Universiẗat, Diss., 2003--Kiel.

Seit mehr als zwei Jahrzehnten ist bekannt, dass nicht nur der Tumor Nekrose Faktor-α (=TNF-α) sondern auch Lymphotoxin-α (=LTα) in Form von Trimeren an TNFR1 und TNFR2 binden kann. Durch diese Fähigkeit an beide Rezeptoren zu binden, haben diese zwei Liganden eine essentielle Rolle in der Entwicklung und dem Verlauf von Autoimmunerkrankungen. Bereits mit Beginn der 1990er Jahren wurde gezeigt, dass LTα nicht nur in Form von Homotrimeren vorliegt, sondern auch mit dem verwandten TNF-Superfamilie Liganden Lymphotoxin β (=LTβ) Heterotrimere bilden kann. Hierbei lagern sich LTα und LTβ in Form von LTα2β und LTαβ2 zusammen. Die initialen Experimente mit diesen Heterotrimeren zeigten bereits Unterschiede von LTα2β und LTαβ2. Während LTα2β wie LTα an den TNFR1 bindet, kann LTαβ2 weder an TNFR1 noch TNFR2 binden und interagiert mit einem eigenen Rezeptor namens Lymphotoxin β Rezeptor (=LTβR). Da bereits zwei Liganden (TNF und LTα) für TNFR1 und TNFR2 bekannt waren, wurde LTα2β bis heute nicht weiter charakterisiert. LTαβ2 hingegen war lange Zeit der einzige bekannte Ligand für den LTβR, weshalb die LTαβ2-LTβR-Interaktion ausführlich untersucht wurde. Diese Arbeit fokusiert sich auf die Charakterisierung von LTα2β. Hierfür wurde die einzige bekannte Eigenschaft aus den 90er Jahren von LTα2β nämlich die Bindung an TNFR1 aufgegriffen und um die Rezeptoren TNFR2 und LTβR erweitert. Diese Arbeit zeigt, dass LTα2β nicht nur an den TNFR1, sondern auch an TNFR2 und schwach an LTβR bindet. Trotz der asymmetrischen Bindestellen kann membrangebundenes LTα2β TNFR1 und TNFR2 nicht nur binden, sondern ist auch in der Lage diese zu aktivieren. Diese Arbeit gibt erste Einblicke in die Komplexizität dieses Heterotrimers indem gezeigt wird, dass LTα2β sowohl in seiner löslichen als auch in seiner membrangebundenen Form den TNFR1 aktivieren kann, während der TNFR2 nur durch das membranständige LTα2β aktiviert wird. Aufgrund der aktivierenden Eigenschaften von membranständigem LTα2β und LTαβ2 auf die murine (=mu) Panc02-Zelllinie wird ein ersten Ausblick auf mögliche weitergehende Experimente in mausbasierten Modellen gegeben. Die erzielten Ergebnisse zeigen, dass mit membranständigem LTα2β ein neuer TNFR2 Agonist gefunden wurde. / Since more than two decades it is known, that not only the Tumor Necrosis Factor-α (=TNF-α) but also lymphotoxin-a (=LTα) bind to TNFR1 and TNFR2. Because of their ability to interact with both of these two receptors, the two ligands play a crucial role in the development and persistence of autoimmune diseases. Already at the beginning of the 1990th, it has been shown that LTα forms homotrimers but is also able to form heterotrimers with the related TNF-Superfamily ligand lymphotoxin-β (=LTβ). Thereby, LTα and LTβ associate to form LTα2β and LTαβ2. Initial experiments already have shown differences between LTα2β and LTαβ2. While LTα2β can bind to TNFR1 like LTα, LTαβ2 can bind neither to TNFR1 nor to TNFR2 but interacts with its own receptor called LTβR. Due to the fact that already two ligands (LTα and TNF) for TNFR1 and TNFR2 were known, LTα2β was not further characterized. Since LTαβ2 was the only known ligand for the LTβR, this LTαβ2-LTβR interaction was further and intensively investigated. This work is focused on LTα2β and its characterization. For this purpose the initial and only finding reported for in the 1990th namely the binding to TNFR1 were taken up and expanded for the interaktion with TNFR2 and LTβR. The results of this work show that LTα2β can not only bind to TNFR1, but also to TNFR2 and weaker to LTβR. Despite its asymmetric binding sites, membrane bound LTα2β was shown not only to bind, but also to be able to activate TNFR1 and TNFR2. This work shows that LTα2β can activate the TNFR1 receptor in its soluble and membrane bound form while TNFR2 can only be activated by membrane bound LTα2β. Due to the activating properties of membrane bound LTα2β and LTαβ2 on murine (=mu) Panc02 cells a first outlook on further experiments in mouse based models will be given. These findings show, that membrane bound LTα2β is a new TNFR2 agonist.

