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Janiszewski, Mariano ; Lopes, Lucia Rossetti ; Carmo, Alípio O. ; Pedro, Marcelo A. ; Brandes, Ralf ; Santos, Célio X. C. ; Laurindo, Francisco R. M.
NAD(P)H oxidase, the main source of reactive oxygen species in vascular cells, is known to be regulated by redox processes and thiols. However, the nature of thiol-dependent regulation has not been established. Protein disulfide isomerase (PDI) is a dithiol/disulfide oxidoreductase chaperone of the thioredoxin superfamily involved in protein processing and translocation. We postulated that PDI regulates NAD(P)H oxidase activity of rabbit aortic smooth muscle cells (VSMCs). Western blotting confirmed robust PDI expression and shift to membrane fraction after incubation with angiotensin II (AII, 100 nm, 6 h). In VSMC membrane fraction, PDI antagonism with bacitracin, scrambled RNase, or neutralizing antibody led to 26-83% inhibition (p < 0.05) of oxidase activity. AII incubation led to significant increase in oxidase activity, accompanied by a 6-fold increase in PDI refolding isomerase activity. AII-induced NAD(P)H oxidase activation was inhibited by 57-71% with antisense oligonucleotide against PDI (PDIasODN). Dihydroethidium fluorescence showed decreased superoxide generation due to PDIasODN. Confocal microscopy showed co-localization between PDI and the oxidase subunits p22(phox), Nox1, and Nox4. Co-immunoprecipitation assays supported spatial association between PDI and oxidase subunits p22(phox), Nox1, and Nox4 in VSMCs. Moreover, in HEK293 cells transfected with green fluorescent protein constructs for Nox1, Nox2, and Nox4, each of these subunits co-immunoprecipitated with PDI. Akt phosphorylation, a known downstream pathway of AII-driven oxidase activation, was significantly reduced by PDIasODN. These results suggest that PDI closely associates with NAD(P)H oxidase and acts as a novel redox-sensitive regulatory protein of such enzyme complex, potentially affecting subunit traffic/assembling.
In der vorliegenden in vitro-Studie wurde der Einfluß von zwei Insertionstechniken auf die zervikale Randqualität von Klasse-II-Kompositrestaurationen unter Zuhilfenahme von Kunststoffmatrizen und Lichtkeilen untersucht. Als weiteren Versuchsparameter wählte man zur Adaptation des Füllungsmaterials neben herkömmlichen Metallinstrumenten zusätzlich modifizierte Biberschwanzpinsel.
Die Photodynamische Therapie (PDT) wird mittlerweile bei einer Vielzahl von Erkrankungen eingesetzt. Ziel dieser Dissertation war die nähere Untersuchung der Kinetik und der Wirkmechanismen der Photosensibilisatoren Methylenblau und disulfoniertem Aluminiumphthalocyanin. Zuerst klärten wir die Frage der Toxizität des Methylenblaus. Wir ermittelten dabei die für die nachfolgenden Versuche nötigen Dosen und Höchstdosen des Methylenblaus in Bezug auf die humane Keratozyten-Linie HaCat und periphere mononukleäre Zellen. Für disulfoniertes Aluminiumphthalocyanin stützten wir uns auf vorhandene Publikationen. Als Lichtquelle benützten wir die PDT Lampe der Firma Waldmann, die ein homogenes Lichtspektrum von 600 bis 700 nm erzeugt, so dass das Wirkungsmaximum aller gängigen Photosensibilisatoren abgedeckt ist. Ausserdem liefert diese Lampe eine gleichmässige Energiedichte über eine größere Fläche, die die Reproduzierbarkeit der Ergebnisse gewährleistet.
