Biologische Hochschulschriften (Goethe-Universität; nur lokal zugänglich)
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The research presented in this thesis characterizes U2AF homology motifs (UHM) and their interactions with UHM ligand motifs (ULM) in the context of splicing regulation. UHM domains are a subgroup of RNA recognition motifs (RRM) originally discovered in the proteins U2AF65 and U2AF35. Whereas canonical RRMs are usually involved in binding of RNA, UHM domains bind tryptophan containing linear protein motifs (ULM) instead. In the first article, we analyze the complex network of interactions between splicing factors and RNA that initiate the assembly of the spliceosome at the 3´ splice site of an intron. The protein U2AF65 binds a pyrimidine-rich element in introns and recruits U2snRNP by binding its protein component SF3b155. My contribution was to define the binding site of the protein U2AF65 to the intrinsically unstructured N-terminus of the scaffolding protein SF3b155. I could show that the UHM domain of U2AF65 recognizes a ULM in SF3b155, and that this binding site is not overlapping with the binding sites of other splicing factors, like p14, to SF3b155. As the U2AF65-UHM:SF3b155-ULM interaction is mutually exclusive with an interaction between U2AF65-UHM and a ULM in the splicing factor SF1, which was reported to initially recognize the branch point sequence, my results provide the molecular details on how SF3b155 replaces SF1 during spliceosomal reorganizations. In the second article, we show that overexpression of the UHM domain of the splicing factor SPF45 induces exon 6 skipping in the pre-mRNA of Fas (CD95/APO-1). I provide evidence for in vitro binding of SPF45-UHM to ULM sequences in the splicing factors U2AF65, SF1, and SF3b155. I crystallized free and SF3b155-bound SPF45 UHM and solved both structures by X-ray crystallography. The analysis of the complex interface and sequence differences in the ULMs allowed me to design mutations of SPF45-UHM, which selectively inhibit binding to distinct ULMs. After assessing the ULM binding properties in vitro, we could show that the activity of SPF45-UHM in influencing the splicing pattern of Fas relies on interactions with SF3b155 and/or SF1, but that an interaction with U2AF65 is dispensable. A mechanism for the activity of SPF45-UHM could thus be engaging in ULM interactions and thus interfering with the network of interactions that initiate the assembly of the spliceosome at the 3´splice site, as described above. In the third article, we describe an unusual flexible homodimerization mode of the UHM in the splicing factor Puf60, which enables simultaneous interactions with ULM sequences on other splicing factors. I could show that the NMR relaxation properties of Puf60-UHM are inconsistent with a model of a rigid dimer, but rather indicate a dimerization via a flexible linker. I identified a flexible loop in the peptide backbone of Puf60-UHM, and showed that mutiation of acidic residues in this loop impairs the dimerization. To analyze the dimerization interface in further detail, I solved the structure of Puf60-UHM by X-ray crystallography. The acidic residues in the flexible loop of one UHM dimer subunit mediate the dimerization by contacting basic residues on the β-sheet surface of the other dimer subunit. Differences in the four dimer interfaces observed for the eight molecules in the asymmetric unit of the crystal support the model of an undescribed, flexible mode of dimerization, and thus complement the NMR relaxation data. Furthermore, I could show that the Puf60-UHM dimer and U2AF65-UHM contact different ULM sequences on the SF3b155 N-terminus in vitro, thus providing a possible explanation for the mutual cooperative activation of Puf60 and U2AF65 in splicing assays described in the literature. The fourth article is a review about recent research on the recognition of DNA double strand breaks (DSB) by covalent histone modifications. The p53 binding protein 1 (53BP1) is a DSB sensor and a checkpoint protein for mitosis. Recent crystallographic evidence indicates that 53BP1 recognizes DSB sites by binding histone H4 dimetylated at lysine 20 (H4-K20). We provide a comprehensive overview of the atomic resolution structures that revealed how proteins can specifically recognize histone tail modifications, especially methylated lysines, to read the information stored in what is called the histone code.
