Preservation of Alpha-3 Neuronal Nicotinic Acetylcholine Receptor Expression in Sympathetic Ganglia After Brain Death

The goal of this study was to evaluate if the immunohistochemical expression of alpha-3 neuronal nicotinic acetylcholine receptor subunit in sympathetic ganglia remains stable after brain death, determining the possible use of sympathetic thoracic ganglia from subjects after brain death as study group. The third left sympathetic ganglion was resected from patients divided in two groups: BD-organ donors after brain death and CON-patients submitted to sympathectomy for hyperhidrosis (control group). Immunohistochemical staining for alpha-3 neuronal nicotinic acetylcholine receptor subunit was performed; strong and weak expression areas were quantified in both groups. The BD group showed strong alpha-3 neuronal nicotinic acetylcholine receptor expression in 6.55% of the total area, whereas the CON group showed strong expression in 5.91% (p = 0.78). Weak expression was found in 6.47% of brain-dead subjects and in 7.23% of control subjects (p = 0.31). Brain death did not affect the results of the immunohistochemical analysis of sympathetic ganglia, and its use as study group is feasible.

Structural Insights into Human Peroxisome Proliferator Activated Receptor Delta (PPAR-Delta) Selective Ligand Binding

Peroxisome proliferator activated receptors (PPARs delta, alpha and gamma) are closely related transcription factors that exert distinct effects on fatty acid and glucose metabolism, cardiac disease, inflammatory response and other processes. Several groups developed PPAR subtype specific modulators to trigger desirable effects of particular PPARs without harmful side effects associated with activation of other subtypes. Presently, however, many compounds that bind to one of the PPARs cross-react with others and rational strategies to obtain highly selective PPAR modulators are far from clear. GW0742 is a synthetic ligand that binds PPAR delta more than 300-fold more tightly than PPAR alpha or PPAR gamma but the structural basis of PPAR delta: GW0742 interactions and reasons for strong selectivity are not clear. Here we report the crystal structure of the PPAR delta:GW0742 complex. Comparisons of the PPAR delta:GW0742 complex with published structures of PPARs in complex with alpha and gamma selective agonists and pan agonists suggests that two residues (Val312 and Ile328) in the buried hormone binding pocket play special roles in PPAR delta selective binding and experimental and computational analysis of effects of mutations in these residues confirms this and suggests that bulky substituents that line the PPAR alpha and gamma ligand binding pockets as structural barriers for GW0742 binding. This analysis suggests general strategies for selective PPAR delta ligand design.

Toll-like receptor 2/MyD88 signaling mediates zymosan-induced joint hypernociception in mice: Participation of TNF-alpha, IL-1 beta and CXCL1/KC

Arthritic pain is a serious health problem that affects a large number of patients. Toll-like receptors (TLRs) activation within the joints has been implicated in pathophysiology of arthritis. However, their role in the genesis of arthritic pain needs to be demonstrated. In the present study, it was addressed the participation of TLR2 and TLR4 and their adaptor molecule MyD88 in the genesis of joint hypernociception (a decrease in the nociceptive threshold) during zymosan-induced arthritis. Zymosan injected in the tibio-tarsal joint induced mechanical hypernociception in C57BL/6 wild type mice that was reduced in TLR2 and MyD88 null mice. On the other hand, zymosan-induced hypernociception was similar in C3H/HePas and C3H/Hej mice (TLR4 mutant mice). Zymosan-induced joint hypernociception was also reduced in TNFR1 null mice and in mice treated with IL-1 receptor antagonist or with an antagonist of CXCR1/2. Moreover, the joint production of TNF-alpha, IL-1 beta and CXCL1/KC by zymosan was dependent on TLR2/MyD88 signaling. Investigating the mechanisms by which TNF-alpha, IL-1 beta and CXCL1/KC mediate joint hypernociception, joint administration of these cytokines produced mechanical hypernociception, and they act in an interdependent manner. In last instance, their hypernociceptive effects were dependent on the production of hypernociceptive mediators, prostaglandins and sympathetic amines. These results indicate that in zymosan-induced experimental arthritis, TLR2/MyD88 is involved in the cascade of events of joint hypernociception through a mechanism dependent on cytokines and chemokines production. Thus, TLR2/MyD88 signaling might be a target for the development of novel drugs to control pain in arthritis. (C) 2011 Elsevier B.V. All rights reserved.