In der Arbeit konnte gezeigt werden, dass es für die photodynamische Therapie mit Methylenblau und disulfoniertem Aluminiumphthalocyanin eine Dosis gibt mit der man sowohl Keratinozyten als auch Leukozyten in ihrer Proliferation hemmen kann, ohne eine zytotoxische Wirkung auszulösen. Für Keratinozyten ergab sich dabei ein Anstieg der Proliferationshemmung bei 5 µM Methylenblau und 2stündiger Inkubationszeit bei 200 J/cm², die Toxizität zeigte sich bei 5µM Methylenblau und 4stündiger Inkubationszeit und bereits bei 100 J/cm² maximal. Demgegenüber ergab sich bei stimulierten Leukozyten bereits bei 1µM Methylenblau und 2 Stunden Inkubationszeit ein starker proliferativer Effekt, bei 5µM Methylenblau und 2 Stunden Inkubationszeit zeigte sich dagegen ein deutliche Toxizität. Hierbei fand sich ab 0,5 J/cm² eine zunehmende Proliferationshemmung und Toxizität. Insgesamt war bei Keratinozyten die Differenz bzgl. antiproliferativer und zytotoxischer Dosis geringer als bei Leukozyten. Letztere zeigten sich dabei auch empfindlicher, besonders wenn man die Leukozyten zuvor stimulierte. Daraus ergibt sich ein Potential für den therapeutischen Einsatz der Photodynamischen Therapie bei entzündlichen Dermatosen.
Als mögliche Wege indirekt toxischer Wirkung wurde in der Folge die Stimulation des nukleären Transkriptionsfaktors NF-ΚB, die Bildung von Stickstoffmonoxid (NO) und der protektive Effekt von α-Liponsäure untersucht. Dass der nukleäre Transkriptionsfactor NF-ΚB durch Photodynamische Therapie mit Methylenblau aktiviert werden kann, ist bereits gezeigt worden, so dass wir diese Versuche nicht wiederholten. Die Photodynamische Therapie mit dem Photosensibilisator Methylenblau wirkt also sowohl direkt als auch indirekt toxisch. In unseren Versuchen beschränkten wir uns im weiteren auf die Wirkung des Photosensibilisators disulfoniertes Aluminiumphthalocyanin auf den nukleären Transkriptionsfaktor NF-ΚB. Mittels Gelelektrophorese konnten wir keine Aktivierung von NF-ΚB zeigen. Die Photodynamische Therapie mit dem Photosensibilisator disulfoniertem Aluminiumphthalocyanin wirkt also nur auf direkt toxischem Weg. Bezüglich der Stickstoffmonoxid-Bildung fand sich bei beiden Photosensibilisatoren in den von uns verwendeten Konzentrationen und Inkubabionszeiten kein Nitritnachweis. Auch bei α-Liponsäure ergab sich bei Keratinozyten weder ein pro- noch antiproliferativer Effekt und somt kein Anhalt auf eine indirekte toxische Wirkung.
Der klinische Einsatz der Photodynamischen Therapie erscheint vor dem Hintergrund der erarbeiteten Daten bei entzündlichen Dermatosen möglich, weil infiltrierende aktivierte Leukozyten sensibler gegenüber PDT sind als das umliegende Gewebe, wie hier beispielhaft für Keratinozyten gezeigt wurde.