(1) Die genomweite Expressionsanalyse von salzadaptierten Zellen von M. mazei Gö1 identifizierte eine Reihe von salzregulierten Genen. Neben den beiden Operone ota und abl, die für die Akkumulierung von Glycin-Betain und Ne-Azetyl-b-Lysin verantwortlich sind, konnte ein ABC-Transporter (MM0953), der in seiner Genumgebung weitere Transporter sowie Proteine mit konservierten S-Layer-Domänen aufweist, als salzreguliert erkannt werden. Dies deutet auf ein S-Layer-Exportsystem hin, das eine Rolle in salzadaptierten Zellen spielen könnte. (2) Eine genomweite Expressionsanalyse von Zellen von M. mazei Gö1 zu unterschiedlichen Zeitpunkten nach einem hyperosmotischen Schock auf 400 mM NaCl ermöglichte Einblicke in den Verlauf der Genexpression. Die Erhöhung der externen Osmolarität resultierte in der erhöhten Expression von Genen, die für die Aufnahme und Biosynthese von kompatiblen Soluten verantwortlich sind sowie von Genen deren Produkte regulatorische Funktion haben könnten. (3) Genomweite Expressionsanalysen von Zellen von M. mazei Gö1 nach einem hypoosmotischen Schock zeigten erhöhte Expression von Genen, die an der Regulation und an der generellen Stressantwort beteiligt sind. Gene, deren Produkte im Stoffwechsel wichtig sind – besonders Gene, die für Methylamin-Corrinoid-Methyltransferasen kodieren – erscheinen stark reprimiert. (4) Die Bestimmung der intrazellulären Ionenkonzentrationen zeigte ein unspezifisches Einströmen von den Ionen, die den osmotischen Schock auslösen sofort nach dem Schock, sowie den Ausstrom derselben Ionen im Verlauf von 5 Minuten. Die Ionenkonzentrationen der Ionen, die den Schock auslösten, blieben intrazellulär erhöht. Das Ein- und Ausströmen der Ionen nach einem hyperosmotischen Stress ist nicht energieabhängig. (5) M. mazei akkumulierte nach einem hyperosmotischen Schock kein K+, zeigte aber eine erhöhte intrazelluläre Konzentration dieses Ions, wenn die Zellen in Medium mit erhöhter Osmolarität angezogen wurden. (6) Durch hyperosmotische Schocks mit verschiedenen Salzen und Zuckern konnte gezeigt werden, dass die kurzzeitige Akkumulation von Ionen keine gerichtete Antwort auf den osmotischen Stress ist. (7) Es konnte weiters gezeigt werden, dass Zellen von M. mazei Gö1, die mit dem kompatiblen Solut Betain inkubiert wurden, nach einem hyperosmotischen Schock K+ akkumulieren. Dies bedeutet möglicherweise eine K+-abhängige Regulation des Glycin-Betain-Transporters. (8) Die Funktion der drei im Genom kodierten Na+/H+-Antiporter konnte auf transkriptioneller Ebene nicht geklärt werden. Trotzdem zeigt ein Hydrophobizitätsplot des Proteins eine mögliche Beteiligung von Nha1 (MM0294) an der Osmoregulation durch eine hydrophile C-terminale Domäne. (9) Nach einem hyperosmotischen Schock von 38,5 auf 400 mM NaCl erhöhte sich die intrazelluläre Konzentration an Glutamat, das in M. mazei als kompatibles Solut fungiert, bereits nach drei Stunden. Zellen, die bereits an die erhöhte Salzkonzentration adaptiert waren, enthielten 1,4 μmol Glutamat/mg Protein. (10) Die Glutamin-Synthetase zeigte eine erhöhte Transkription nach einem hyperosmotischen Schock. Das Protein wird aber nicht salzabhängig produziert und zeigt keine Enzymaktivität. Die Biosynthese des Solutes über eine Glutamat-Dehydrogenase ist die wahrscheinliche Alternative. (11) Aufgrund der generierten Expressionsprofile und der physiologischen Daten konnte ein Modell der Osmoadaptation in Methanosarcina mazei Gö1 erstellt werden.
Paleoecology is the study of organismal interactions with the environment in the geological past. Organisms are influenced in their distribution and abundance by abiotic factors such as temperature and precipitation. A change in these factors, for example by major climatic shifts, would then affect the communities of organisms. Studying this hypothesized causal link between climatic and faunal change is especially interesting for the Plio-Pleistocene of East Africa due to the fact that our own ancestors also inhabited these regions. Both the Turkana basin in Kenya and the Lake Albert region in Uganda offer unique opportunities to investigate these paleoecological issues. Their late Miocene through Pleistocene deposits provide a very good record of climatic, vegetation and faunal change in East Africa (Pickford et al. 1993, Leakey et al. 1995, 1998, McDougall & Feibel 2003, Wynn 2004). This study focuses on the mammal family Bovidae as they are good indicator of vegetation and environment (e.g. Vrba 1980, 1995, Shipman & Harris 1988, Bobe & Eck 2001, Bobe & Behrensmeyer 2004, Bobe et al. 2007). Bovidae are quite species-rich and inhabit a wide range of habitats from tropical rain forests to deserts which predicates their array of morphological adaptations (ecovariables) to these environments. Diet is the ecovariable that is most to climate and thus habitat change. Therefore, the fossil Bovidae are especially suitable for reconstructing past environments. The objective of this thesis is to test the hypothesis that, from the late Miocene through the Holocene, Africa has experienced an overall increase in aridity and concomitant pulses of habitat change. The hypothesis predicts that increasing aridity causes a likewise growth in the abundance of taxa adapted to open arid environments. In particular, an increase in bovid grazers should be observed in combination with a decrease of bovid browsers. To test this hypothesis, I examine the fossil bovid communities