No description available.

Effects of norepinephrine (NE) on macrophage cytokine release are complex because the cells have both α2 and β2 adrenergic receptors, which mediate opposing actions. Furthermore, β2-adrenergic agonists are reported to have both stimulatory and inhibitory effects on interleukin-6 (IL-6). This study was designed to clarify the autocrine role of macrophage-derived NE on IL-6 production in activated peritoneal macrophages. Effects of NE on IL-6 production in the RAW264.7 macrophage cell-line also were investigated. Treatment of activated peritoneal macrophages with endotoxin, the α2-adrenergic antagonists yohimbine or RS79948 revealed that the α2-adrenergic receptor mediates a stimulatory autocrine action of catecholamines on IL-6 production. When peritoneal macrophages were treated with the β2 antagonist ICI-118,551 (ICI), there was both inhibition and stimulation of IL-6. Treatment of RAW264.7 macrophages with high and low concentrations of NE and various concentrations of ICI provided evidence that the concentration of NE determines whether the β2-adrenergic receptor mediates stimulation or inhibition of IL-6 production.

Die Subtypselektivität von Liganden für einzelne Rezeptoren, deren Aktivierung und die anschließende Signalweiterleitung sind bis heute weitestgehend ungeklärt. Die hier synthetisierten Liganden-Gruppen sollen helfen, die verschiedenen Prozesse am muskarinischen Rezeptor und seinen Subtypen zu verstehen. Die Einzelprojekte werden im Folgenden vorgestellt. 1) Um mittels FRET-Mikroskopie den Einfluss von allosteren Modulatoren, die sich von W84 bzw. Naphmethonium ableiten, in Bezug auf die Konformationsänderung aktivierter Rezeptoren untersuchen zu können, wurden die bekannten allosteren Bausteine sowie eine Reihe neuer Derivate synthetisiert. Alle untersuchten Substanzen zeigten einen hemmenden Effekt auf die mit dem Agonisten Iper-oxo vorstimulierten Rezeptoren. Das heißt, die Verbindungen ließen sich als negative allostere Modulatoren charakterisieren. 2) Da Iperoxo aufgrund seiner großen agonistischen Aktivität ein interessantes Werkzeug für die Grundlagenforschung darstellt, war es von großer Bedeutung, eine schnelle und reproduzierbare Synthese zu gewährleisten. Ausgehend von Propargylalkohol wurde in einer Mannich-Reaktion 4-Dimethylamino-but-2-en-1-ol gebildet, was mit dem zuvor hergestellten 3-Nitro-Δ2-isoxazolin zur Iperoxo-Base umgesetzt wurde. Neben einer deutlichen Ausbeutesteigerung ist nun die Reproduzierbarkeit im Gegensatz zu der von Dallanoce et al. publizierten Synthese gewährleistet. 3) Um den Einfluss der Kettenlänge der Hybride Iper-6-Phth und Iper-6-Naph auf die agonistische Aktivität und Subtypselektivität der Substanzen untersuchen zu können, wurde versucht, Hybride verschiedener Kettenlängen herzustellen. Dabei konnte Iper-4-Phth erhalten werden. 4) Weiterhin sollte der Einfluss der Alkylkette am Stickstoff-Atom auf die Wirksamkeit in Bezug auf Affinität und Zellantwort des Iperoxo-Moleküls ohne allosteren Modulator analysiert werden. Hierzu wurden die N-alkylierten Iperoxo-Derivate mit den Kettenlängen C2 bis C10 synthetisiert.. Mithilfe von Radioligand-Bindungsstudien und der dynamischen Massenumverteilung sollte die konformative Änderung des Rezeptors durch Aktivierung und die Signalweiterleitung untersucht werden. Durch Verlängerung der N-Alkylkette zeigte sich ein Wirksamkeitsverlust, d. h. die Dosis-Wirkungs-Kurven der prozentualen Zellantwort wurden im Vergleich zu denen des Iperoxos nach rechts verschoben. In Untersuchungen an der Rezeptor-Mutante CHO-hM2-Y1043.33A zeigte sich zudem, dass für den maximalen Effekt eine deutlich höhere Konzentration der Iperoxo-Derivate benötigt wird, wobei dieser Wirksamkeitsverlust im Vergleich zum Rezeptor-Wildtyp für Iperoxo selbst am stärksten ausgeprägt ist. Allerdings weisen Iperoxo und seine N-alkylierten Derivate an dieser Mutante eine höhere intrinsische Aktivität auf als die Kontrollverbindungen Oxotremorin M, C1-IP-C1 und Acetylcholin. Weiterhin konnte gezeigt werden, dass Iperoxo ebenso wie Oxotremorin, Acetylcholin, aber auch die kurzkettigen Iperoxo-Derivate neben dem Gi-Signalweg auch den Gs-Weg aktivieren können, wohingegen die langkettigen Derivate eine Gi-Signalwegs-Selektivität aufweisen. 5) In Analogie zu den N-alkylierten-Iperoxo-Derivaten sollten Untersuchungen mit den Antagonisten N-Alkyl-Atropin und -Scopolamin durchgeführt werden. In beiden Fällen zeigte das N-Methyl-Derivat eine höhere Affinität zum Rezeptor als der jeweilige Antagonist selbst, was auf die durch Alkylierung generierte positive Ladung zurückzuführen ist. Durch Verlängerung der Alkylketten ist jeweils eine Abnahme der Affinität zu beobachten. Die Affinität findet ebenfalls in beiden Fällen mit dem Butyl-Derivat ihr Minimum. Der anfängliche Affinitätsverlust lässt sich durch die zunehmende sterische Hinderung des Moleküls erklären, der spätere Anstieg bei einer Alkylkette länger als C4 deutet auf eine Wechselwirkung dieser langen Alkylkette mit einer weiteren (allosteren) Bindungsstelle hin. 6) Ausgehend von Iperoxo sollten dualstere Liganden entwickelt werden, die durch geeignete allostere Modulatoren selektiv nur einen Rezeptor-Subtyp adressieren und diesen durch Iperoxo aktivieren sollten. Als allostere Bausteine sollten die M4-selektiven Thienopyridine und die M1-selektiven Chinolone verwendet werden. 