The glycine receptor (GlyR) is a ligand-gated ion channel member of the cys-loop receptor superfamily, responsible for inhibitory neurotransmission in the brain and spinal cord. Both glycine and the partial agonist taurine act as endogenous ligands of the GlyR. Taurine-activated GlyR may have a role in the rewarding effects of drugs of abuse, such as ethanol. As a partial agonist, taurine has a decreased efficacy relative to glycine, resulting in a decreased maximum response. We investigated the effects of ethanol, anesthetics, inhalants, and zinc to determine if these allosteric modulators could increase the efficacy of the taurine-activated GlyR. Whole cell recordings of wild type GlyR revealed that each of the allosteric modulators potentiated currents generated by saturating concentrations of taurine but not glycine, implying an increase in efficacy. Zinc is found at GlyR-potentiating concentrations throughout the nervous system, so we examined the combinatorial effects of these allosteric modulators with zinc to mimic in vivo conditions. Whole cell recordings revealed that zinc potentiation of saturating taurine-generated currents decreased further potentiation by another allosteric modulator, indicating no synergistic effects on efficacy. We next investigated the actions of ethanol and isoflurane on the taurine-activated GlyR at the single channel level, finding that both allosteric modulators stabilized the channel open state, increasing the efficacy of the taurine-activated GlyR. We previously identified a mutation in the ligand-binding domain of the GlyR (D97R) that produces spontaneously activating channels, on which taurine has increased efficacy. We identified a residue, R131, as a possible binding partner of D97 in forming an electrostatic interaction that holds the channel in the closed state. We found that disruption of this interaction results in greatly increased taurine efficacy, indicating that efficacy for partial agonists may be determined by agonist ability to break this bond early in the activation process following binding. Thus we find differential mechanisms of allosteric modulation and efficacy determinations for the GlyR when activated by taurine vs. glycine. / text

The glycine receptor (GlyR) is a ligand-gated ion channel member of the cys-loop receptor superfamily, responsible for inhibitory neurotransmission in the brain and spinal cord. Both glycine and the partial agonist taurine act as endogenous ligands of the GlyR. Taurine-activated GlyR may have a role in the rewarding effects of drugs of abuse, such as ethanol. As a partial agonist, taurine has a decreased efficacy relative to glycine, resulting in a decreased maximum response. We investigated the effects of ethanol, anesthetics, inhalants, and zinc to determine if these allosteric modulators could increase the efficacy of the taurine-activated GlyR. Whole cell recordings of wild type GlyR revealed that each of the allosteric modulators potentiated currents generated by saturating concentrations of taurine but not glycine, implying an increase in efficacy. Zinc is found at GlyR-potentiating concentrations throughout the nervous system, so we examined the combinatorial effects of these allosteric modulators with zinc to mimic in vivo conditions. Whole cell recordings revealed that zinc potentiation of saturating taurine-generated currents decreased further potentiation by another allosteric modulator, indicating no synergistic effects on efficacy. We next investigated the actions of ethanol and isoflurane on the taurine-activated GlyR at the single channel level, finding that both allosteric modulators stabilized the channel open state, increasing the efficacy of the taurine-activated GlyR. We previously identified a mutation in the ligand-binding domain of the GlyR (D97R) that produces spontaneously activating channels, on which taurine has increased efficacy. We identified a residue, R131, as a possible binding partner of D97 in forming an electrostatic interaction that holds the channel in the closed state. We found that disruption of this interaction results in greatly increased taurine efficacy, indicating that efficacy for partial agonists may be determined by agonist ability to break this bond early in the activation process following binding. Thus we find differential mechanisms of allosteric modulation and efficacy determinations for the GlyR when activated by taurine vs. glycine. / text

We have shown previously [Hosohata, K., Logan, J.K., Varga, E., Burkey, T.H., Vanderah, T.W., Porreca, F., Hruby, V.J., Roeske, W.R., Yamamura, H.I., 2000. The role of the G protein γ2 subunit in opioid antinociception in mice. Eur. J. Pharmacol. 392, R9-R11] that intracerebroventricular (i.c.v.) treatment of mice with a phosphorothioate oligodeoxynucleotide antisense to the γ2 subunit (Gγ2) of the heterotrimeric G proteins (antisense ODN) significantly attenuates antinociception by a δ-opioid receptor agonist. In the present study, we examined the involvement of Gγ2 in antinociception mediated by other (μ- or κ-opioid, cannabinoid, α2-adrenoreceptor) analgesic agents in a warm (55°C) water tail-flick test in mice. Interestingly, i.c.v. treatment with the antisense ODN attenuated antinociception by each analgesic agent. Missense phosphorothioate oligodeoxynucleotide treatment, on the other hand, had no effect on antinociception mediated by these agonists. The antinociceptive response recovered in 6 days after the last antisense ODN injection, indicating a lack of nonspecific tissue damage in the animals. These results suggest a pervasive role for the G protein γ2 subunits in supraspinal antinociception.