Vortex dynamics in the high-temperature superconductors YBa 2 Cu 3 O 7 and Bi 2 Sr 2 CaCu 2 O 8 + delta in low- and high-dissipative transport measurements

This thesis investigates phenomena of vortex dynamics in type II superconductors depending on the dimensionality of the flux-line system and the strength of the driving force. In the low dissipative regime of Bi_2Sr_2CaCu_2O_{8+delta} (BSCCO) the influence of oxygen stoichiometry on flux-line tension was examined. An entanglement crossover of the vortex system at low magnetic fields was identified and a comprehensive B-T phase diagram of solid and fluid phases derived.In YBa_2Cu_3O_7 (YBCO) extremely long (>100 mm) high-quality measurement bridges allowed to extend the electric-field window in transport measurements by up to three orders of magnitude. Complementing analyses of the data conclusively produced dynamic exponents of the glass transition z~9 considerably higher than theoretically predicted and previously reported. In high-dissipative measurements a voltage instability appearing in the current-voltage characteristics of type II superconductors was observed for the first time in BSCCO and shown to result from a Larkin-Ovchinnikov flux-flow vortex instability under the influence of quasi-particle heating. However, in an analogous investigation of YBCO the instability was found to appear only in the temperature and magnetic-field regime of the vortex-glass state. Rapid-pulse measurements fully confirmed this correlation of vortex glass and instability in YBCO and revealed a constant rise time (~µs).

Magnetic properties of selected 3d- and 4f-systems on alpha-Al 2 O 3 substrates

In this work the growth and the magnetic properties of the transition metals molybdenum, niobium, and iron and of the highly-magnetostrictive C15 Laves phases of the RFe2 compounds (R: Rare earth metals: here Tb, Dy, and Tb{0.3}Dy{0.7} deposited on alpha-Al2O3 (sapphire) substrates are analyzed. Next to (11-20) (a-plane) oriented sapphire substrates mainly (10-10) (m-plane) oriented substrates were used. These show a pronounced facetting after high temperature annealing in air. Atomic force microscopy (AFM) measurements reveal a dependence of the height, width, and angle of the facets with the annealing temperature. The observed deviations of the facet angles with respect to the theoretical values of the sapphire (10-1-2) and (10-11) surfaces are explained by cross section high resolution transmission electron microscopy (HR-TEM) measurements. These show the plain formation of the (10-11) surface while the second, energy reduced (10-1-2) facet has a curved shape given by atomic steps of (10-1-2) layers and is formed completely solely at the facet ridges and valleys. Thin films of Mo and Nb, respectively, deposited by means of molecular beam epitaxy (MBE) reveal a non-twinned, (211)-oriented epitaxial growth as well on non-faceted as on faceted sapphire m-plane, as was shown by X-Ray and TEM evaluations. In the case of faceted sapphire the two bcc crystals overgrow the facets homogeneously. Here, the bcc (111) surface is nearly parallel to the sapphire (10-11) facet and the Mo/Nb (100) surface is nearly parallel to the sapphire (10-1-2) surface. (211)-oriented Nb templates on sapphire m-plane can be used for the non-twinned, (211)-oriented growth of RFe2 films by means of MBE. Again, the quality of the RFe2 films grown on faceted sapphire is almost equal to films on the non-faceted substrate. For comparison thin RFe2 films of the established (110) and (111) orientation were prepared. Magnetic and magnetoelastic measurements performed in a self designed setup reveal a high quality of the samples. No difference between samples with undulated and flat morphology can be observed. In addition to the preparation of covering, undulating thin films on faceted sapphire m-plane nanoscopic structures of Nb and Fe were prepared by shallow incidence MBE. The formation of the nanostructures can be explained by a shadowing of the atomic beam due to the facets in addition to de-wetting effects of the metals on the heated sapphire surface. Accordingly, the nanostructures form at the facet ridges and overgrow them. The morphology of the structures can be varied by deposition conditions as was shown for Fe. The shape of the structures vary from pearl-necklet strung spherical nanodots with a diameter of a few 10 nm to oval nanodots of a few 100 nm length to continuous nanowires. Magnetization measurements reveal uniaxial magnetic anisotropy with the easy axis of magnetization parallel to the facet ridges. The shape of the hysteresis is depending on the morphology of the structures. The magnetization reversal processes of the spherical and oval nanodots were simulated by micromagnetic modelling and can be explained by the formation of magnetic vortices.