7) Zur Fluoreszenzmarkierung sollte Iperoxo-Base zudem mit dem Farbstoff Py-1 umgesetzt werden. / The subtype-selectivity of ligands for receptors and the corresponding subtypes, their activation and the subsequent signalling is still largely unknown. The synthesized groups of ligands, described here were designed to understand the various processes at the muscarinic receptor subtypes. The single projects will be discussed below. 1) In order to examine the influence of allosteric modulators derived from W84 and Naphmethonium on the conformation of an activated receptor by means of FRET-microscopy, the allosteric building blocks were synthesized. All substances showed an inhibitory effect on the receptors pre-treated with the agonist iperoxo. That is to say that they behave as negative allosteric modulators. 2) Due to its high potency iperoxo is an important tool for basic research. In the past the availability of this compound was limited because of the elaborate chromatography and low reproducibility of the existing synthesis developed by Dallanoce et al.109 Thus a new synthesis pathway was established. By means of a Mannich reaction and using 2-propyn-1-ol alcohol as a starting material the amino-butinol was obtained. Product was converted to iperoxo-base by the reaction with 3-nitro-Δ2-isoxazolin. Besides reducing the reaction time and increasing the overall yield - compared to the known synthesis of Dallanoce et al. - the reproducibility was now ensured. 3) To investigate the influence of the chain-length in the hybrid compounds iper-6-phth and iper-6-naph on the agonistic activity and the subtype-selectivity, efforts were made to develop derivatives with different N-alkyl-chains. The synthesis of iper-4-phth was successful. 4) Furthermore the influence of the N-alkyl-chain-length on the potency of iperoxo having no allosteric building block was to be examined. Therefore the iperoxo-base was N-alkylated with different bromoalkanes (ethyl-decyl). It was aimed to study the change of the conformation on receptor activation and signalling by means of radioligand binding studies und dynamic mass redistribution. The elongation of the N-alkyl-chain length resulted in a loss of potency compared to iperoxo. All iperoxo derivatives lose potency at the CHO-hM2-Y1043.33A-receptor-mutant compared to the receptor wildtype, which was especially pronounced with iperoxo itself. However, for this receptor-mutant iperoxo and its N-alkylated derivatives had a higher intrinsic activity than the control compounds oxotremorine-M, C1-IP-C1 and acetylcholine. Beyond this, it could be demonstrated that iperoxo, as well as oxotremorine M, acetylcholine and the short-chain derivatives have the ability to activate not only the Gi-, but also the GS-signal-pathway, whereas the long-chain derivatives show selectivity for the Gi-pathway. 5) In analogy to these agonistic derivatives the antagonists atropine and scopolamine were N-alkylated. In both cases radioligand binding studies revealed that the methyl derivatives had a higher affinity to the receptor than both antagonists themselves. This might be caused by the generated positively charged nitrogen atom. The extension of the chain length leads to a loss of affinity, which has a minimum at the butyl-derivatives in both antagonists. The initial loss of affinity is ascribed to the steric hindrance and the subsequent gain of affinity for derivatives with a longer chain length indicates an interaction of this alkyl-chain with a second (allosteric) binding-site. For the special arrangement in the binding pocket it is essential to know the exact configuration of the ligand. The synthesized N-alkylated antagonists were examined via NMR-spectroscopy. By using the NOE-effect, the position of the methyl-groups and the alkyl-chains, respectively, were revealed. Contrary to expectations all derivatives bear their methyl-group in axial position. 6) On the basis of iperoxo, dualsteric ligands should be developed which selectively address only one muscarinic subtype, using a suitable allosteric modulator and simultaneously activate the receptor via iperoxo. As allosteric building blocks the M4-selective thienopyridines and the M1-selective quinolones should be used. 7) Besides radioligand binding studies the introduction of fluorescence markers provides an alternative to investigate the affinity of GPCR ligands. For this reason iperoxo should be coupled with the fluorescence marker py-1.