from each stratigraphic member of Lake Turkana (Lothagam, Kanapoi, West Turkana and Koobi Fora) and Lake Albert (Nkondo-Kaiso region) and through a taxonomic and a functional perspective reconstruct the paleoenvironments and -climates from approximately 8 to 0.6 Ma. This study is the first to use taxonomic and ecomorphological data together to reconstruct the paleoenvironments of the Turkana basin and the Nkondo-Kaiso region of Lake Albert. In a first analysis, mesowear, as introduced by Fortelius & Solounias (2000), is used to gather information about the diet of bovids. As a result of my preliminary investigations on upper vs. lower molars of recent species, the sample of fossil bovid specimens from the Turkana basin and Lake Albert were found to be unsuitable to reveal a meaningful diet reconstruction. Therefore, the bovids are assigned to diet categories based on literature. For each member of the time period from 8.0 to 0.6 Ma, I provide a detailed characterization of the bovid fauna in terms of α- and β- diversity both on tribe and diet level based on presence-absence as well as for the Turkana basin on abundance data. Statistical comparisons between the fossil bovid communities and those in modern protected areas with known vegetation and climatic conditions have yielded modern analogues for each stratigraphic member. Following that I provide paleoclimatic conditions such as assumed mean annual temperature for each member. Based on abundance of diet categories in the bovid communities, the paleoclimate of the Turkana basin was in general cooler and considerably more humid during the late Miocene to the Pleistocene than today. The mean annual temperature at Lothagam is assumed as 22.2 °C, the annual precipitation as 685 mm for 8.0 – 6.54 Ma and 4.9 – 3.4 Ma. The intervening time period is characterized by a slightly lower mean annual temperature and precipitation (20.3 °C, 583 mm). From 4.17 to 4.07 Ma Kanapoi faced 21.3 °C and 592 mm rainfall. In the eastern part of the basin the climate was warmer and more humid (3.4 – 2.68 Ma: 26.2, 961 mm; 2.68 – 1.3 Ma: 27.1 °C, 935 mm) from 3.4 to 1.3 Ma than in the preceeding eras. In the western part, the climate became warmer and more humid ~500,000 years later and was more variable than that in the eastern basin. From 2.94 to 2.52 Ma the mean annual temperature was 26.2 °C and the annual precipitation 961 mm. Between 2.34 and 1.6 Ma the climate again cooled and became drier as before 2.94 Ma. A second shift to higher temperature and precipitation occurred after 1.6 Ma (27.1 °C, 935 mm) lasted until 1.34 Ma. The results of the bovid community analyses do not support the hypothesis of increasing aridity in Eastern Africa during the late Mio- to Pleistocene. Instead, the results show that the bovid communities differed much over time and on a relatively small spatial scale. Regional paleovegetation and paleoclimate exhibit fluctuations through the studied time period at western Turkana and differences between the western and eastern part of the Turkana basin. This is indicative of a patchy habitat distribution both on temporal and spatial levels. Increased climate variability predicts an increase in landscape complexity as proposed by the ‘variability selection hypothesis’ (Potts 1998a+b). Therefore, this thesis research supports the hypothesis of increased landscape complexity on the spatial level. This study has important implications for future research. First, an analysis based on ecovariable characteristics such as diet may be preferred to a taxonomic analysis. Second, abundance data should be used for an ecovariable analysis because the results then provide more precise information on the paleovegetation and –climate than just the presence of these adaptations in the faunal community. Lastly, as this study is based on one mammal family, further studies on other mammal groups should be conducted to increase the database of exploited resource by the entire faunal community. Most significantly this study provides a basis for new interpretations of faunal community distributions. It also raises the question whether small scale spatial community variability is also to be expected at other fossil sites. If so then this methodology has important implications for reconstructions of paleovegetation and paleoclimate.
Pharmakokinetische Charakterisierung der Terpenlaktone aus Ginkgo biloba im ZNS am Tiermodell
(2010)
Ginkgo biloba (Gb), eine der am besten untersuchten pharmazeutisch-medizinisch genutzten Pflanzen, wird heute in Form von Spezialextrakten im Sinne einer evidenzbasierten Phytopharmakatherapie eingesetzt. Grundlage hierfür sind die genaue Spezifikation der Zusammensetzung des Spezialextraktes in Bezug auf die wirksamkeitsbestimmenden Inhaltsstoffe, balstbare klinische Daten, das Erforschen des molekularen Wirkmechanismus‘ des Gesamtextraktes aber auch der Einzelbestandteile und die Pharmakokinetik im Targetgewebe. Heute werden im Sinne einer evidenzbasierten Phytopharmakatherapie lediglich Extrakte verwendet, die der Monographie der Komission E entsprechen (22 - 27% Flavonoide, 5 - 7% Terpenlaktone und weniger als 5 ppm Ginkgolsäuren). Der am besten klinisch und pharmakologisch untersuchte Gb-Spezialextrakt ist EGb 761® (Tebonin®), der im zentralen Fokus der vorliegenden Arbeit steht. Die im Jahr 2008 vom IQWiG veröffentlichte Metaanalyse zur Klinik von EGb 761® hat in äußerst detaillierter Form