Um die Rolle von potentiell schmerzauslösenden Substanzen bei der Entstehung von menschlichem Muskelschmerz und von muskulärer Hyperalgesie zu beurteilen, wurde bei dieser Arbeit das DOMS Muskelschmerzmodell und das hypertone NaCl Muskelschmerzmodell in Kombination mit der Mikrodialysetechnik verwendet. Dabei wurden bei 10 gesunden, untrainierten Probanden metabolische Änderungen im Glucosestoffwechsel (Glucose, Laktat) und Fettstoffwechsel (Glycerol), Änderung der Glutamat Freisetzung und Änderungen von inflammatorischen Mediatoren (PGE2, NO, Substanz P) in den schmerzhaften und in den Kontrollmuskeln untersucht. Studienbegleitend erfolgte zur Beurteilung der Effektivität der Übungen und des dabei entstandenen Muskelschadens die Bestimmung von Serum CK, Serum Laktat, des Muskelumfangs und der Muskeldruckschmerzschwelle (PPT). Die Probanden gaben regelmäßig die Schmerzintensität auf einer visuellen Analogskala (VAS) an. Die DOMS Muskelschmerzen wurden 24 Stunden vor dem Beginn der Mikrodialyse durch konzentrisch/ exzentrische Kontraktionen der Wadenmuskulatur im Verum Bein ausgelöst. Während der Mikrodialyse erfolgte die Schmerzstimulation der Wadenmuskulatur durch Plantar- und Dorsalflexion des Fußes. Die Schmerzauslösung beim hypertonen NaCl Modell geschah während der Mikrodialyse durch sequentielle Injektionen von hypertoner NaCl Lösung ( 5 ∗ 200 µl 5.8% NaCl Lösung in 2 Minuten Intervallen) in den Bizepsmuskel am Oberarm. Die Zuordnung der Behandlung (Verum vs. Kontrollmuskel) erfolgte jeweils nach dem Zufallsprinzip.
Direkt nach den DOMS Übungen kam es zu einem signifikanten Anstieg von Laktat im Serum, nach 24 Stunden zu einem signifikanten Ansteigen der CK Aktivität und einer Zunahme des Muskelumfangs. Mit beiden Modellen konnte zuverlässig ein Muskelschmerz erzeugt werden, wobei die Schmerzintensität bei wiederholter Stimulierung abnahm und dies im DOMS Modell stärker ausgeprägt war. Eine mechanische Hyperalgesie konnte nur an den Waden beobachtet werden, die dort aber beidseitig auftrat und damit eine Art „zentraler Übererregbarkeit“ vermuten lässt. Die Dialysatkonzentrationen von Glutamat, PGE2 und Substanz P zeigten aufgrund der Schmerzstimulation im DOMS Bein einen lokalen Anstieg (Glutamat 125 ± 20 µM [p=0.005], PGE2 239 ± 45 pg/ml, Substanz P 64 ± 11 pg/ml). Dabei traten im Kontrollbein keine signifikanten Änderungen auf. Während der Mikrodialyseperiode war die NO Konzentration im DOMS Bein signifikant geringer als im Kontrollbein (p = 0.02), zeigte dabei aber keine Beeinflussung durch die Schmerzstimulation. Gleichzeitig war dabei die Laktatkonzentration im DOMS Bein im Vergleich zum Kontrollbein erhöht. Die Glucose- und Glycerolkonzentrationen wiesen durch die Schmerzauslösung keine bedeutenden Veränderungen auf.
Im Bizepsmuskel kam es infolge der hypertonen NaCl Injektionen zu einem signifikanten Anstieg der Glutamat Konzentration im Dialysat (50 ± 3 µM, p = 0.003), wobei diese im Kontrollmuskel konstant blieb. Die Injektionen hatten aber keinen Einfluss auf die Werte von Glucose, Laktat, Glycerol, NO, PGE2, des Muskelumfangs und der PPT.
Möglicherweise ist ein inflammatorischer Prozess an den peripheren Mechanismen der Muskelschmerzentstehung beim DOMS Modell beteiligt. Die Injektion von hypertoner NaCl Lösung löst den Muskelschmerz vermutlich direkt durch die hohe extrazelluläre Natrium Konzentration aus, wobei es zu einer Depolarisation der Nozizeptormembran mit einer nachfolgenden Glutamat Freisetzung aus den aktivierten Nozizeptoren kommt. Die Vorteile dieses Modells sind die Wiederholbarkeit und die kurze Dauer des Muskelschmerzes. Die dem ausgelösten Schmerz zugrundeliegenden Mechanismen ähneln jedoch nicht den Mechanismen die dem klinischen Muskelschmerz zugrunde liegen. Deshalb könnte es sein, dass die Bedeutungen der Ergebnisse aus diesem Modell relativ beschränkt sind und die Nützlichkeit insbesondere für pharmakologische Studien damit auch eingeschränkt ist.