On the cellular stress response to Staphylococcus aureus alpha-toxin

Staphylococcus aureus alpha-hemolysin was the first bacterial toxin recognized to form pores in the plasma membrane of eukaryotic cells. It is secreted as a water-soluble monomer that upon contact with target membranes forms an amphiphatic heptameric beta-barrel which perforates the bilayer. As a consequence, red cells undergo colloidosmotic lyses, while some nucleated cells may succumb to necrosis or programmed cell death. However, most cells are capable of repairing a limited number of membrane lesions, and then respond with productive transcriptional activation of NF-kB. In the present study, by using microarray and semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR), data from a previously performed serial analysis of gene expression (SAGE) were extended and verified, revealing that immediate early genes (IEGs) such as c-fos, c-jun and egr-1 are strongly induced at 2-8 h after transient toxin treatment. Activating protein 1 (AP-1: c-Fos, c-Jun) binding activity was increased accordingly. As IEGs are activated by growth factors, these findings led to the discovery that -toxin promotes cell cycle progression of perforated cells in an EGFR-dependent fashion. Although the amount of c-fos mRNA rose rapidly after toxin treatment, c-Fos protein expression was observed only after a lag of about 3 h. Since translation consumes much ATP, which transiently drops after transient membrane perforation, the suspicion arised that membrane-perforation caused global, but temporary downregulation of translation. In fact, eIF2α became heavily phosphorylated minutes after cells had been confronted with the toxin, resulting in shutdown of protein synthesis before cellular ATP levels reached the nadir. GCN2 emerged as a candidate eIF2α kinase, since its expression rapidly increased in toxin-treated cells. Two hours after toxin treatment, GADD34 transcripts, encoding a protein that targets the catalytic subunit of protein phosphatase 1 (PP1) to the endoplasmic reticulum, were overexpressed. This was followed by dephosphorylation of eIF2α and resumption of protein synthesis. Addition of tautomycetin, a specific inhibitor of PP1, led to marked hyperphosphorylation of eIF2α and significantly reduced the drop of ATP-levels in toxin-treated cells. A novel link between two major stress-induced signalling pathways emerged when it was found that both translational arrest and restart were under the control of stress-activated protein kinase (SAPK) p38. The data provide an explanation for the indispensible role of p38 for defence against the archetypal threat of membrane perforation by agents that produce small transmembrane-pores.

1,2-Additionen deprotonierter Alpha-Aminonitrile: Umgepolte Imine als vielseitige Synthesebausteine