belastbare Daten zur Wirksamkeit dieses Extraktes beschrieben. Es kann festgehalten werden, dass ein Einsatz dieses Spezialextraktes im Rahmen der Therapie einer beginnenden Demenz zu befürworten ist. Basis des klinischen Einsatzes des EGb 761® sind in vitro und in vivo pharmakologische Untersuchungen. Es werden unterschiedliche Gesamtkonzepte zur Wirkung von EGb 761® bzw. Einzeleffekte der Inhaltsstoffe im ZNS diskutiert. Konsensfähig sind heute sicher die Mitochondrien-stabilisierende Wirkung der Terpenlaktone und ein antioxidativer Effekt der Flavonoide. Bb zeigt zusätzlich deutlich protektive Effekte in Bezug auf durch zerebrovaskuläre Ereignisse geschädigte Hirnareale. Darüber hinaus ist die Wirkung der Flavonoide auf die monoaminerge Neurotransmission aktueller Gegenstand der Forschung. Basis jeglicher pharmakologischen Betrachtung ist das pharmakokinetische Verhalten der wirksamen Inhaltsstoffe im Target-Gewebe. Nachdem die ZNS-Bioverfügbarkeit der Flavonoide nachgewiesen wurde, hat die vorliegende Arbeit das zentrale Ziel, die pharmakokinetische Charakteristik der Terpenlaktone aus Gb im ZNS zu untersuchen. Zur quantitativen Analyse der Terpenlaktone (GKA, GKB, GKC und Bb) in biologischen Matrices (Hirn-Homogenat, Plasma) und Hirn-Dialysat-Pufferlösung (aCSF-Puffer) wurde eine LC-MS-Analytik-Methode entwickelt und validiert. Unter Verwendung einer 250x4 mm, Multo High 100 RP18, 5 μm (CS-Chromatographie Service GmbH)-Säule und einer isokratischen Auftrennung mittels einer mobilen Phase bestehend aus 60% 0,1%-iger Ameisensäure und 40%-igem Methanol konnten alle vier genannten Terpenlaktone simultan innerhalb von 20 Minuten analysiert werden. Die beschriebene LC-MS(TOF)-Methode verfügt über eine ausreichende Sensitivität, um die Analyten im nanomolaren Bereich zu quantifizieren (z.B. LOQ Bb in aCSF-Puffer: 0,25 pg/μl; LOQ Bb in Hirnhomogenat: 1 ng/ml). Die Aufarbeitung der Plasma- bzw. Hirn-Homogenat-Proben erfolgte durch eine flüssig-flüssig-Extraktion mit Hilfe von Extrelut®-Säulen; die Hirn-Dialysat-Proben bedurften keiner Probenaufarbeitung. Mit Hilfe der beschriebenen Analytik-Methode war es möglich, GKA, GKB, GKC und Bb in Plasma und Hirnhomogenat von Ratten nach oraler Gabe von 600 mg/kg Körpergewicht EGb 761® bzw. einer vergleichbaren Menge der Reinsubstanzen zu bestimmen. Im Rahmen dieses Projektes wurde ein direkter Vergleich der erhalten Plasma-Konzentrationen nach Extrakt- bzw. Reinsubstanzgabe gezogen, wobei der Extrakt die höhere AUC (für GKA u. Bb) und daher bessere Bioverfügbarkeit aufwies. Es konnten in Plasma und Gehirngewebe sowohl GKA als auch GKB und Bb in nativer nicht metabolisierter Form nachgewiesen werden. GKC konnte weder in Plasma noch in Hirngewebe bestimmt werden, was die in der Literatur diskutierte These einer schnellen Metabolisierung (Methylierung) stärkt. Die Terpenlaktone sind im Plasma sehr schnell angeflutet und zeigten ein ebenfalls zügiges Abfallen, so dass 24 Stunden nach oraler Applikation keine Konzentrationen mehr zu detektieren waren. Bei der Untersuchung der Hirn-Gewebspiegel von GKA, GKB und Bb zeigten sich keine Unterschiede nach Gabe von Extrakt bzw. Reinsubstanz. Die Substanzen fluteten im Vergleich zum Plasma etwas verzögert an, fielen aber auch bis 24 Stunden nach Applikation wieder unter die Nachweisgrenze. Die Konzentrations-Zeit-Kurven ähnelten in ihrer Form stark denen aus Plasma, waren jedoch zeitlich nach rechts verschoben, so dass ausgeschlossen werden kann, dass es sich im Hirngewebe um Artefakt aus Restblut handelt. Wesentliches Resultat dieser Untersuchungen war, dass erstmalig nach oraler Gabe von EGb 761® gezeigt wurde, dass deutliche Gewebespiegel im Gehirn von Ratten zu erzielen sind und damit diese Substanzen im Target-Gewebe die postulierten pharmakologischen Wirkungen ausüben können. Aufbauend auf diesen Ergebnissen wurden mit Hilfe der Mikrodialyse-Technik und der bereits beschriebenen LC-MS-Analytik-Methode weitere pharmakokinetische Untersuchungen am Maus-Modell durchgeführt. Es konnte zunächst rein technisch im Rahmen von Wiederfindungsuntersuchungen gezeigt werden, dass die im Dialysat bestimmte Menge Bb ca. 6% der tatsächlich im Extrazellularraum des Maus-Hirns vorliegenden Bb-Konzentration entspricht. Weiterhin zeigten diese Versuche, dass Bb kaum an Plasma-Proteine bindet, da keine signifikanten Unterschiede bei der Dialyse von Bb aus Puffer, Blut oder Plasma zu sehen waren. In einem ersten Tierversuch an gesunden Mäusen konnten die pharmakokinetischen Charakteristika von Bb, die in der Fütterungsstudie an Ratten bestimmt wurden, reproduziert werden, obwohl es sich um einen völlig divergenten Versuchsaufbau, unterschiedliche Tierspezies und nicht um die gleichen Applikationsformen handelt. Diese Tatsache unterstreicht die Aussage beider Studien. Als zusätzliche Aussage ergibt sich aus dem Versuchsaufbau, dass Bb frei und biologisch aktiv im Extrazellularraum vorliegt und nicht z.B. in Membranen gebunden ist. Die Möglichkeit mittels Mikrodialyse und LC-MS-Technik Bb im Extrazellularraum definierter Hirnregionen nachzuweisen, erlaubte eine pharmakokinetische Charakterisierung von Bb in vom Schlaganfall geschädigten Hirngewebe. Es zeigte sich, dass bei Gabe von 10 mg/Kg Bb eine Stunde vor dem Schlaganfall die Bb-Konzentrationen zwar deutlich abfallen, aber dann relativ konstant bleiben, was durch einen fehlenden Abtransport durch die unterbrochene Blutversorgung zu erklären ist.