Der Neurotransmitter Glutamat ist an den peripheren Mechanismen der Muskelschmerzentstehung beteiligt, da die Glutamat Freisetzung direkt mit dem Muskelschmerz beim DOMS Modell und beim Hypertonen NaCl Modell assoziiert war. Die beim DOMS Modell erhöhten Konzentrationen von Laktat, PGE2, sowie die Änderungen von Substanz P und die erniedrigten NO Konzentrationen könnten auch zu der Entstehung von Muskelschmerz beitragen.
Der beobachtete Rückgang der Schmerzintensität bei wiederholter Stimulierung lässt auf eine Art „Gewöhnung“ schließen, die bei Anwendung des DOMS Modells für pharmakologische Untersuchungen einen Nachteil darstellen könnte.
Ziel: Anliegen des Kooperationsprojektes der Klinik für Nephrologie und der Klinik für Psychosomatische Medizin und Psychotherapie ist die internistische und eine umfassende psychologische Untersuchung von152 Lebendnierenspendern, die ihre Niere zwischen 1973 und 2001 in der Universitätsklinik Frankfurt am Main spendeten. Im Rahmen dieser Studie werden aus der oben genannten Arbeitsgruppe heraus, mehrere Arbeiten und Publikationen entstehen. Ziel der vorliegenden Arbeit ist die Untersuchung der 152 Frankfurter Lebendnierenspender in Bezug auf psychosomatische und psychosoziale Aspekte des Erlebens und der Verarbeitung der Lebendnierentransplantation und ihrer Folgen. In der bisherigen empirischen Forschung zu psychischen und psychosomatischen Folgen einer Lebendnierentransplantation wurden beim Spender eher wenige und wenn, dann im Ausmaß begrenzte psychische Komplikationen berichtet. In der Regel sind die psychische Verarbeitung sowie die psycho-sozialen Auswirkungen einer Lebendnierentransplantation insgesamt positiv zu bewerten.
Methode: N= 152 Lebendnierenspender werden internistisch und psychologisch untersucht. Die psychologische Untersuchung verwendet ein breites Spektrum von Erhebungsmethoden. Neben vier standardisierten testpsychologischen Fragebögen wird ein semistrukturiertes ca. einstündiges Interview mit den Spendern geführt. Die vorliegende Arbeit befasst sich gesondert mit dem halbstrukturierten Interview. Die Erlebnisberichte der Spender werden mittels eines eigens erstellten Kategoriensystems ausgewertet.
Ergebnisse: Abschluss der Datenerhebung der vorliegenden Arbeit ist der 15. Februar 2002. Sieben Spender verstarben vor Beginn der Studie, jedoch nicht an den Folgen der Einnierigkeit.Drei Spender waren nicht auffindbar. 19 Spender wurden wegen Wohnsitz im Ausland und/oder Mangel an deutschen Sprachkenntnissen vom psychologischen Interview ausgeschlossen. Von den 123 in Frage kommenden Untersuchungsteilnehmern haben wir mit 100 Spendern Interviews führen können, was einer vergleichsweise hohen Rücklaufquote von 81,3% entspricht. Die meisten Spender trafen ihre Entscheidung sofort (84%) und bereuten ihre Spende im Nachhinein nicht.
Nahezu alle Spender (97%) würden die Entscheidung ihre Niere spenden zu wollen heute wieder treffen. Die meisten Spender bewerten die Spende als ein positives Ereignis vergleichbar mit einer Lebensrettung oder einer Geburt. Einige Spender berichten durch die Spende eine Steigerung ihres Selbstwertgefühls und Selbstbewusstseins erfahren zu haben. 75% der Spender schildern durch die Spende keine Veränderung in der Beziehung zu dem Empfänger erlebt zu haben, bei 23% habe sich die Beziehung verbessert. 3% geben an, die Beziehung zu bestimmten Familienmitgliedern sei nach der Spende schlechter geworden. 3% der Spender bereuen gespendet zu haben. 8% empfanden Druck im Entscheidungsprozess. 5% hatten starke Angst vor der Operation oder dem Leben mit einer Niere. Insgesamt 6% der Spender berichten über langfristige psychische Komplikationen (Verdacht auf: 2% Anpassungsstörung, 2% Angststörung, 1% Depression, 1% Burnout). 11% wünschen sich eine professionelle psychologische Vor- und/oder Nachbetreuung.
