktionsmechanismus wurde dieVinorin-Synthase kristallisiert. Die bis jetzt erhaltenenKristalle (Kristallgröße in µm x: 150, y: 200, z: 200)gehören der Raumgruppe P212121 (orthorhombisch primitiv) anund beugen bis 3.3 Å. Da es bis jetzt keine Kristallstruktureines zur Vinorin-Synthase homologen Proteins gibt, konntedie Struktur noch nicht vollständig aufgeklärt werden. ZurLösung des Phasenproblems wird mit der Methode der multiplenanomalen Dispersion (MAD) jetzt versucht, die ersteKristallstruktur in dieser Enzymfamilie aufzuklären.

[Homicide by bow and arrow]

Since the invention of firearms, arrow wounds represent a rarity in the daily routine of forensic pathologists. In the present paper, we describe a homicide by a broadhead arrow shot from a compound bow in a domestic environment. Based on this homicide, we discuss the characteristics of the lesion caused by broadhead arrows and field-tip arrows. We look critically at the free saleability of this equipment mostly used for sport shooting in Germany.

The “Out of Africa Tribe” (II): Paleolithic warriors with big canoes and protective weapons

It is generally difficult to establish a timeline for the appearance of different technologies and tools during human cultural evolution. Here I use stochastic character mapping of discrete traits using human mtDNA phylogenies rooted to the Reconstructed Sapiens Reference Sequence (RSRS) as a model to address this question. The analysis reveals that the ancestral state of Homo sapiens was hunting, using material innovations that included bows and arrows, stone axes and spears. However, around 80,000 y before present, a transition occurred, from this ancestral hunting tradition, toward the invention of protective weapons such as shields, the appearance of ritual fighting as a socially accepted behavior and the construction of war canoes for the fast transport of large numbers of warriors. This model suggests a major cultural change, during the Palaeolithic, from hunters to warriors. Moreover, in the light of the recent Out of Africa Theory, it suggests that the “Out of Africa Tribe” was a tribe of warriors that had developed protective weapons such as shields and used big war canoes to travel the sea coast and big rivers in raiding expeditions.

Searching for the majority: algorithms of voluntary control.

Voluntary control of information processing is crucial to allocate resources and prioritize the processes that are most important under a given situation; the algorithms underlying such control, however, are often not clear. We investigated possible algorithms of control for the performance of the majority function, in which participants searched for and identified one of two alternative categories (left or right pointing arrows) as composing the majority in each stimulus set. We manipulated the amount (set size of 1, 3, and 5) and content (ratio of left and right pointing arrows within a set) of the inputs to test competing hypotheses regarding mental operations for information processing. Using a novel measure based on computational load, we found that reaction time was best predicted by a grouping search algorithm as compared to alternative algorithms (i.e., exhaustive or self-terminating search). The grouping search algorithm involves sampling and resampling of the inputs before a decision is reached. These findings highlight the importance of investigating the implications of voluntary control via algorithms of mental operations.

Baylor History of Medicine Society Schedule 2006-2007

After Western Medicine: From Hippocrates to Xavier Bichat (H. Tristram Engelhardt, Jr.) Who Goes First? The Story of Self-Experimentation in Medicine (David Sears, M.D.) Exercise and Health: From Pre-History to the Present (Carlos Valbonna, M.D.) Supernaturalism to Rationalism and the Man Between (Student Essay Contest Winners) (Don Lassus) The Fog of War’s Silver Lining: The Lasting Impact of Military Medicine (Student Essay Contest Winners) (Ajit Vyas) From Drummers to Detail Men: Medicine and the pharmaceutical industry in the United States, 1900-1960 (Howard Brody) Eyewash and Thunderbolts: The Medical Adventures of Lewis and Clark (Herbert M. Swick) Angry Arrows and Satin Dresses: Tales from the Annals of Plague (Herbert M. Swick) The Greatest Books in the History of Neurology (Robert Gordon) Franklin Delano Roosevelt’s Paralytic Illness: What was the cause? (Armond S. Goldman)

A refined Pliocene to early Pleistocene chronostratigraphic frame at ODP Hole 653A (West Mediterranean)