Phykologie - Blütezeiten und Ruhepausen einer Wissenschaft im Nebenamt (Fallbeispiel Grünalgen)
(2002)
Phykologie (Algenforschung) ist der Öffentlichkeit dem Namen nach und als wissenschaftliche Disziplin kaum geläufig. Innerhalb der Botanik (Biologie) besteht die Algenkunde gleichsam "implizit" als eine Vielfalt von Entwicklungssträngen, die selten gebündelt sind, vielmehr leicht immer wieder abreißen können, denen sich aber immer wieder auch neue Stränge hinzugesellen. Die Phykologie erforscht, im Gegensatz etwa zum Studium der Blütenpflanzen, nur wenige aus dem Alltagsleben vertraute Gewächse. Ihre Untersuchungsobjekte wurden (und werden immer noch) erst nach und nach entdeckt, weshalb sich die Vorstellung, was eine Alge ist, ständig wandelt. Viele sind ohne Mikroskop gar nicht wahrzunehmen. Forschungsgegenstand und Forschungsaufgabe bilden durch ihre fortwährende Umgestaltung die Leitgedanken der Botanik und Biologie überhaupt ab, wobei die Beziehung reziprok ist. Eine lebhafte Fluktuation von ausübenden Personen und Paradigmata ist ebenso charakteristisch für die Phykologie wie eine ausgeprägte Inter- bzw. Transdisziplinarität und ein hohes aktives internes Innovationspotential. Am Beispiel der traditionellen, heute so nicht mehr bestehenden, »Chlorophyta« wird untersucht, wie diese Sippe zunächst für makroskopisch sichtbare Meeresalgen etabliert wurde. Dann wurden ihr schrittweise verschiedene binnenländische Formenkreise von Algen zugeordnet, bis hin zu den grünen Phytoflagellaten. In der jüngsten Entwicklungsphase haben sich die "grünen Algen" als heterogener Komplex mehrerer voneinander unabhängiger Entwicklungslinien erwiesen, die bis auf eine über das Niveau der Algen nicht hinausgekommen sind, wohl aber zum Landleben fähige Vertreter haben. Als Wurzelbereich der echten Landpflanzen erwies sich überraschend ein kleiner Verwandtschaftsbereich der Algenklasse der Charophyceae.
This study comprises a survey on ecology, morphology and taxonomy of parasitic fungi infecting Pteridophytes and Orchidaceae found by the author on several field trips to Western Panama as part of the project plant parasitic micro-fungi of Western Panama (ppMP). In Panama, approximately 9500 species of vascular plants are found. Of these, Orchidaceae are with ca. 1150 (ca. 12%) species by far the most speciose family. The Pteridophytes in Panama comprise ca. 940 species in 31 families. Most fungal pathogens on Orchidaceae in tropical regions were described from plants in culture or from material intercepted at borders by plant quarantine services and not from their natural habitats. Therefore, little is known about distribution and ecology of these pathogens in their natural range. The author determined and classified several hundred Orchidaceae-species and Pteridophytes at the sites selected in the context of the project. This work facilitated the identification of many host plants (at least to genus-level) even in sterile condition in the field. About 65 species of Pucciniales are known to infest Orchidaceae and ca. 38% of them are described from tropical America. All available types of Pucciniales on Orchidaceae in tropical America were studied and compared with 91 specimens of rust fungi on orchids collected by the author in Panama. Several hundred additional specimens housed in the BPI, almost all intercepted from plant quarantine services, were used for comparison. As result of this work, it is suggested to combine Uromyces stenorrhynchi Henn. to Sphenospora and, as this is the oldest epithet, to synonymize S. kevorkianii Linder, S. mera Cumm. and S. saphena Cumm. with it. Further, it could be demonstrated that Uredo aurantiaca Montemartini, U. cyrtopodii Syd. & P. Syd., U. epidendri Henn., U. guacae Mayor, U. gynandrearum Corda, U. lynchii (Berk.) Plowr., U. neopustulata Cumm. (≡U. pustulata Henn.), U. nigropuncta Henn., U. oncidii Henn., U. ornithidii F. Kern., Cif. & Thurst., and presumably U. scabies Cke., are anamorphs of this variable species. U. gynandrearum is the oldest anamorph-name for all these taxa. Therefore, it can be established that this rust infects more than 80 species of Orchidaceae in three subfamilies. In total, the anamorph of this species was collected by the author on 17 different species of Orchidaceae in Panama which, apart from one species, are all new hosts to science. The molecular data obtained by the author confirm this view, although more data, especially from material from the whole range of distribution of U. gynandrearum, are necessary. Puccinia spiranthicola Cumm. was found to be a synonym of P. cinnamomea Diet. & Holw. and was found by the author on three different Orchidaceae in two subfamilies. Uredo pleurothallidis Keissl. is now considered a synonym of U. wittmackiana Henn. and the latter as the anamorph of Puccinia oncidii Cumm. In the anamorph genus Uredo, a new species was found infecting at least five different species of Sobralia and Elleanthus (Sobraliinae) at different localities. Molecular data indicate it to be related to the currently polyphyletic Phakopsoraceae. For the rusts with suprastomatal sori on Orchidaceae, now separated from Hemileia and placed in the genus Desmosorus (nom. inval.), the current concept with only one taxon is rejected and the establishment of three subspecies is suggested. The complicated taxonomy is discussed and makes it necessary to validate the genus-name and make a new combination. Another Hemileia-anamorph species was found by the author and is considered to be new to science. This is the first species of this alliance in America on Orchidaceae. Molecular data obtained by the author confirm the separation of Desmosorus from Hemileia