Interaction of Gα₁₂ with Gα₁₃ and Gα_q signaling pathways

The G(12) subfamily of heterotrimeric G-proteins consists of two members, G(12) and G(13). Gene-targeting studies have revealed a role for G(13) in blood vessel development. Mice lacking the a subunit of G(13) die around embryonic day 10 as the result of an angiogenic defect. On the other hand, the physiological role of G(12) is still unclear. To address this issue, we generated Galpha(12)-deficient mice. In contrast to the Galpha(13)-deficient mice, Galpha(12)-deficient mice are viable, fertile, and do not show apparent abnormalities. However, Galpha(12) does not seem to be entirely redundant, because in the offspring generated from Galpha(12)+/-Galpha(13) intercrosses, at least one intact Galpha(12) allele is required for the survival of animals with only one Galpha(13) allele. In addition, Galpha(12) and Galpha(13) showed a difference in mediating cell migratory response to lysophosphatidic acid in embryonic fibroblast cells. Furthermore, mice lacking both Galpha(12) and Galpha(q) die in utero at about embryonic day 13. These data indicate that the Galpha(12)-mediated signaling pathway functionally interacts not only with the Galpha(13)- but also with the Galpha(q/11)-mediated signaling systems

Large chemokine binding spectrum of human and mouse atypical chemokine receptor GPR182 (ACKR5)

Atypical chemokine receptors (ACKRs) play pivotal roles in immune regulation by binding chemokines and regulating their spatial distribution without inducing G-protein activation. Recently, GPR182, provisionally named ACKR5, was identified as a novel ACKR expressed in microvascular and lymphatic endothelial cells, with functions in hematopoietic stem cell homeostasis. Here, we comprehensively investigated the chemokine binding profile of human and mouse GPR182. Competitive binding assays using flow cytometry revealed that besides CXCL10, CXCL12 and CXCL13, also human and mouse CXCL11, CXCL14 and CCL25, as well as human CCL1, CCL11, CCL19, CCL26, XCL1 and mouse CCL22, CCL24, CCL27 and CCL28 bind with an affinity of less than 100 nM to GPR182. In line with the binding affinity observed in vitro, elevated serum levels of CCL22, CCL24, CCL25, and CCL27 were observed in GPR182-deficient mice, underscoring the role of GPR182 in chemokine scavenging. These data show a broader chemokine binding repertoire of GPR182 than previously reported and they will be important for future work exploring the physiological and pathophysiological roles of GPR182, which we propose to be renamed atypical chemokine receptor 5 (ACKR5)

Gα15 and Gα16 Couple a Wide Variety of Receptors to Phospholipase C

The murine G-protein α-subunit Gα15 and its human counterpart Gα16 are expressed in a subset of hematopoietic cells, and they have been shown to regulate β-isoforms of inositide-specific phospholipase C. We studied the ability of a variety of receptors to interact with Gα15 and Gα16 by cotransfecting receptors and G-protein α-subunits in COS-7 cells. Activation of β2 adrenergic and muscarinic M2 receptors in cells expressing the receptors alone or together with Gαq, Gα11, or Gα14 led to a very small stimulation of endogenous phospholipase C. However, when the receptors were coexpressed with Gα15 and Gα16, addition of appropriate ligands caused a severalfold increase in inositol phosphate production which was time- and dose-dependent. A similar activation of phospholipase C was observed when several other receptors which were previously shown to couple to members of the Gi and Gs family were coexpressed with Gα15/16. In addition, stimulation of inositol phosphate formation via receptors naturally coupled to phospholipase C was enhanced by cotransfection of Gα15 and Gα16. These data demonstrate that Gα15 and Gα16 are unique in that they can be activated by a wide variety of G-protein-coupled receptors. The ability of Gα15 and Gα16 to bypass the selectivity of receptor G-protein interaction can be a useful tool to understand the mechanism of receptor-induced G-protein activation. In addition, the promiscuous behavior of Gα15 and Gα16 toward receptors may be helpful in finding ligands corresponding to orphan receptors whose signaling properties are unknown

An Exploratory Investigation of a New Method for Music-Therapeutic Research

MUSIC LISTENING AND MAKING ACTIVATES A multitude of brain structures, the engagement of which is likely to have beneficial effects on the psychological and physiological health of individuals. We first briefly review functional neuroimaging experiments on music and emotion, showing that music-evoked emotions can change activity in virtually all core areas of emotional processing.We then enumerate social functions that are automatically and effortlessly engaged when humans make music. Engagement in these social functions fulfils basic human needs, is part of what makes us human, and is an important source for pleasure and happiness. Finally, we present a new method for music therapy, including an exploratory empirical study on effects of music making. Results show that the music making increased the mood of individuals compared to a control group. This music therapy method is promising in encouraging further development for the treatment of affective disorders, and can be used in both single- and double-blinded studies for empirical, evidence-based medical research