No / A sensitive and rapid high performance liquid chromatography method has been developed and used for the simultaneous determination of formoterol and budesonide in Symbicort Turbuhaler when assessing the aerodynamic characteristics of the emitted dose using Pharmacopoeial methods. This capability results in both time and cost saving. The mobile phase composition was acetonitrile-5 mM sodium dihydrogen orthophosphate, pH 3 (60: 40% v/v), and was passed at 1.5 ml min-1 through a C18 column with a UV detection (wavelength 214 nm). The method was shown to give good analytical performance in terms of linearity, precision (using phenylpropanolamine as an internal standard), sensitivity and solution stability. The intra-day precision for both formoterol and budesonide were 0.75% and 1.11%, respectively (n = 10). The limit of quantitation for formoterol was 10 ¿g L-1 and for budesonide was 120 ¿g L-1, and the limit of detection were 3 and 30 ¿g L-1, for both formoterol and budesonide, respectively. The method has been applied to determine the content of the emitted dose and the fine particle dose of Symbicort Turbuhaler.

Rhodopsin, der Rezeptor der visuellen Kaskade, gehört zu größten Klasse A der G-Protein-koppelnden Rezeptoren (GPCRs) und gilt als Modell-Rezeptor in der GPCR-Forschung. Über 3 % des humanen Genoms kodieren für GPCRs, doch trotz der physiologischen Bedeutung dieser Proteinfamilie sind die fundamentalen Mechanismen, mit denen diese Rezeptoren extrazelluläre Signale in das Zellinnere weiterleiten noch nicht verstanden. In der vorliegenden Dissertation werden Aspekte des Aktivierungsmechanismus von Rhodopsin sowie der Kopplung und Aktivierung des G-Proteins Transduzin untersucht. Die Arbeit ist in drei Schwerpunkte unterteilt: I. Es wurde ein in GPCR’s hochkonserviertes NPxxYx(5,6)F Motiv (Aminosäuresequenz Asn-Pro-x-x-Tyr-x(5,6)-Phe) in der siebten und achten Helix charakterisiert. In diesem konservierten Motiv sind mehrere für die Ausbildung der aktiven Rezeptorkonformation wichtige Funktionen vereint: Verknüpfung zu einem Wasserstoffbrückennetzwerk, Helixflexibilität sowie die exakte Positionierung der achten Helix. Letzteres hat nicht nur bei der Rezeptoraktivierung sondern auch bei der nachfolgenden Interaktion mit dem G-Protein eine Bedeutung. II. Anhand von chimären Rezeptoren, bei denen Teile der achten Helix durch homologe Sequenzen des beta2-adrenergen Rezeptors ausgetauscht wurden, wurde die Rolle der achten Helix bei der Rezeptor-Aktivierung und Bindung des G-Proteins untersucht. Auch bei dieser Studie wurde gezeigt, dass die exakte Positionierung der achten Helix essentiell für die Interaktion mit dem G-Protein ist. Zudem wurde ein bezüglich der G-Protein-Aktivierung funktionsfähiger chimärer Rezeptor gefunden, was auf einen übergeordneten Mechanismus bei der Aktivierung von G-Proteinen durch GPCRs hindeutet. III. Die Funktion des ß-Ionon-Rings des Retinals beim Aktivierungsmechanismus von Rhodopsin wurde an einem Retinal studiert, bei welchem Teile des Retinal-Rings fehlten (azyklisches Retinal). Auch diesem azyklischen Retinal können Eigenschaften eines partiellen Agonisten zugeschrieben werden. Beim Vergleich zu Pigmenten mit dem nativen 11-cis-Retinal wurden starke Analogien bei der initialen Energieaufnahme durch die Retinal-Isomerisierung sowie bei der Weiterleitung der Lichtenergie ins Protein gefunden. Allerdings wird die Energie schlechter auf das Protein übertragen, wodurch wesentlich weniger der aktiven G-Protein bindenden Rezeptorkonformation gebildet wird. Als wichtigste Funktion des Retinal-Rings wurde die Aufrechterhaltung der aktiven Meta-II-Konformation identifiziert. / Rhodopsin, the receptor of the visual cascade, belongs to the largest group A of G-protein coupled receptors (GPCRs) and can be seen as a model receptor in GPCR research. More than 3 % of the human genome code for GPCRs. But despite their physiological relevance, the detailed mechanism of signal transduction from extra cellular signal to different cellular pathways remains to be fully understood. Different aspects of receptor activation and the coupling and activation of the G-protein transducin are investigated in this dissertation. The thesis focuses on the following three subjects: I. A NPxxYx(5,6)F motif (amino acid sequence Asn-Pro-x-x-Tyr-x(5,6)-Phe) has been characterized for rhodopsin. It is localized in helix VII and VIII and is highly conserved throughout the GPCR family. Various roles for rhodopsin activation are combined in this motif: linkage to a hydrogen-bond network, helix flexibility and the exact positioning of helix VIII. The latter is not only relevant for the activation of the receptor but also for interaction with its G-protein. II. The role of helix VIII for receptor activation and G-protein coupling was studied on chimeric receptors, in which parts of helix VIII were exchanged against homologous sequences of the beta2 adrenergic receptor. This study confirmed the importance of helix VIII’s position for G-protein coupling. Furthermore, a chimeric receptor was found, which was fully functional concerning G-protein activation. This indicates that GPCRs might use a single, generic mechanism for G-protein activation. III. The role of the ß-ionone-ring for the activation mechanism of rhodopsin was studied by means of an acyclic retinal, which lacks four carbon atoms of the ß-ionone-ring. This modified retinal could be classified as a partial agonist for rhodopsin. Energy input by retinal isomerization and formation of the G-protein binding Meta-II conformation were found to be very similar to rhodopsin when bound to its native 11-cis-retinal. However, the lack of the ring structure resulted in a lower amount of Meta-II and a fast decay of activity. It was concluded that the main role of the ring structure is to maintain the active state of rhodopsin.