Diskussion: Die Ergebnisse der Untersuchung weisen insgesamt auf eine langfristig positive psychische Verarbeitung, sowie auf positive psychosoziale Auswirkungen einer Lebendnierentransplantation hin. Es gibt eine inhomogene Subgruppe mit kleiner Personenanzahl, die negative Erfahrungen mit der Lebendnierenspende machte. Dieser wird gesondert Beachtung geschenkt und die Bereitstellung von adäquaten Beratungs- und Hilfsangeboten diskutiert.
Oberoi-Khanuja, Tripat Kaur ; Karreman, Christiaan ; Larisch, Sarit ; Rapp, Ulf Rüdiger ; Rajalingam, Krishnaraj
Inhibitor of apoptosis (IAPs) proteins are characterized by the presence of evolutionarily conserved baculoviral inhibitor of apoptosis repeat (BIR) domains, predominantly known for their role in inhibiting caspases and, thereby, apoptosis. We have shown previously that multi-BIR domain-containing IAPs, cellular IAPs, and X-linked IAP can control tumor cell migration by directly regulating the protein stability of C-RAF kinase. Here, we extend our observations to a single BIR domain containing IAP family member melanoma-IAP (ML-IAP). We show that ML-IAP can directly bind to C-RAF and that ML-IAP depletion leads to an increase in C-RAF protein levels, MAPK activation, and cell migration in melanoma cells. Thus, our results unveil a thus far unknown role for ML-IAP in controlling C-RAF stability and cell migration.
Oertel, Bruno Georg ; Kettner, Mattias ; Scholich, Klaus ; Renné, Christoph ; Roskam, Bianca ; Geisslinger, Gerd ; Schmidt, Peter Harald ; Lötsch, Jörn
The single nucleotide polymorphism 118A>G of the human micro-opioid receptor gene OPRM1, which leads to an exchange of the amino acid asparagine (N) to aspartic acid (D) at position 40 of the extracellular receptor region, alters the in vivo effects of opioids to different degrees in pain-processing brain regions. The most pronounced N40D effects were found in brain regions involved in the sensory processing of pain intensity. Using the mu-opioid receptor-specific agonist DAMGO, we analyzed the micro-opioid receptor signaling, expression, and binding affinity in human brain tissue sampled postmortem from the secondary somatosensory area (SII) and from the ventral posterior part of the lateral thalamus, two regions involved in the sensory processing and transmission of nociceptive information. We show that the main effect of the N40D micro-opioid receptor variant is a reduction of the agonist-induced receptor signaling efficacy. In the SII region of homo- and heterozygous carriers of the variant 118G allele (n=18), DAMGO was only 62% as efficient (p=0.002) as in homozygous carriers of the wild-type 118A allele (n=15). In contrast, the number of [3H]DAMGO binding sites was unaffected. Hence, the micro-opioid receptor G-protein coupling efficacy in SII of carriers of the 118G variant was only 58% as efficient as in homozygous carriers of the 118A allele (p<0.001). The thalamus was unaffected by the OPRM1 118A>G SNP. In conclusion, we provide a molecular basis for the reduced clinical effects of opioid analgesics in carriers of mu-opioid receptor variant N40D.