Verhalten dünnwandiger austenitischer Rohre bei Wasserstoff-Sauerstoff-Detonation

Beim Auftreten oder bei der Verwendung von Wasserstoff in sauerstoffhaltiger Umgebung ist eines der Hauptrisiken dessen leichte Entzündbarkeit, wobei die Gasreaktionen mit sehr unterschiedlichen Geschwindigkeiten und Gasdruckentwicklungen ablaufen. In der Folge können zum Beispiel in druckführenden Komponenten technischer Anlagen extreme mechanische und auch thermische Belastungen auftreten. Die vorliegende Arbeit befasst sich mit der sicherheitstechnischen Bewertung austenitischer Rohrleitungen unter Beanspruchung durch Wasserstoff-Sauerstoff-Reaktionen. Derzeit bekannte Ansätze für die Auslegung von Rohrleitungen unter derartigen Belastungsszenarien sind entweder auf rein elastische Rohrreaktionen beschränkt oder aber sie berücksichtigen die gasdynamischen Vorgänge nicht in ausreichendem Maße. Neben der genauen Kenntnis der vielfältigen komplexen Belastungsgrößen und deren Wechselwirkung mit der Struktur ist aber auch das Verständnis des Materialverhaltens und der Bruchmechanismen der Komponentenstruktur unter den auftretenden Beanspruchungsgeschwindigkeiten- und -temperaturen von zentraler Bedeutung. Zur Beschreibung der Vorgänge wurden von Stadtmüller und Offermanns durchgeführte Detonationsversuche herangezogen. Ziel dieser Versuche war die Untersuchung von Detonationsvorgängen in Deckelsprühleitungen von Siedewasserreaktoren. Die Versuche wurden daher an für diese Anwendung relevanten austenitischen Rohren (Werkstoffnummer 1.4541) mit den nominalen Abmessungen Da x s = (114,30 x 6,02) mm durchgeführt. Die Untersuchungen wurden im Rahmen dieser Arbeit durch weitere Versuche mit identischem Außendurchmesser, aber veränderter Wanddicke ergänzt. Die Auswertung der Versuche hat ergeben, dass geringere Mengen an reaktionsfähigem Gas zu einer heftigeren Rohrreaktion führen können als es bei höheren Mengen der Fall ist. Diese Beobachtung kann auf den Einfluss der so genannten überkomprimierten Detonation zurückgeführt werden. Von einer überkomprimierten Detonation spricht man, wenn die mit Unterschallgeschwindigkeit ablaufende deflagrative Verbrennung vor ihrer Flammenfront Frischgas vorkomprimiert und der Umschlag in eine Detonation (DDT) innerhalb dieser vorkomprimierten Zone stattfindet. Die Deflagrationsstrecke wird mit abnehmender Konzentration reaktionsfähiger Gase länger, so dass die Vorkompression zunimmt. Dies hat zur Folge, dass am Umschlagpunkt zur Detonation höhere Druckspitzen und Dehnraten auftreten, als bei einer höheren Gaskonzentration. Die Analyse der Bruchstücke hat ergeben, dass es, abhängig von Gasmischung und Rohrwanddicke, zur Ausbildung so genannter adiabatischer Scherbänder kommen kann. Adiabatische Scherbänder sind sehr schmale Zonen, in denen es zu intensiv lokalisierten Scherdeformationen kommt. Zurückzuführen ist diese Lokalisierung auf die Umwandlung eines erheblichen Teils der Formänderungsarbeit in Wärme. Da der Vorgang bei hoher Belastungsgeschwindigkeit weitgehend adiabat abläuft, d. h. die Zeit für eine Diffusion der Wärme nicht ausreicht, kommt es zu einer thermischen Entfestigung welche schließlich die Kaltverfestigung überschreitet. Aus dieser Erkenntnis heraus wurde ein Schädigungsmodell verwendet, das die Werkstoffschädigung über einen weiten Bereich verschiedener Spannungszustände abbilden kann. Es hat sich jedoch gezeigt, dass die Schädigung des Untersuchungswerkstoffs in starkem Maße auch vom Lode-Winkel beeinflusst wird. Dieser Umstand wurde in einem vereinfachten Ansatz berücksichtigt. Durch Kleinprobenversuche wurde die kritische Dehnung in Abhängigkeit von Spannungsmehrachsigkeit und Lode-Winkel bestimmt. Es erfolgte eine numerisch gestützte Auswahl geeigneter Probenformen und die Festsetzung entsprechender Versuchsanordnungen. Zur konstitutiven Beschreibung des Werkstoffverhaltens unter den auftretenden Beanspruchungsgeschwindigkeiten- und -temperaturen wurden geeignete Materialmodelle ausgewählt. Die erforderlichen Parameter wurden experimentell durch Kleinprobenzug- und Druckversuche evaluiert und numerisch verifiziert. Mit Hilfe der konstitutiven Werkstoffbeschreibung und des Schädigungsmodells wurden numerische Simulationen der Detonationsversuche durchgeführt. Die räumlichen und zeitlichen Druckverläufe wurden am Institut für Kern- und Energietechnik am Karlsruher Institut für Technologie numerisch ermittelt, gelten jedoch nur für unnachgiebige Rohrleitungen. Zur Berücksichtigung der Interaktion mit den stark plastifizierenden Rohren aus [1] wurde ein einfacher Korrekturansatz über die Isentropenbeziehung des Gases herangezogen. Die Simulationen der Detonationsversuche haben ergeben, dass im Bereich der stabilen Detonation die plastische Verformung gegenüber dem Experiment überschätzt wird. Diese Beobachtung ist vermutlich hauptsächlich vereinfachten Annahmen bei der Gas-Strukturinteraktion geschuldet. Beispielsweise wird bei dem verwendeten Ansatz sowohl der axiale Druckausgleich als auch die radiale Strömung während der Rohraufweitung vernachlässigt. Eine direkte Kopplung der verwendeten gas- und strukturmechanischen Berechnungsmethoden könnte hier zu einer Verbesserung führen. Weiterhin konnte das Versagen von Rohren infolge der Ausbildung adiabatischer Scherbänder in befriedigender Übereinstimmung mit dem Experiment simuliert werden