and the position of the new species. For rusts on Pteridophytes, a new species of Milesia, (teleomorph: Milesina) and a new anamorphic species of Uredinopsis was found, both on hosts hitherto not known. In Calidion, the presumable anamorph-genus of Uncol, the species C. cf. cenicafeae Salazar & Buriticá was found on several new hosts. Further, the teleomorph was found. Morphologically, this teleomorph did not agree with the description of Uncol by the author of the genus, although the anamorph characteristics left no doubt that it is Calidion. Apparently, the description of Uncol is inadequate, but cannot be improved, as the type is unavailable. Molecular data obtained by the author show this species to be closest to Desmosorus. For Uredo superficialis Speg., the anamorph of Desmella, nine new hosts in eight different fern families were found by the author and the collaborators of the ppMP-project. Ecological data indicate that this species includes different host specific races, which, however could not be distinguished morphologically. For all these rusts, a thorough discussion of the ecology in their habitats is given. In total, 21 LSU rDNA sequences from 6 different rust species on Orchidaceae and Pteridophytes were obtained and analyzed with the Maximum Parsimony and Minimum Evolution method. Here, the position of several groups could be confirmed, and some anamorphs could be assigned to different teleomorphic relationships. Within the Ascomycota and their anamorphs, several hitherto unknown species and species not known from these hosts or not known from Panama were found and analyzed. On Orchidaceae, the following fungi belonging to the Ascomycota are described, illustrated and discussed: In the Phyllachorales, a hitherto not known Phyllachora sp. was found on Oncidium warszewiczii Rchb. f. and was compared with the other species of this order currently known from Orchidaceae. In the Asterinaceae s. l. Lembosia cf. epidendri Meir. Silva & O. R. Pereia was found on Maxillaria crassifolia (Lindl.) Rchb. f., which is a new host and new host alliance for this fungus hitherto only known from Brazil. The fungus is described and compared with all species of Asterinaceae currently known on Orchidaceae. In the Meliolaceae, Meliola orchidacearum Cif. was found on Camaridium biolleyi (Schltr.) Schltr. and an Epidendrum sp. which are new hosts and new host alliances of this fungus which was hitherto only known from the Caribbean Islands. It is described, illustrated and compared with the type. In the Glomerellaceae, Glomerella cingulata and its anamorph Colletotrichum gloeosporioides were found on several hosts. The species is illustrated, described and compared with data from literature. In the anamorphic Mycosphaerellaceae, Pseudocercospora odontoglossii (Prill. & Delacr.) U. Braun, a species currently only known from culture, was found on the new host Pleurothallis imraei Lindl. It is illustrated, described and compared with data from literature. On ferns, the following other fungi are described, illustrated and discussed: A conspicuous undescribed form of Polycyclus was found by the author on Elaphoglossum ciliatum (C. Presl.) T. Moore (Dryopteridaceae) and Serpocaulon loriceum (L.) A. R. Sm. (Polypodiaceae). A conspectus of Parmulariaceae infecting ferns is given and demonstrated that Polycyclina should be synonymized under Polycyclus. Summing up, it can be assessed, especially for the Pucciniales, that the most speciose plant family in Panama carries remarkable few species of specific parasites, and that many of them seem to be distributed over a wide range of species which often are not closely related. One reason amongst others seems to be that parasites need a minimum density of host plants in a habitat to survive. As orchid species often occur with only few (and often small) individual plants at a given locality, the probability for a specific pathogen to infect a plant gets too low, hence high diversity by low abundance of hosts might be an impediment for specific pathogens. In this case, unspecific parasites, or such which are infecting larger alliances, are in advantage. Other reasons could be specific traits of orchids, like succulence and mycotrophy which might hamper fungal infections.
5-Lipoxygenase (5-LO) catalyzes the two initial steps in the biosynthesis of leukotrienes, a group of inflammatory lipid mediators derived from arachidonic acid. Here, the regulation of 5-LO mRNA expression by alternative splicing and nonsense-mediated mRNA decay (NMD) was investigated. In the present study, the identification of two truncated transcripts and four novel 5-LO splice variants containing premature termination codons (PTC) was reported. The characterization of one of the splice variants, 5-LOΔ3, revealed that it is a target for NMD since knockdown of the NMD factors UPF1, UPF2 and UPF3b in the human monocytic cell line Mono Mac 6 (MM6) altered the expression of 5-LOΔ3 mRNA up to 2-fold in a cell differentiation-dependent manner suggesting that cell differentiation alters the composition or function of the NMD complex. In contrast, the mature 5-LO mRNA transcript was not affected by UPF knockdown. Thus, the data suggest that the coupling of alternative splicing and NMD is involved in the regulation of 5-LO gene expression.
RT-PCR analysis of different cell types revealed the existence of a large number of 5-LO splice variants. The most interesting splice variants were observed in BL41-E95A cells, which give a raise to novel 5-LO protein isoforms. This leads to the hypothesis of a novel regulatory mechanism in which the dimerization of 5-LO with 5-LO isoforms might regulate the 5-LO activity.
