cGMP-dependent protein kinase regulates Rap1 signaling in platelets : poster presentation

cGMP- and cAMP-dependent protein kinases (cGK and cAK) mediate the inhibitory effects of endothelium-derived messenger molecules nitric oxide and prostacyclin on platelets. To understand the mechanisms involved in platelet inhibition we searched for new substrates of cGK and cAK. We identified Rap1GAP2, the only GTPase-activating protein of Rap1 in platelets. Rap1 is a guanine-nucleotide binding protein that controls integrin activity, platelet adhesion and aggregation. Rap1GAP2 is required to turn over Rap1-GTP to Rap1-GDP resulting in the inactivation of integrins and reduced cellular adhesion. Using phospho-specific antibodies we demonstrate phosphorylation of endogenous Rap1GAP2 on serine 7 by cGK and cAK in intact platelets. Yeast-two-hybrid screening revealed an interaction of the phosphoserine/-threonine binding adapter protein 14-3-3 with Rap1GAP2, and we mapped the 14-3-3 binding site to the N-terminus of Rap1GAP2 close to the cGK/cAK phosphorylation site. We could show that 14-3-3 binding to Rap1GAP2 requires phosphorylation of serine 9. Platelet activation by ADP and thrombin treatment induces Rap1GAP2 serine 9 phosphorylation and enhances the attachment of 14-3-3 to Rap1GAP2. In contrast, phosphorylation of serine 7 by cGK/cAK leads to the detachment of 14-3-3. Furthermore, Rap1GAP2 serine 7 phosphorylation correlates with the inhibition of Rap1-GTP formation by cGMP and cAMP in platelets. Cell adhesion experiments provide additional evidence that Rap1GAP2 is activated by the detachment of 14-3-3. Point mutants of Rap1GAP2 deficient in 14-3-3 binding inhibit Rap1-mediated cell adhesion significantly stronger than a Rap1GAP2 mutant that binds 14-3-3 constitutively. Our findings define a novel regulatory mechanism that might contribute to both platelet activation and endothelial inhibition of platelet adhesion and aggregation

L'individuation selon Brentano

Brentano, expert et continuateur de l’ontologie aristotélicienne, traite du problème métaphysique de l’individuation dans un petit nombre d’écrits (réunis dans le recueil intitulé Kategorienlehre ). Il retient pour ce problème une solution ordinairement rejetée, l’individuation par le lieu (qui serait de l’ordre de l’accident dans la perspective d’Aristote et comme tel impropre à fournir le principe de l’individuation ). À ce titre, il occupe une position originale. La notion de lieu, cependant, tout comme plus généralement la notion de l’accident, ressort tout à fait transformée des analyses de Brentano : le lieu devient une détermination substantielle, non accidentelle, des corps. Dans des écrits tardifs, Brentano étend son analyse du lieu à une cosmologie qui n’admet qu’une seule substance corporelle étendue, dont les corps particuliers sont les accidents.Brentano, both expert and continuator of the Aristotelian ontology, devotes his attention to the metaphysical problem of individuation in a small number of writings collected under the title Kategorienlehre. For this problem, he adopts a solution which gets ordinarily rejected, individuation through place (place would be among the accidents within the Aristotelian perspective and as such unsuitable as the principle of individuation). Hence he occupies an original position on this issue. The notion of place, however, as well as the notion of accident more generally, gets completely transformed in the Brentanian analysis : place becomes not an accidental but a substantial determination. In some late writings, Brentano extends his analysis of place to a cosmology which admits a unique extended bodily substance, of which particular bodies are only the accidents

Papst Silvester II. (Gerbert) als Lehrer und Staatsmann

von Karl SchulteßProgr.-Nr. 72

Molekulare Evolution der Intermediärfilament-Proteine des Flussneunauges Lampetra fluviatilis

Über cDNA-Banken und RT-PCR wurden erstmals 15 Intermediärfilament-Proteine (IF-Proteine) des Flussneunauges Lampetra fluviatilis (Agnatha, kieferlose Wirbeltiere) kloniert und sequenziert: drei Typ I-Keratine, vier Typ II-Keratine, fünf keratinartige IF-Proteine (drei Kγ, zwei Kα), die Typ III-Proteine Vimentin und Desmin sowie ein Typ IV-Neurofilament-Protein (NF).Die IF-Proteine wurden aus verschiedenen Organen isoliert und durch zweidimensionale Polyacrylamid-Gelelektrophorese (2D-PAGE) aufgetrennt. Biochemische sowie massenspektrometrische Analysen anhand der 2D-PAGE ermöglichten in Kombination mit den Sequenzdaten die Identifizierung von Vimentin, Desmin sowie aller sequenzierten Keratine bis auf zwei der fünf Kα/Kγ-Proteine. Die meisten Keratine ließen sich darüber hinaus in die Kategorien „E“ (von „epidermal“) und „S“ (von „simple epithelial“) einteilen.Von den sequenzierten Keratinen ist das IIS-Keratin K8 wahrscheinlich ortholog zu den bekannten K8-Sequenzen höherer Vertebraten. Die Bezeichnung K18 für das einzige IS-Keratin des Neunauges in Anlehnung an das IS-Keratin K18 des Menschen basiert auf der stets beobachteten Koexpression mit K8 in einfachen Epithelien.Die Sequenz des Neunaugen-Vimentins zeigt große Übereinstimmungen mit den bekannten Desminsequenzen der Vertebraten. Die keratinartigen Proteine Kα und Kγ sind bis jetzt nur von Agnathen (Neunaugen und Schleimaale) bekannt.In molekularen Stammbäumen können K8, K18, Vimentin, Desmin und das NF_L des Neunauges gut als Außengruppe definiert werden.15 intermediate filament proteins (IF-proteins) of the river-lamprey Lampetra fluviatilis have been cloned and sequenced by means of cDNA libraries and RT-PCR: three type I-keratins, four type II-keratins, five keratin-like IF-proteins (three Kγ, two Kα), the type III-proteins vimentin and desmin as well as a type IV-neurofilament-protein (NF).After isolating the IF-proteins from different tissues, they were separated by 2D-PAGE. Biochemical and mass-spectrometric analysis in combination with sequence data enabled the identification of vimentin and desmin, as well as all sequenced keratins, except two of the five keratin-like proteins in the 2D-PAGE. Most keratins can be divided into the categories “E” (“epidermal”) and “S” (“simple epithelia”). The IIS Keratin K8 is probable an ortholog to the known K8-sequences of higher vertebrates. The designation K18, for the only IS keratin of the lamprey was chosen because of coexpression with K8 in simple epithelia.The sequence of lamprey vimentin shows good identity with all known vertebrate desmins. To date, the keratin-like proteins Kα and Kγ are only known to be present in agnathans (lampreys and hagfishes). In molecular phylogenetic trees, K8, K18, vimentin, desmin and the NF_L of the lamprey serve as well defined outgroups

Herodes Atticus : (101 - 177 nach Chr. Geb.)

von Carl SchulteßProgr.-Nr. 85

Die Abfassungszeit des platonischen Theätet

von Fritz Schultes