Von aromatischen Aldehyden abgeleitete α-Aminonitrile können ohne die Anwendung von Schutzgruppen in α-Position deprotoniert werden, wenn keine lithiumhaltigen Basen verwendet werden. Ziel der vorliegenden Arbeit war es, die Reaktionen deprotonierter α-Aminonitrile mit Elektrophilen zu untersuchen. Die Addition von α-Aminocarbanionen an Imine führt unter intramolekularer Eliminierung von HCN zu Endiaminen, die sich in einer Eintopfsynthese abhängig von der Aufarbeitung in 1,2-Diamine oder 1,2-Diimine umwandeln lassen. Die nach Oxidation durch Luftsauerstoff erhaltenen Diimine können mit dem Reduktionsmittel BH3·THF diastereoselektiv reduziert werden. Es hat sich hier gezeigt, dass durch Zugabe einer katalytischen Menge an NaBH4 hauptsächlich die syn-Diamine erhalten werden, der Zusatz von Phthalsäure wiederum liefert bevorzugt die anti-Produkte. In beiden Fällen wird das Produkt in quantitativer Ausbeute erhalten. So konnte also eine effektive diastereoselektive Reduktionsmethode entwickelt werden, die eine freie Wahl der syn- oder anti-Konfiguration ermöglicht. Um enantiomerenreine 1,2-Diamine zu erhalten, wurden verschiedene Methoden getestet. Sowohl auxiliargesteuerte Synthesen mit einem N-Glycosyl-Aminonitril oder mit chiralen Sulfinyliminen als auch die Reduktion durch chirale Borverbindungen (CBS-Katalysatoren, Triacyloxyborhydrid oder Diisopinocamphenylboran), Transferhydrierungen mit chiralen Difluortitanocen-, Noyori- oder Organophosphat-Katalysatoren sowie enantioselektive Hydrierungen mit chiralen Übergangsmetall-katalysatoren waren jedoch nicht erfolgreich. Die Umsetzung der 1,2-Diimine mit Chlormethylethern oder -estern liefert die entsprechenden unsymmetrischen Imidazoliumsalze. Diese konnten zu N-heterocyclischen Carbenen deprotoniert und erfolgreich als Liganden in Suzuki- und Heck-Reaktionen eingesetzt werden. Durch die 1,2-Addition α-deprotonierter Streckerprodukte und anschließende Reduktion im Eintopfverfahren konnten 1,2-Aminoalkohole in mäßigen bis guten Ausbeuten dargestellt werden. Die Umsetzung von α-Aminocarbanionen mit N-Acyliminen erlaubt zudem die Synthese tetrasubstituierter Imidazole und trisubstituierter Oxazole in drei beziehungsweise vier Stufen: Die zunächst gebildeten α-Amino-α-acylaminopropionitrile können isoliert und in Gegenwart von Base einer Retro-Strecker-Reaktion unterworfen werden. Abhängig vom Substitutionsmuster schließt sich in manchen Fällen nach der Eliminierung von HCN direkt die Cyclisierung zum Imidazol an. Nicht cyclisierte Intermediate lassen sich durch Dehydratisierung mit PCl5 zu Imidazolen umsetzen, aber auch unter sauren Bedingungen zu α-Acylaminoketonen hydrolysieren, welche wiederum durch Einwirkung von PCl5 in Oxazole überführt werden können. Auf diese Weise wurden Imidazole und Oxazole in moderaten bis hohen Gesamtausbeuten hergestellt.

Neue 18 F-markierte Liganden zur Visualisierung des GABA A, alpha 5-Rezeptorstatus mittels PET