Scorpion toxins specific for Na+ and K+ channels, have been studied extensively but relatively little has been done on Ca2+ channel toxins. Toxins in the venom of only two South African scorpions P. transvaalicus and P. granulatus have been found to interact with Ca2+ channels. Kurtoxin isolated from the venom of P. transvaalicus inhibits the T and L-type neuronal Ca2+ channels, whereas KLI and KLII (Kurtoxin-like peptide I and II), isolated from P. granulatus, inhibits T-type Ca2+ channel activity in mouse male germ cells. In this study the effects of fractions isolated from the venom of P. granularus on Cca2+ channels in rat ventricular myocytes were investigated by means of the whole-cell patch clamp technique. Fractions of P. granulatus crude venom were isolated with Sephadex G50 columns (fraction I-IV). Fraction III (PgIII) showed a voltage dependent increase of the inward Ca2+ current and influenced the channel kinetics by shifting the voltage dependence of activation towards more hyperpolarizing membrane potentials and decreased the rate of inactivation and deactivation. The time of the current to reach peak was also delayed. PgIII was further separated by HPLC in an attempt to identify the subfraction/s responsible for the agonistic effect. Subfraction I had an agonistic effect similar to PgIII, whereas subfraction II and III, decreased the Ca2+ current. The observed agonistic effect has not been described in the literature. The identification of new peptide structures with unique functions are important in the field of toxin research. Peptides that target Ca2+ channels can be valuable tools to characterize Ca2+ channels. Ca2+ channels in the heart are implicated in a number of pathological disorders like angina, ischemia, some arrhythmias and hypertension. / Thesis (M.Sc. (Physiology))--North-West University, Potchefstroom Campus, 2005.