Two planktonic foraminiferal oxygen isotope records of ODP Hole 653A (Tyrrhenian Sea) are presented for the time period extending from approximately 0.8 to 3.0 Ma. Six, generally accepted, synchronous bioevents were used to precise the oxygen isotope chronology and to identify the oxygen isotope stages 22 down to 114. Subsequently, this oxygen isotope chronology was used to determine the synchronism or diachronism of various other biostratigraphic events with those recorded in the Singa and Ficarazzi land sections (Italy) and those in other DSDP/ODP sites. New results concern the diachronity of the FOD of the planktonic foraminiferal species N. atlantica, G.truncatulinoides truncatulinoides and G. inflata between ODP Hole 653A and the Italian landsections. Because many species entered the Mediterranean in short term fluxes, strongly related to the southward migration of cool North Atlantic surface waters, their time distribution through the Pliocene-Pleistocene generally corresponds to alternated intervals of presence and absence. This should explain most of the apparently diachronous appearances and disappearances. Alternating presence-absence patterns are of less importance for the various nannofossil events. The LOD of D. surculus occurs during the transition of stage 100 to 101 in both ODP Hole 653A and the Singa section, which is in perfect agreement with the disappearance of this species from the open ocean. The LOD of D. pentaradiatus in the Mediterranean occurs in stages 100-99, which seems to be consistent with the extinction of this species in the southern Hemisphere. G. oceanica, which corresponds to the 4 µm < Gephyrocapsa spp <5.5 µm is recorded in stages 65 to 64 at ODP Hole 653A. The Gephyrocapsa spp. >5.5 µm first occurred in stage 51 at Hole 653A, which fits within the uncertainty interval for this event stretching from stage 51 to 47 in the open ocean and seems therefore a useful tool for conventional biostratigraphy in the Mediterranean.

Cuentos medievales : "La dama del castillo del gavilán" y el "Ejemplo de las flechas" Juan de Mandevilla, Libro de las maravillas del mundo

A partir de los últimos estudios acerca de la cuentística medieval, tendientes a ampliar el corpus de relatos insertos no sólo en colecciones de cuentos sino en textos muy variados, se rastrean y clasifican los incluidos en el Libro de las maravillas del mundo de Juan de Mandevilla. Asimismo, se realiza el análisis puntual de dos cuentos: "La dama del castillo del gavilán" y el "Ejemplo de las flechas".

Cuentos medievales : "La dama del castillo del gavilán" y el "Ejemplo de las flechas" Juan de Mandevilla, Libro de las maravillas del mundo

A partir de los últimos estudios acerca de la cuentística medieval, tendientes a ampliar el corpus de relatos insertos no sólo en colecciones de cuentos sino en textos muy variados, se rastrean y clasifican los incluidos en el Libro de las maravillas del mundo de Juan de Mandevilla. Asimismo, se realiza el análisis puntual de dos cuentos: "La dama del castillo del gavilán" y el "Ejemplo de las flechas".

Cuentos medievales : "La dama del castillo del gavilán" y el "Ejemplo de las flechas" Juan de Mandevilla, Libro de las maravillas del mundo

A partir de los últimos estudios acerca de la cuentística medieval, tendientes a ampliar el corpus de relatos insertos no sólo en colecciones de cuentos sino en textos muy variados, se rastrean y clasifican los incluidos en el Libro de las maravillas del mundo de Juan de Mandevilla. Asimismo, se realiza el análisis puntual de dos cuentos: "La dama del castillo del gavilán" y el "Ejemplo de las flechas".

Infrastructure (Resilience-oriented) Modelling Language: I®ML A proposal for modelling infrastructures and their connections

The modelling of critical infrastructures (CIs) is an important issue that needs to be properly addressed, for several reasons. It is a basic support for making decisions about operation and risk reduction. It might help in understanding high-level states at the system-of-systems layer, which are not ready evident to the organisations that manage the lower level technical systems. Moreover, it is also indispensable for setting a common reference between operator and authorities, for agreeing on the incident scenarios that might affect those infrastructures. So far, critical infrastructures have been modelled ad-hoc, on the basis of knowledge and practice derived from less complex systems. As there is no theoretical framework, most of these efforts proceed without clear guides and goals and using informally defined schemas based mostly on boxes and arrows. Different CIs (electricity grid, telecommunications networks, emergency support, etc) have been modelled using particular schemas that were not directly translatable from one CI to another. If there is a desire to build a science of CIs it is because there are some observable commonalities that different CIs share. Up until now, however, those commonalities were not adequately compiled or categorized, so building models of CIs that are rooted on such commonalities was not possible. This report explores the issue of which elements underlie every CI and how those elements can be used to develop a modelling language that will enable CI modelling and, subsequently, analysis of CI interactions, with a special focus on resilience