Die heutige Verfügbarkeit der molekularen Bildgebung ermöglicht einen signifikanten Einfluss auf die Diagnostik und die Therapiekontrolle von neurodegenerativen Erkrankungen, die unter anderem durch Fehlsteuerungen im GABAergen System auftreten können. Die Visualisierung und Quantifizierung des GABAA-alpha5-Subtyps durch PET könnte dabei zu einem besseren Verständnis von Erkrankungen wie Alzheimer und traumatischen Neurosen (emotionales Langzeitgedächtnis) beitragen. Ferner eröffnen GABAA/alpha5-subtypselektive Liganden die Möglichkeit, wesentliche Grundlagen der elementaren Vorgänge von Lernen und Erinnern zu untersuchen. 7,8,9,10-Tetrahydro-(7,10-ethan)-1,2,4-triazol[3,4-alpha]phthalazine stellen sich als vielverspre-chende Leitstrukturen zur Entwicklung neuer 18F-markierter alpha5-subtypselektiver GABAA-Rezeptorliganden für die PET dar. Um diese neuartigen Substanzen hinsichtlich ihrer Potenz als GABAA-alpha5-subtypselektive Radioliganden zu verifizieren, wurden zunächst die entsprechenden 19F-Derivate TC07-TC12 synthetisiert. Diese Referenzverbindungen wurden in Rezeptor-bindungsassays und in Autoradiographien mit [3H]Ro 15-4513 als zu verdrängender Radioligand evaluiert. In beiden Experimenten als auch in in vivo-Verdrängungsexperimenten an Ratten konnte eine hohe Affinität im nanomolaren Bereich als auch eine hohe Selektivität bezüglich der GABAA/alpha5-Untereinheit für einige der dargestellten Referenzverbindungen nachgewiesen werden. Gemäß diesen vielversprechenden Ergebnissen wurden verschiedene Markie-rungsvorläufer für eine 18F-Direktmarkierung der relevantesten Substanz TC07 in einer mehrstufigen organischen Synthese dargestellt. Die anschließende 18F-Markierung erfolgte über eine nukleophile Substitution mit [18F]Fluorid. Die Reaktionsparameter wurden hinsichtlich Reaktionstemperatur und dauer, Markierungsvorläuferkonzentration, Basenabhängigkeit und verschiedenen Markierungsmethoden optimiert. Daraus resultierend konnte [18F]TC07 mit bis zu 45 % radiochemischer Ausbeute erhalten werden. Die zerfallskorrigierte, gesamtradiochemische Ausbeute von nca [18F]TC07 in isotonischer NaCl-Lösung betrug 15 %. Basierend auf den bisher erhaltenen Ergebnissen wurde der Radioligand in in vitro-, ex vivo- und in vivo µPET-Experimenten evaluiert. Die zunächst durchgeführten in vitro-Experimente deuteten auf eine homogene Verteilung der Aktivität hin und zeigten keine spezifische Anreicherung. Diese Ergebnisse wurden sowohl in ex vivo- als auch in in vivo-µPET-Studien bestätigt. Auch hier konnte nur eine niedrige Aktivitätsanreicherung, eine homogene Verteilung im gesamten Gehirn und keine Übereinstimmung mit der bekannten GABAA/alpha5-Subtypverteilung gefunden werden. Eine im Anschluss durchgeführte Metabolismusstudie zeigte eine langsame Metabolisierungsrate des [18F]TC07 und auch eine Organverteilungsstudie zeigte keine außergewöhnlichen Anreicherungen. Aus den erhaltenen Ergebnissen kann geschlossen werden, dass der Radioligand [18F]TC07 kein geeigneter Tracer zur in vivo-Visualisierung der alpha5-Untereinheit des GABAA-Rezeptors ist.

Algebraische Zyklen auf abelschen Varietäten der Dimension 4 mit Polarisierung von Typ (1,2,2,2)