Die GnRH-Agonisten-Applikation zur Downregulation vor IvF ist "gold standard", überwiegend im sogenannten langen Protokoll. Die Behandlung soll den vorzeitigen LH-Anstieg mit vorzeitiger Ovulation verhindern. Die unerwünschten Wirkungen sind dosisabhängig und rechtfertigen die Suche nach der optimal niedrigen Dosis des GnRH-Agonisten. Mit dieser Fragestellung wurde eine prospektive randomisierte Dosisfindungsstudie durchgeführt. 200 sterile Frauen zwischen 18 und 38 Jahren erhielten vor der IvF-Behandlung im langen Protokoll die Standarddosis von 4,12 mg Triptorelinazetat-Depot (1 Amp. i.m. = Gruppe B: n = 100) versus 2,1 mg Triptorelinazetat Depot (1/2 Amp. i.m = Gruppe A. n = 100) zur Downregulation. Folgende Parameter wurden bestimmt: E2, LH, Progesteron. Die Behandlungsergebnisse wurden korreliert mittels der Anzahl der gewonnenen Oocyten, der fertilisierten Oocyten, der transferierten Embryonen und der Schwangerschaftsraten pro Embryotransfer. Abgebrochene IvF-Zyklen wurden einzeln analysiert. Bezüglich der Hormonwerte waren beide Gruppen ohne signifikanten Unterschied. In der Gruppe der Patientinnen mit der halbierten Dosis (A) kam es nur in einem Fall zu einer vorzeitigen Luteinisierung, in der Standartdosisgruppe (B) in keinem Fall. Wegen low response wurde in Gruppe A in 5 Fällen die Therapie abgebrochen, versus 3 Fälle in Gruppe B (ns). Ebenfalls vergleichbar war das IvF-outcome, nur die ET-Rate pro begonnener Stimulation zeigte einen signifikanten Unterschied: 88 % (A) versus 96 % (B), p / The GnRH agonist application for the downregulation prior to IVF is 'gold standard', mainly in the so-called long protocol. This should avoid premature ovulations. The dose-dependent, undesired effects justify the search for the optimal low dose of the GnRH agonist. A prospective randomised dose-finding study was carried out in this respect. Among 200 sterile women (18 and 38 years) for the planned IVF and/or IVF / ICSI treatment in the long protocol, n = 100 in group A received 2.1 mg Triptorelinacetate depot (1/2 amp., i.m.) and n = 100 in group B the standard dose of 4.12 mg (1 amp., i.m.) for the downregulation. The hormone values E2, LH, progesterone were determined. The treatment results were compared by means of the number and quality of the oocoytes, the embryo transfers and the pregnancy rates. Cancelled IVF cycles were analysed. With respect to the hormone values, neither of the two groups showed significant differences. A premature luteinization occurred in group A (reduced dose) in only one case; in the standard dose of group B, none occurred. Due to the low response, the therapy was cancelled in 5 cases in group A, in comparison to 3 cases in group B (ns). The IVF outcome showed a comparable result. The only significant difference was the ET rate per started stimulation (p