The 5-LO protein expression was reduced on translational level in UPF1 knock down cells, suggesting that UPF1 has a positive influence on 5-LO translation. Therefore, a mass spectrometry based proteomics study was started to identify compartment specific protein expression changes upon UPF1 knockdown in differentiated and undifferentiated MM6 cells. The proteomics analysis demonstrated that the knockdown of UPF1 results in numerous protein changes in the microsomal fraction (~ 21%) but not in the soluble fraction (< 1%). Western blot data confirmed the trend of the proteomics analysis. This data suggest that UPF1 is a critical gene expression regulator in a compartment specific way. During differentiation by TGFβ and calcitriol the majority of UPF1 regulated proteins was adjusted to normal level. It appears that that not only the NMD mechanism alters its composition during differentiation. Also the gene expression regulation on translational level by UPF1 seems to be also cell differentiation dependent. An interesting group of UPF1 target genes represent the downregulated proteins. qRT-PCR analysis of randomly chosen genes revealed no effect on mRNA expression upon UPF1 knockdown, suggesting that UPF1 positively influences the translation of these genes. Computational sequence analysis identified a conserved C-rich sequence which might be a hnRNP E2-binding site. hnRNP E2 has been characterized as a translational repressor in myeloid cells. Western blot analysis revealed a differentiation independent up regulation of hnRNP E2 by UPF1 knockdown. Additionally, microRNA-328 (miR-328) has been described as an RNA decoy modulating hnRNP E2 regulation. Due to this, stem loop qRT-PCR showed an up regulation of miR-328 in TGFβ and calcitriol differentiated MM6 cells. Based on this data we suggest a model in which downregulation of UPF1 increases hnRNP E2 expression, leading to translation inhibition. During differentiation, miRNA-328 is upregulated thereby competing with hnRNP E2 leading to an efficient translation
Since combinatorial chemistry and high throughput screening have become a common technique in the drug discovery phase the number of compounds being considered has increased frequently. These structures are often characterized by high molecular weight, high lipophilicity and low solubility in aqueous and physiological media. Due to the generally poor bioavailability, new in vitro techniques were needed for screening of pharmacokinetic properties. An important parameter for these screening methods is the implementation at an early state of drug discovery phase, to find potential lead structures, before investment costs become significant. The established in vitro methods for the prediction of membrane interaction are not reliable especially for poorly soluble compounds. A new method that is fast and easy to use, requires only small amounts of NCE and which can provide more reliable predictions is needed. In this study, a new screening technique based on surface activity profiling for the prediction of oral drug absorption was evaluated with special emphasis on the predictability of biological membrane interaction of poorly soluble drug compounds. It was demonstrated that drug absorption through a bilayer membrane can be modeled by the orientation of compounds at the air/water interface. Thus amphilicity of a drug is generally related to both oral absorption and blood brain barrier penetration. In turn, amphiphilicity is influenced by the lipophilicity, size and charge distribution of a drug. Surface activity profiling was determined by analysis of surface pressure profiles using the Gibbs adsorption isotherm. The surface activity measurements were carried out using a multichannel tensiometer Delta 8, which was developed by Kibron to be utilized in conjugation high throughput screening in early drug discovery processes. For this study two test sets were analyzed, one for the prediction of gastrointestinal wall interaction and the second for the prediction of the penetration behavior at the blood brain barrier. Both test sets consist of drug compounds with a wide range of absorption properties and consist mainly of compounds with poor water solubility. Since the drugs characteristics varied, they were classified according to water solubility and surface activity and a sample preparation method for each group was established. For the prediction of oral drug absorption, three different methods were established to model the interaction of compound and gastrointestinal wall. For drug compounds with solubility above 1mmol/L the traditional shake-flask method enabled the determination of the amphiphilic properties of drug compounds in pure aqueous media. Compounds with solubility below 1mmol/L tend to not to exhibit any increase in surface activity. Thus surface tension measurements of compounds, which exhibited a limited surface activity due to poor aqueous solubility, were conducted from stock solutions prepared with various organic solvents. Mainly polar organic solvents were used. A mixture of DMSO and DMF resulted in the best combination of properties: the intensive solubility enhancing effect of DMF and the lower intrinsic surface activity of DMSO. The polar solvent ruptured the water clusters, so that highly lipophilic structures had a higher affinity to the solvent and higher concentrations could be obtained. For these compounds higher maximum surface pressure were generated than was possible in pure aqueous media. The surface pressure data were correlated with the fraction absorbed values in vivo. However it was found that poor water solubility is not the only limiting step to exhibiting any surface activity. Some compounds were showed no surface activity in either solvent system. Therefore a micelle vehicle method was established using short chain phospholipids to mimic the gastrointestinal wall. It could be concluded from the results, that non surface active drugs can interact with the phospholipids micelle vehicle in a way analogous to their interaction with the membrane bilayer. The relative critical micelle concentration was correlated with the fraction absorbed of this test set. A sample preparation schema based on the three types of drugs was established. This schema enabled us to predict the absorbance of slightly soluble and poorly soluble drugs with acceptable reliability for early compound screening. For the prediction of blood brain barrier penetration using surface activity profiling as analyzing method, a test set with very poorly soluble characteristics was chosen. The sample preparation method was based on a strictly aqueous approach using the ‘shake flask’ method. The surface tension measurements enabled correlation of the amphiphilic properties of the very poorly soluble drug compounds with BBB uptake. From the aqueous surface pressure profiles and the determination of physicochemical parameters, it was found that blood brain barrier is more likely when a drug provides a small cross-sectional area, As, at the interface. The cross-sectional area is the only parameter which is independent from the maximal concentration in aqueous media and it is particularly suitable for lower solubility compounds. In summary, it was shown that amphilicity is related to biological membrane interaction in the human body and that surface activity profiling with appropriate sample preparation can be used as a reliable screening tool for the prediction of oral drug absorption of poorly soluble drugs. Furthermore an in vitro screening method of blood-brain-barrier penetration was established.
Summary:
1) Three types of forest, evergreen seasonal forest, heath forest and Melaleuca swamp forest, were distinguished and studied in the vicinity of Cheko in southwestern Cambodia, where moist tropical climate with a pronounced dry season in three winter months prevails.