Diese Arbeit besch"aftigt sich mit algebraischen Zyklen auf komplexen abelschen Variet"aten der Dimension 4. Ziel der Arbeit ist ein nicht-triviales Element in $Griff^{3,2}(A^4)$ zu konstruieren. Hier bezeichnet $A^4$ die emph{generische} abelsche Variet"at der Dimension 4 mit Polarisierung von Typ $(1,2,2,2)$. Die ersten drei Kapitel sind eine Wiederholung von elementaren Definitionen und Begriffen und daher eine Festlegung der Notation. In diesen erinnern wir an elementare Eigenschaften der von Saito definierten Filtrierungen $F_S$ und $Z$ auf den Chowgruppen (vgl. cite{Sa0} und cite{Sa}). Wir wiederholen auch eine Beziehung zwischen der $F_S$-Filtrierung und der Zerlegung von Beauville der Chowgruppen (vgl. cite{Be2} und cite{DeMu}), welche aus cite{Mu} stammt. Die wichtigsten Begriffe in diesem Teil sind die emph{h"ohere Griffiths' Gruppen} und die emph{infinitesimalen Invarianten h"oherer Ordnung}. Dann besch"aftigen wir uns mit emph{verallgemeinerten Prym-Variet"aten} bez"uglich $(2:1)$ "Uberlagerungen von Kurven. Wir geben ihre Konstruktion und wichtige geometrische Eigenschaften und berechnen den Typ ihrer Polarisierung. Kapitel ref{p-moduli} enth"alt ein Resultat aus cite{BCV} "uber die Dominanz der Abbildung $p(3,2):mathcal R(3,2)longrightarrow mathcal A_4(1,2,2,2)$. Dieses Resultat ist von Relevanz f"ur uns, weil es besagt, dass die generische abelsche Variet"at der Dimension 4 mit Polarisierung von Typ $(1,2,2,2)$ eine verallgemeinerte Prym-Variet"at bez"uglich eine $(2:1)$ "Uberlagerung einer Kurve vom Geschlecht $7$ "uber eine Kurve vom Geschlecht $3$ ist. Der zweite Teil der Dissertation ist die eigentliche Arbeit und ist auf folgende Weise strukturiert: Kapitel ref{Deg} enth"alt die Konstruktion der Degeneration von $A^4$. Das bedeutet, dass wir in diesem Kapitel eine Familie $Xlongrightarrow S$ von verallgemeinerten Prym-Variet"aten konstruieren, sodass die klassifizierende Abbildung $Slongrightarrow mathcal A_4(1,2,2,2)$ dominant ist. Desweiteren wird ein relativer Zykel $Y/S$ auf $X/S$ konstruiert zusammen mit einer Untervariet"at $Tsubset S$, sodass wir eine explizite Beschreibung der Einbettung $Yvert _Thookrightarrow Xvert _T$ angeben k"onnen. Das letzte und wichtigste Kapitel enth"ahlt Folgendes: Wir beweisen dass, die emph{ infinitesimale Invariante zweiter Ordnung} $delta _2(alpha)$ von $alpha$ nicht trivial ist. Hier bezeichnet $alpha$ die Komponente von $Y$ in $Ch^3_{(2)}(X/S)$ unter der Beauville-Zerlegung. Damit und mit Hilfe der Ergebnissen aus Kapitel ref{Cohm} k"onnen wir zeigen, dass [ 0neq [alpha ] in Griff ^{3,2}(X/S) . ] Wir k"onnen diese Aussage verfeinern und zeigen (vgl. Theorem ref{a4}) begin{theorem}label{maintheorem} F"ur $sin S$ generisch gilt [ 0neq [alpha _s ]in Griff ^{3,2}(A^4) , ] wobei $A^4$ die generische abelsche Variet"at der Dimension $4$ mit Polarisierung vom Typ $(1,2,2,2)$ ist. end{theorem}

Histamine increases sphingosine kinase-1 expression and activity in the human arterial endothelial cell line EA.hy 926 by a PKC-alpha-dependent mechanism

Sphingosine 1-phosphate (S1P) is a potent mitogenic signal generated from sphingosine by the action of sphingosine kinases (SKs). In this study, we show that in the human arterial endothelial cell line EA.hy 926 histamine induces a time-dependent upregulation of the SK-1 mRNA and protein expression which is followed by increased SK-1 activity. A similar upregulation of SK-1 is also observed with the direct protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA). In contrast, SK-2 activity is not affected by neither histamine nor TPA. The increased SK-1 protein expression is due to stimulated de novo synthesis since cycloheximide inhibited the delayed SK-1 protein upregulation. Moreover, the increased SK-1 mRNA expression results from an increased promoter activation by histamine and TPA. In mechanistic terms, the transcriptional upregulation of SK-1 is dependent on PKC and the extracellular signal-regulated protein kinase (ERK) cascade since staurosporine and the MEK inhibitor U0126 abolish the TPA-induced SK-1 induction. Furthermore, the histamine effect is abolished by the H1-receptor antagonist diphenhydramine, but not by the H2-receptor antagonist cimetidine. Parallel to the induction of SK-1, histamine and TPA stimulate an increased migration of endothelial cells, which is prevented by depletion of the SK-1 by small interfering RNA (siRNA). To appoint this specific cell response to a specific PKC isoenzyme, siRNA of PKC-alpha, -delta, and -epsilon were used to selectively downregulate the respective isoforms. Interestingly, only depletion of PKC-alpha leads to a complete loss of TPA- and histamine-triggered SK-1 induction and cell migration. In summary, these data show that PKC-alpha activation in endothelial cells by histamine-activated H1-receptors, or by direct PKC activators leads to a sustained upregulation of the SK-1 protein expression and activity which, in turn, is critically involved in the mechanism of endothelial cell migration.