Biglycan, a nitric oxide-regulated gene, affects adhesion, growth, and survival of mesangial cells
(2003)
Schäfer, Liliana ; Beck, Karl-Friedrich ; Raslik, Igor ; Walpen, Sebastian ; Mihalik, Daniel ; Mičegová, Miroslava ; Macakova, Katarina ; Schönherr, Elke ; Seidler, Daniela Gabriele ; Varga, Georg ; Schaefer, Roland M. ; Kresse, Hans ; Pfeilschifter, Josef
During glomerular inflammation mesangial cells are the major source and target of nitric oxide that pro-foundly influences proliferation, adhesion, and death of mesangial cells. The effect of nitric oxide on the mRNA expression pattern of cultured rat mesangial cells was therefore investigated by RNA-arbitrarily-primed polymerase chain reaction. Employing this approach, biglycan expression turned out to be down-regulated time- and dose-dependently either by interleukin-1beta-stimulated endogenous nitric oxide production or by direct application of the exogenous nitric oxide donor, diethylenetriamine nitric oxide. There was a corresponding decline in the rate of biglycan biosynthesis and in the steady state level of this proteoglycan. In vivo, in a model of mesangioproliferative glomerulonephritis up-regulation of inducible nitric-oxide synthase mRNA was associated with reduced expression of biglycan in isolated glomeruli. Biglycan expression could be normalized, both in vitro and in vivo, by using a specific inhibitor of the inducible nitric-oxide synthase, l-N6-(l-iminoethyl)-l-lysine dihydrochloride. Further studies showed that biglycan inhibited cell adhesion on type I collagen and fibronectin because of its binding to these substrates. More importantly, biglycan protected mesangial cells from apoptosis by decreasing caspase-3 activity, and it counteracted the proliferative effects of platelet-derived growth factor-BB. These findings indicate a signaling role of biglycan and describe a novel pathomechanism by which nitric oxide modulates the course of renal glomerular disease through regulation of biglycan expression.
Blaukat, Andree ; Pizard, Anne ; Breit, Andreas ; Wernstedt, Christer ; Alhenc-Gelas, François ; Müller-Esterl, Werner ; Đikić, Ivan
Reversible phosphorylation plays important roles in G protein-coupled receptor signaling, desensitization, and endocytosis, yet the precise location and role of in vivo phosphorylation sites is unknown for most receptors. Using metabolic 32P labeling and phosphopeptide sequencing we provide a complete phosphorylation map of the human bradykinin B2 receptor in its native cellular environment. We identified three serine residues, Ser(339), Ser(346), and Ser(348), at the C-terminal tail as principal phosphorylation sites. Constitutive phosphorylation occurs at Ser(348), while ligand-induced phosphorylation is found at Ser(339) and Ser(346)/Ser(348) that could be executed by several G protein-coupled receptor kinases. In addition, we found a protein kinase C-dependent phosphorylation of Ser(346) that was mutually exclusive with the basal phosphorylation at Ser(348) and therefore may be implicated in differential regulation of B2 receptor activation. Functional analysis of receptor mutants revealed that a low phosphorylation stoichiometry is sufficient to initiate receptor sequestration while a clustered phosphorylation around Ser(346) is necessary for desensitization of the B2 receptor-induced phospholipase C activation. This was further supported by the specifically reduced Ser(346)/Ser(348) phosphorylation observed upon stimulation with a nondesensitizing B2 receptor agonist. The differential usage of clustered phosphoacceptor sites points to distinct roles of multiple kinases in controlling G protein-coupled receptor function.
Ludwig, Jürgen ; Kerscher, Stefan ; Brandt, Ulrich ; Pfeiffer, Kathy ; Getlawi, Fariha ; Apps, David K. ; Schägger, Hermann
Vacuolar proton-translocating ATPase (holoATPase and free membrane sector) was isolated from bovine chromaffin granules by blue native polyacrylamide gel electrophoresis. A 5-fold excess of membrane sector over holoenzyme was determined in isolated chromaffin granule membranes. M9.2, a novel extremely hydrophobic 9.2-kDa protein comprising 80 amino acids, was detected in the membrane sector. It shows sequence and structural similarity to Vma21p, a yeast protein required for assembly of vacuolar ATPase. A second membrane sector-associated protein (M8-9) was identified and characterized by amino-terminal protein sequencing.