Tratamiento N-categorial de la lógica de la programación

En el contexto de las técnicas de validación de programas, de las estructuras de axiomatización de la programación planteadas por Hoare y del desarrollo de la Lógica de la Programación que Dijkstra realiza con el operador wp, hemos dotado a los elementos esenciales que intervienen en dicho desarrollo de una estructura que permite su estudio en el ámbito de las categorias. En este trabajo se demuestra que el conjunto de precondiciones de un fragmento de programa anotado tiene estructura de N-categoría y que sucede lo mismo con el conjunto de postcondiciones. Más aún, se ha puesto de manifiesto (y probado) que el operador wp actúa como un funtor entre estos pares de N-categorías. Además, los conjuntos de guardas surgen de un modo natural en la lógica de la programación, han sido tratados desde el punto de vista de la semántica denotacional (Wlrth para el PASCAL y Scott y otros, después, en un enfoque más general) y presentados por Manes y Arbib en su semántica parcialmente aditiva. Pues bien, en este trabajo se demuestra que los conjuntos de guardas tienen también estructura de N-categoría y gue cualguier N-categoría dotada de una suma definida adecuadamente tiene estructura de conjunto de guardas, de tal modo, además, gue el preorden inducido por la suma en el conjunto de guardas coincide con la flecha de la N-categoría. Esta suma es, en concreto, la disyunción exclusiva, lo que adicionalmente supone una sorprendentemente sencilla definición alternativa a la suma de las categorías parcialmente aditivas definida por Manes y Arbib. Con todo esto, se aportan herramientas conceptuales para entender mejor y resolver más eficientemente los problemas que tiene planteados la lógica de la programación, pues se dispone de un punto de vista distinto y nuevo y de toda una familia de instrumentos adicionales.---ABSTRACT---In the context of program validation techniques, Hoare's systems for programming and Dijkstra's development of the logic of programming, based on the operator wp, we have endowed the essential features of this development with a structure that permits to study them in the frame of category theory. In this thesis we show that the set of preconditions of an annotated program segment is an N-category, and the same happens for the set of postconditions. Even more, it is shown that the operator wp acts as a functor between those pairs of Ncategories. Furthermore, guard sets come out in a natural way in the logic of programming, they have been considered from a denotational semantics point of view (Wirth for Pascal and afterwards Scott and al. in a more general setting) and they have been embodied by Manes and Arbib in their partially additive semantics. Then, it is shown in this thesis that the above mentioned guard sets also have the structure of an N-category and that any N-category with an appropiately defined sum has the structure of a guard set in such a way that, besides, the preorder defined in the guard set by the sum operation coincides with the arrows of the N-category. This sum is just the exclusive or of Boolean Logic and this fact adds a surprisingly simple alternative definition for the sum operation in Manes and Arbib partially additive categories. The present work, in summary, makes a contribution of conceptual tools for a better understanding and a more efficient solution of the problems posed to the logic of programming and it does so by providing a new different point of view and a whole family of additional techniques.

Do students confuse dimensionality and “directionality”?

The aim of this research is to understand the way in which students struggle with the distinction between dimensionality and “directionality” and if this type of potential confusion could be a factor affecting students’ tendency toward improper linear reasoning in the context of the relations between length and area of geometrical figures. 131 9th grade students were confronted with a multiple-choice test consisting of six problems related to the perimeter or the area of an enlarged geometrical figure, then some interviews were carried out to obtain qualitative data in relation to students’ reasoning. Results indicate that more than one fifth of the students’ answers could be characterized as based on directional thinking, suggesting that students struggled with the distinction between dimensionality and “directionality”. A single arrow showing one direction (image provided to the students) seemed to strengthen the tendency toward improper linear reasoning for the area problems. Two arrows showing two directions helped students to see a quadratic relation for the area problems.

Telhîs-i resâ'il ül-rumāt

Mustafa Kânî bin Muhammed.

Symposium on work zone traffic control. Proceedings.

Federal Highway Administration, Office of Implementation, Washington, D.C.

Effectiveness of flashing arrow boards during moving maintenance operations. Final report.

Federal Highway Administration, Washington, D.C.