Thrombin Protease-activated Receptor-1 Signals through Gq- and G13-initiated MAPK Cascades Regulating c-Jun Expression to Induce Cell Transformation

Although the ability of G protein-coupled receptors to stimulate normal and aberrant cell growth has been intensely investigated, the precise nature of the molecular mechanisms underlying their transforming potential are still not fully understood. In this study, we have taken advantage of the potent mitogenic effect of thrombin and the focus-forming activity of one of its receptors, protease-activated receptor-1, to dissect how this receptor coupled to Gi, Gq/11, and G12/13 transduces signals from the membrane to the nucleus to initiate transcriptional events involved in cell transformation. Using endogenous and transfected thrombin receptors in NIH 3T3 cells, ectopic expression of muscarinic receptors coupled to Gq and Gi, and chimeric G protein subunits and murine fibroblasts deficient in Gq/11, and G12/13, we show here that, although coupling to Gi is sufficient to induce ERK activation, the ability to couple to Gq and/or G13 is necessary to induce c-jun expression and cell transformation. Furthermore, we show that Gq and G13 can initiate the activation of MAPK cascades, including JNK, p38, and ERK5, which in turn regulate the activity of transcription factors controlling expression from the c-jun promoter. We also present evidence that c-Jun and the kinases regulating its expression are integral components of the transforming pathway initiated by protease-activated receptor-1

Calcium-Dependent Persistent Facilitation of Spike Backpropagation in the CA1 Pyramidal Neurons

Sodium-dependent action potentials initiated near the soma are known to backpropagate over the dendrites of CA1 pyramidal neurons in an activity-dependent manner. Consequently, later spikes in a train have smaller amplitude when recorded in the apical dendrites. We found that depolarization and resultant Ca²⁺ influx into dendrites caused a persistent facilitation of spike backpropagation. Dendritic patch recordings were made from CA1 pyramidal neurons in mouse hippocampal slices under blockade of fast excitatory and inhibitory synaptic inputs. Trains of 10 backpropagating action potentials induced by antidromic stimulation showed a clear decrement in the amplitude of later spikes when recorded in the middle apical dendrites. After several depolarizing current pulses, the amplitude of later spikes increased persistently, and all spikes in a train became almost equal in size. BAPTA (10 mM) contained in the pipette or low-Ca^(2+) perfusing solution abolished this depolarization-induced facilitation, indicating that Ca²⁺ influx is required. This facilitation was present in Gα_q knock-out mice that lack the previously reported muscarinic receptor-mediated enhancement of spike backpropagation. Therefore, these two forms of facilitation are clearly distinct in their intracellular mechanisms. Intracellular injection of either calmodulin binding domain (100 μM) or Ca²⁺/calmodulin-kinase II (CaMKII) inhibitor 281–301 (10 μM) blocked the depolarization-induced facilitation. Bath application of a membrane-permeable CaMKII inhibitor KN-93 (10 μM) also blocked the facilitation, but KN-92 (10 μM), an inactive isomer of KN-93, had no effect. These results suggest that increases in [Ca²⁺)]_i cause persistent facilitation of spike backpropagation in the apical dendrite of CA1 pyramidal neuron by CaMKII-dependent mechanisms