2) These three forest types respectively occupied deep latosol derived from sandstone, very sandy soil around the swamp forest, and deep deposit of silica sand with underground hardpan in shallow valleys.
3) Total plant biomass was estimated by the allometric method based on some 140 sample trees (DBH24.5 cm) which were felled in four sample plots (two 50 mX50 m plots in the evergreen seasonal forest, and each one 20 m x 50 m plot in the other two types). Biomass of ground vegetation was estimated separately by similar technique and clipping.
4) The biomass of evergreen seasonal forest was estimated as follows. Stem 215 ton/ha, branch 99 ton/ha, root 61 ton/ha, leaf 7.3 ton/ha, leaf area index 7.4 ha/ha, density of trees over 4.5 cm DBH 1,280/ha, relative basal area of Whole stand 3.19 o/oo.
5) The biomass of heath forest was as follows. Stem 111 ton/ha, branch 35 ton/ha·, root 19 ton/ha, leaf 7.7 ton/ha, leaf area index 7.1 ha/ha, tree density 2,570/ha, relative basal area 2.3 o/oo.
6) The biomass of M elaleuca swamp forest was as follows. Stem 7.4 ton/ha, branch 3.9 ton/ha, root 2.6 ton/ha, leaf 0.79 ton/ha, leaf area index 0.37, undergrowth of sedge 2.57 ton/ha, tree density 200/ha, relative basal area of trees 0.35 o/oo.
7) It was found that the biomass of small trees (4.5 cm>DBH>1 cm) and ground vegetation (4.5 cm <= DBH) was so unevenly distributed over the forest floor that a few hundred square meters of sample area would be needed for estimating them at a moderate level of statistical reliability.
8) The estimated biomass of the evergreen seasonal forest was compared with the data hitherto obtained in moist tropical forests of Cote d'Ivoire and Thailand. The forest of Cheko was found to have the biomass equivalent to other rain forests, but to be characterized by a specific DBH-tree height curve, a rather small leaf area index and a high value of leaf area/leaf weight ratio.
G-protein coupled receptors (GPCRs) comprise the largest superfamily of cell surface receptors and possess a signature motif of seven transmembrane helices. The endothelin B (ETB) receptor is a member of rhodopsin like GPCR family. It plays an important role in vasodilation and is found in the membranes of the endothelial cells enveloping blood vessels. Knowledge of the three-dimensional structure of G-protein coupled receptors in general would significantly add to our understanding of their molecular mechanisms and would be useful in the search for new specific drugs. However, three-dimensional structural analysis will require milligram quantities of pure and homogeneous protein. This dissertation is a study of the production, biochemical characterization and preliminary structural studies of the human ETB G-protein coupled receptor. The present work aimed at elucidating the structure and mechanistic details of function of the receptor by using a combination of X-ray crystallographic and NMR methods for collecting structural data. To obtain homogenous and monodisperse receptor protein preparation for structural and functional studies, we implemented the baculovirus expression system for the production of ETB receptor for the present work. The two step affinity purification ensured capture of full-length receptor. Silver stained SDS-PAGE of the purified receptor-ligand complex indicated greater than 90% protein purity. Based on previous reports, we used the high affinity ligand (endothelin -1) binding to the receptor for co-crystallization of receptor-ligand complex by locking the receptor in the activated conformation. As a prerequisite for 3D crystallization trials, the stability of the detergent solubilized receptor-ligand complex was assessed with respect to pH, temperature and time. Receptor-ligand complex did not show any degradation and aggregation over 6 days at 4°C and 18°C. Interestingly, change of pH suggested that receptor-ligand complex is unstable at lower pH due to possible charge induced conformational changes. In our work, we introduced the idea of using fluorophore labeled ligand for simple visual recognition of the receptor-ligand complex during purification and crystallization. On the other hand, we alternatively used biotinylated endothelin-1 to produce an adequate amount of ligand bound receptor complex, thus ensuring homogeneity of the purified complex for use in structural studies. Thus far, preliminary crystals have been obtained for both the unlabelled ET-1 and fluorophore labeled ET-1 complexed with ETB receptor. Moreover, we performed the systematic investigation of the protein/peptide binding partner for the receptor-ligand complex with the chief aims of stabilizing structure and increasing the possibilities of 3D-crystal contacts. Thus subsequent to formation of receptor-ligand complex, the additional in vitro formation of a ternary arrestin-receptor-ligand complex was also attempted for use in structural studies. We successfully demonstrated that arrestin mutant (R169E) forms a tight complex with ETB receptor regardless of its phosphorylation state. A second approach to get insight into the ETB receptor ligand binding site relied on the use of spin isotope labeled ET-1 ligand peptide by employing solid state MAS NMR method. Preliminary data provided compelling evidence that the C-terminal region of the peptide is immobilized in an ordered environment and presumably bound to the receptor. This indicates that the approach is feasible, although there are difficulties in sample preparation for further spectral measurements and data collection which are currently being discussed in ongoing investigations. At this point of our research work, we initiated a collaborative effort to obtain high yields of pure, active receptor without post translational modifications, from an E. coli cell lysate based in vitro expression system. We successfully optimized the production of homogenous and monodisperse endothelin B receptor in mg amounts. Thus this could potentially provide an alternative source of high quality receptor production in large quantities for immediate crystallization trials. Thus we hope that the results from these investigations can be applied in a more general sense to the production and crystallization of other G protein-coupled receptors.