A GABA(A) receptor of defined subunit composition and positioning: concatenation of five subunits

We show that the five subunits of a gamma-aminobutyric acid type A receptor (GABA(A) receptor) can be concatenated to yield a functional receptor. This concatenated receptor alpha(1)-beta(2)-alpha(1)-gamma(2)-beta(2) has the advantage of a known subunit arrangement. Most of its functional properties are not significantly different from a receptor formed by individual subunits. Extent of expression amounted to about 40% of that of non-concatenated receptors in Xenopus oocytes, after injection of oocytes with comparable amounts of cRNA coding for concatenated and non-concatenated receptors. The ability to express receptors consisting of five subunits enables detailed studies of GABA(A) receptor subtype selective compounds.

Two alpha subunits and one beta subunit of meprin zinc-endopeptidases are differentially expressed in the zebrafish Danio rerio

Meprins are members of the astacin family of metalloproteases expressed in epithelial tissues, intestinal leukocytes and certain cancer cells. In mammals, there are two homologous subunits, which form complex glycosylated disulfide-bonded homo- and heterooligomers. Both human meprin alpha and meprin beta cleave several basement membrane components, suggesting a role in epithelial differentiation and cell migration. There is also evidence that meprin beta is involved in immune defence owing to its capability of activating interleukin-1beta and the diminished mobility of intestinal leukocytes in meprin beta-knockout mice. Here we show for the first time by reverse transcription PCR, immunoblotting and immunofluorescence analyses that meprins are expressed not only in mammals, but also in the zebrafish Danio rerio. In contrast to the human, mouse and rat enzymes, zebrafish meprins are encoded by three genes, corresponding to two homologous alpha subunits and one beta subunit. Observations at both the mRNA and protein level indicate a broad distribution of meprins in zebrafish. However, there are strikingly different expression patterns of the three subunits, which is consistent with meprin expression in mammals. Hence, D. rerio appears to be a suitable model to gain insight into the basic physiological functions of meprin metalloproteases.

Two neighboring residues of loop A of the alpha1 subunit point towards the benzodiazepine binding site of GABAA receptors

Benzodiazepines are widely used drugs exerting sedative, anxiolytic, muscle relaxant, and anticonvulsant effects by acting through specific high affinity binding sites on some GABA(A) receptors. It is important to understand how these ligands are positioned in this binding site. We are especially interested here in the conformation of loop A of the alpha(1)beta(2)gamma(2) GABA(A) receptor containing a key residue for the interaction of benzodiazepines: alpha(1)H101. We describe a direct interaction of alpha(1)N102 with a diazepam- and an imidazobenzodiazepine-derivative. Our observations help to better understand the conformation of this region of the benzodiazepine pocket in GABA(A) receptor.

Protein kinase C alpha-dependent phosphorylation of the mRNA-stabilizing factor HuR: implications for posttranscriptional regulation of cyclooxygenase-2

In this study, we investigated the molecular mechanisms underlying the ATP analogue adenosine-5'-O-(3-thio)triphosphate-induced nucleocytoplasmic shuttling of the mRNA stabilizing factor HuR in human (h) mesangial cells (MC). Using synthetic protein kinase C (PKC) inhibitors and small interfering RNA approaches, we demonstrated that knockdown of PKC alpha efficiently blocked the ATP-dependent nuclear HuR export to the cytoplasm. The functional importance of PKC alpha in HuR shuttling is highlighted by the high cytosolic HuR content detected in hMC stably overexpressing PKC alpha compared with mock-transfected cells. The ATP-induced recruitment of HuR to the cytoplasm is preceded by a direct interaction of PKC alpha with nuclear HuR and accompanied by increased Ser phosphorylation as demonstrated by coimmunoprecipitation experiments. Mapping of putative PKC target sites identified serines 158 and 221 as being indispensable for HuR phosphorylation by PKC alpha. RNA pull-down assay and RNA electrophoretic mobility shift assay demonstrated that the HuR shuttling by ATP is accompanied by an increased HuR binding to cyclooxygenase (COX)-2 mRNA. Physiologically, the ATP-dependent increase in RNA binding is linked with an augmentation in COX-2 mRNA stability and subsequent increase in prostaglandin E(2) synthesis. Regulation of HuR via PKC alpha-dependent phosphorylation emphasizes the importance of posttranslational modification for stimulus-dependent HuR shuttling.