Die Akromegalie - Folge eines Wachstumshormon (STH) sezernierenden Hypophysentumors - ist eine seltene Erkrankung. Bei früher Diagnose ist die Akromegalie gut behandelbar. Unbehandelt - oder zu spät behandelt - führt sie zu hoher Co-Morbidität und verkürzt das Leben. Endokrinologische Therapieziele wurden kürzlich definiert: Heilung bei STH / Acromegaly, caused by a growth hormone (GH)-secreting pituitary adenoma, is a rare disease. If diagnosed early therapeutic results are good. However, untreated or treated belatedly, acromegaly is associated with a high co-morbidity and reduced life-expectancy. The therapeutic goals have recently been defined as follows: complete remission or cure as GH

No description available.

No / A novel high-performance liquid chromatography (HPLC) assay for the estimation of formoterol in urine samples was developed and validated. A solid phase extraction (SPE) using Oasis HLB was optimised to isolate formoterol from a urine matrix followed by HPLC with UV detection. This extraction procedure concentrated the final analyte forty times so that UV detection can be used to determine even a low concentration of formoterol in urine samples. The urinary assay was performed in accordance with FDA and ICH regulations for the validation of bioanalytical samples. The samples were injected onto a C18 Spherisorb® (250 mm x 4.6 mm x 5 ¿m) analytical column maintained at 30 °C. The mobile phase consisted of 5 mM of potassium dihydrogen orthophosphate buffer (adjusted to pH 3 with ortho phosphoric acid):acetonitrile (ACN) (70:30, v/v), and the formoterol peak was detected at wavelength 214 nm. The extraction recovery of formoterol from the urine sample was >95%. The calibration curve was linear (r2=0.99) over formoterol concentrations ranging from 1.5 to 25 ng/mL (n=6). The method had an accuracy of >92% and intra and inter-day precision CV% of <3.9% and <2.2%, respectively, at three different concentrations low, medium and high (10, 15, 20 ng/mL). The limit of quantification (LOQ) for formoterol was found to be 1.50 ng/mL. The accuracy and precision at the LOQ level were 95% and %CV <3.7% (n = 10), respectively. The method reported is simple, reliable, precise, and accurate and has the capacity to be used for determination of formoterol in urine samples.

Master of Science / Department of Animal Sciences and Industry / Bradley J. Johnson / Zilpaterol hydrochloride (ZH) is a growth promotant that is approved for use in finishing cattle to improve growth performance and increase lean tissue accumulation. Little is known about the combined effects of ZH with anabolic steroid hormone implants. There is also little published data on the effect these growth promotants have on genes that play a role in skeletal muscle synthesis and degradation. Therefore, two separate studies were conducted to address these issues. The first study evaluated the effects of ZH and the steroid implant Revalor-S (RS) on animal performance and skeletal muscle gene expression in feedlot steers. Four treatments were used to analyze the effects of RS implanted 58 days before ZH, which was fed for 30 days with a 3 day withdrawal. It was determined that ZH and RS additively contribute to improved live and carcass performance; however these compounds had different effects on the abundance of the receptors for ZH as well as the abundance of myosin heavy chain (MHC) mRNA in skeletal muscle of feedlot steers. It was also determined that ZH can cause a transition in the abundance of MHC mRNA isoforms in skeletal muscle that are available for the translation of larger, faster, more glycolytic fiber types of MHC. The second study evaluated the effects of two types of anabolic steroid hormones on myosin heavy chain gene expression. Four treatments were used to measure the effects of trenbolone acetate (TBA) and estradiol (E[subscript]2) on performance and the amount of MHC mRNA in skeletal muscle of finishing steers. It was determined that anabolic steroid implants improve live animal performance, however there was no alteration in the abundance of MHC mRNA in skeletal muscle of feedlot steer for 28 days after implantation; however there was an increase in intermediate fiber type IIA of MHC mRNA in skeletal muscle with increasing days on feed. From these studies we concluded that ZH and anabolic steroids do have an effect on growth performance; however they may differ in the distinct mechanism of action utilized to enhance lean tissue deposition in feedlot steers.