Plexin-B1/RhoGEF–mediated RhoA activation involves the receptor tyrosine kinase ErbB-2

Plexins are widely expressed transmembrane proteins that mediate the effects of semaphorins. The molecular mechanisms of plexin-mediated signal transduction are still rather unclear. Plexin-B1 has recently been shown to mediate activation of RhoA through a stable interaction with the Rho guanine nucleotide exchange factors PDZ-RhoGEF and LARG. However, it is unclear how the activity of plexin-B1 and its downstream effectors is regulated by its ligand Sema4D. Here, we show that plexin-B family members stably associate with the receptor tyrosine kinase ErbB-2. Binding of Sema4D to plexin-B1 stimulates the intrinsic tyrosine kinase activity of ErbB-2, resulting in the phosphorylation of both plexin-B1 and ErbB-2. A dominant-negative form of ErbB-2 blocks Sema4D-induced RhoA activation as well as axonal growth cone collapse in primary hippocampal neurons. Our data indicate that ErbB-2 is an important component of the plexin-B receptor system and that ErbB-2–mediated phosphorylation of plexin-B1 is critically involved in Sema4D-induced RhoA activation, which underlies cellular phenomena downstream of plexin-B1, including axonal growth cone collapse

Previously differentiated medial vascular smooth muscle cells contribute to neointima formation following vascular injury

Background The origins of neointimal smooth muscle cells that arise following vascular injury remains controversial. Studies have suggested that these cells may arise from previously differentiated medial vascular smooth muscle cells, resident stem cells or blood born progenitors. In the current study we examined the contribution of the previously differentiated vascular smooth muscle cells to the neointima that forms following carotid artery ligation. Methods We utilized transgenic mice harboring a cre recombinase-dependent reporter gene (mTmG). These mice express membrane targeted tandem dimer Tomato (mTomato) prior to cre-mediated excision and membrane targeted EGFP (mEGFP) following excision. The mTmG mice were crossed with transgenic mice expressing either smooth muscle myosin heavy chain (Myh11) or smooth muscle α-actin (Acta2) driven tamoxifen regulated cre recombinase. Following treatment of adult mice with tamoxifen these mice express mEGFP exclusively in differentiated smooth muscle cells. Subsequently vascular injury was induced in the mice by carotid artery ligation and the contribution of mEGFP positive cells to the neointima determined. Results Analysis of the cellular composition of the neointima that forms following injury revealed that mEGFP positive cells derived from either Mhy11 or Acta2 tagged medial vascular smooth muscle cells contribute to the majority of neointima formation (79 ± 17% and 81 ± 12%, respectively). Conclusion These data demonstrate that the majority of the neointima that forms following carotid ligation is derived from previously differentiated medial vascular smooth muscle cells

Activation of G12/G13 Results in Shape Change and Rho/Rho-Kinase–mediated Myosin Light Chain Phosphorylation in Mouse Platelets

Platelets respond to various stimuli with rapid changes in shape followed by aggregation and secretion of their granule contents. Platelets lacking the α-subunit of the heterotrimeric G protein Gq do not aggregate and degranulate but still undergo shape change after activation through thromboxane-A2 (TXA2) or thrombin receptors. In contrast to thrombin, the TXA2 mimetic U46619 led to the selective activation of G12 and G13 in Gαq-deficient platelets indicating that these G proteins mediate TXA2 receptor-induced shape change. TXA2 receptor-mediated activation of G12/G13 resulted in tyrosine phosphorylation of pp72syk and stimulation of pp60c-src as well as in phosphorylation of myosin light chain (MLC) in Gαq-deficient platelets. Both MLC phosphorylation and shape change induced through G12/G13 in the absence of Gαq were inhibited by the C3 exoenzyme from Clostridium botulinum, by the Rho-kinase inhibitor Y-27632 and by cAMP-analogue Sp-5,6-DCl-cBIMPS. These data indicate that G12/G13 couple receptors to tyrosine kinases as well as to the Rho/Rho-kinase–mediated regulation of MLC phosphorylation. We provide evidence that G12/G13-mediated Rho/Rho-kinase–dependent regulation of MLC phosphorylation participates in receptor-induced platelet shape change