Scorpion toxins specific for Na+ and K+ channels, have been studied extensively but relatively little has been done on Ca2+ channel toxins. Toxins in the venom of only two South African scorpions P. transvaalicus and P. granulatus have been found to interact with Ca2+ channels. Kurtoxin isolated from the venom of P. transvaalicus inhibits the T and L-type neuronal Ca2+ channels, whereas KLI and KLII (Kurtoxin-like peptide I and II), isolated from P. granulatus, inhibits T-type Ca2+ channel activity in mouse male germ cells. In this study the effects of fractions isolated from the venom of P. granularus on Cca2+ channels in rat ventricular myocytes were investigated by means of the whole-cell patch clamp technique. Fractions of P. granulatus crude venom were isolated with Sephadex G50 columns (fraction I-IV). Fraction III (PgIII) showed a voltage dependent increase of the inward Ca2+ current and influenced the channel kinetics by shifting the voltage dependence of activation towards more hyperpolarizing membrane potentials and decreased the rate of inactivation and deactivation. The time of the current to reach peak was also delayed. PgIII was further separated by HPLC in an attempt to identify the subfraction/s responsible for the agonistic effect. Subfraction I had an agonistic effect similar to PgIII, whereas subfraction II and III, decreased the Ca2+ current. The observed agonistic effect has not been described in the literature. The identification of new peptide structures with unique functions are important in the field of toxin research. Peptides that target Ca2+ channels can be valuable tools to characterize Ca2+ channels. Ca2+ channels in the heart are implicated in a number of pathological disorders like angina, ischemia, some arrhythmias and hypertension. / Thesis (M.Sc. (Physiology))--North-West University, Potchefstroom Campus, 2005.

Universiẗat, Diss., 2006--Frankfurt (Main). / Erscheinungsjahr an der Haupttitelstelle: 2005.

Thesis (doctoral)--Universität, Marburg, 1999.

In this study, 20 young steers received no beta-adrenergic agonist (C), 100 animals all received zilpaterol hydrochloride, with 1 group only receiving zilpaterol (Z) while the other 4 groups received zilpaterol and vitamin D3 at the following levels and durations before slaughter: 7 million IU Vit D3 /animal/day for 3 days (3D7M); 7 million IU Vit D3/animal/day for 6 days (6D7M); 7 million IU Vit D3/animal/day for six days with 7 days no supplementation (6D7M7N) and 1 million IU Vit D3/animal/day for 9 days (9D1M). Left carcass sides were electrically stimulated (ES) and the right side not electrically stimulated (NES). Samples were aged for 3 or 14 days post mortem. Parameters included Warner Bratzler shear force (WBSF), myofibril filament length (MFL), sarcomere length and calpastatin and calpain enzyme activities. For drip loss and instrumental colour measurements, samples were analysed fresh (1 day post mortem) or vacuum-aged for 14 days post mortem. Both ES-treatment and prolonged aging reduced WBSF (P < 0.001). Treatments 6D7M, 6D7M7N and Z remained significantly tougher than C (P < 0.001), while 3D7M and 9D1M improved WBSF under NES conditions. ES was shown to be more effective at alleviating beta-adrenergic agonist induced toughness than high vitamin D3 supplementation. Aging increased drip loss, lightness, redness and yellowness while ES increased drip loss. In general, Z showed increased drip loss, lighter meat, and reduced redness. Vitamin D3 supplementation could not consistently overcome the adverse effects of zilpaterol hydrochloride in feedlot steers. / Thesis (PhD)--University of Pretoria, 2012. / Animal and Wildlife Sciences / unrestricted