73 research outputs found
Pasteurella multocida Toxin Activates Various Heterotrimeric G Proteins by Deamidation
Pasteurella multocida produces a 146-kDa protein toxin (Pasteurella multocida toxin, PMT), which stimulates diverse cellular signal transduction pathways by activating heterotrimeric G proteins. PMT deamidates a conserved glutamine residue of the α-subunit of heterotrimeric G proteins that is essential for GTP-hydrolysis, thereby arresting the G protein in the active state. The toxin substrates are Gαq Gα13 and the Gαi-family proteins. Activation of these α-subunits causes stimulation of phospholipase CÎČ, Rho-guanine nucleotide exchange factors or inhibition of adenylyl cyclase. This article provides the current knowledge on PMT concerning the structure-function analysis based on the crystal structure and recently elucidated molecular mode of action. Furthermore, the impact of PMT on cellular signaling is discussed
The two-pore channel TPC1 is required for efficient protein processing through early and recycling endosomes
Two-pore channels (TPCs) are localized in endo-lysosomal compartments and assumed to play an important role for vesicular fusion and endosomal trafficking. Recently, it has been shown that both TPC1 and 2 were required for host cell entry and pathogenicity of Ebola viruses. Here, we investigate the cellular function of TPC1 using protein toxins as model substrates for distinct endosomal processing routes. Toxin uptake and activation through early endosomes but not processing through other compartments were reduced in TPC1 knockout cells. Detailed co-localization studies with subcellular markers confirmed predominant localization of TPC1 to early and recycling endosomes. Proteomic analysis of native TPC1 channels finally identified direct interaction with a distinct set of syntaxins involved in fusion of intracellular vesicles. Together, our results demonstrate a general role of TPC1 for uptake and processing of proteins in early and recycling endosomes, likely by providing high local Ca2+ concentrations required for SNARE-mediated vesicle fusion
Chemokine CCL9 Is Upregulated Early in Chronic Kidney Disease and Counteracts Kidney Inflammation and Fibrosis
Inflammation and fibrosis play an important pathophysiological role in chronic kidney
disease (CKD), with pro-inflammatory mediators and leukocytes promoting organ damage with
subsequent fibrosis. Since chemokines are the main regulators of leukocyte chemotaxis and tissue
inflammation, we performed systemic chemokine profiling in early CKD in mice. This revealed
(C-C motif) ligands 6 and 9 (CCL6 and CCL9) as the most upregulated chemokines, with significantly higher levels of both chemokines in blood (CCL6: 3â4 fold; CCL9: 3â5 fold) as well as kidney
as confirmed by Enzyme-linked Immunosorbent Assay (ELISA) in two additional CKD models.
Chemokine treatment in a mouse model of early adenine-induced CKD almost completely abolished
the CKD-induced infiltration of macrophages and myeloid cells in the kidney without impact on circulating leukocyte numbers. The other way around, especially CCL9-blockade aggravated monocyte
and macrophage accumulation in kidney during CKD development, without impact on the ratio
of M1-to-M2 macrophages. In parallel, CCL9-blockade raised serum creatinine and urea levels as
readouts of kidney dysfunction. It also exacerbated CKD-induced expression of collagen (3.2-fold)
and the pro-inflammatory chemokines CCL2 (1.8-fold) and CCL3 (2.1-fold) in kidney. Altogether, this
study reveals for the first time that chemokines CCL6 and CCL9 are upregulated early in experimental
CKD, with CCL9-blockade during CKD initiation enhancing kidney inflammation and fibrosis
Phospho-proteomic analyses of B-Raf protein complexes reveal new regulatory principles
B-Raf represents a critical physiological regulator of the Ras/RAF/MEK/ERK-pathway and a pharmacological target of growing clinical relevance, in particular in oncology. To understand how B-Raf itself is regulated, we combined mass spectrometry with genetic approaches to map its interactome in MCF-10A cells as well as in B-Raf deficient murine embryonic fibroblasts (MEFs) and B-Raf/Raf-1 double deficient DT40 lymphoma cells complemented with wildtype or mutant B-Raf expression vectors. Using a multi-protease digestion approach, we identified a novel ubiquitination site and provide a detailed B-Raf phospho-map. Importantly, we identify two evolutionary conserved phosphorylation clusters around T401 and S419 in the B-Raf hinge region. SILAC labelling and genetic/biochemical follow-up revealed that these clusters are phosphorylated in the contexts of oncogenic Ras, sorafenib induced Raf dimerization and in the background of the V600E mutation. We further show that the vemurafenib sensitive phosphorylation of the T401 cluster occurs in trans within a Raf dimer. Substitution of the Ser/Thr-residues of this cluster by alanine residues enhances the transforming potential of B-Raf, indicating that these phosphorylation sites suppress its signaling output. Moreover, several B-Raf phosphorylation sites, including T401 and S419, are somatically mutated in tumors, further illustrating the importance of phosphorylation for the regulation of this kinase
Li14Ln5[Si11N19O5]O2F2 with Ln = Ce, Nd-Representatives of a Family of Potential Lithium Ion Conductors
The isotypic layered oxonitridosilicates Li14Ln5[Si11N19O5]O2F2 (Ln = Ce, Nd) have been synthesized using Li as fluxing agent and crystallize in the orthorhombic space group Pmmn (Z = 2, Li14Ce5[Si11N19O5]O2F2: a = 17.178(3), b = 7.6500(15), c = 10.116(2) Ă
, R1 = 0.0409, wR2 = 0.0896; Li14Nd5 Si11N19O5]O2F2: a = 17.126(2), b = 7.6155 15), c = 10.123(2) Ă
, R1 = 0.0419, wR2 = 0.0929). The silicate layers consist of dreier and sechser rings interconnected via common corners, yielding an unprecedented silicate substructure. A topostructural analysis indicates possible 1D ion migration pathways between five crystallographic independent Li positions. The specific Li-ionic conductivity and its temperature dependence were determined by impedance spectroscopy as well as DC polarization/depolarization measurements. The ionic conductivity is on the order of 5 Ă
10â5 S/cm at 300°C, while the activation energy is 0.69 eV. Further adjustments of the defect chemistry (e.g., through doping)can make these compounds interesting candidates for novel oxonitridosilicate based ion conductors
Instruments for reproducible setting of defects in cartilage and harvesting of osteochondral plugs for standardisation of preclinical tests for articular cartilage regeneration
Vergleichende LymphozytenzÀhlung mit dem Coulter Counter und dem Laser Flare-Cell Meter
Es soll gezeigt werden, ob Lymphozyten mit dem Laser Flare-Cell Meter (LFCM) gemessen werden können. Dabei werden die zeitlichen und rĂ€umlichen Parameter der Messung sowie der valide MeĂbereich untersucht. Um Lymphozyten zu messen, wird Vollblut mit Trennmedien bearbeitet und mit dem LFCM ausgewertet, die Gegenkontrolle erfolgt mit dem Coulter Counter. Reproduzierbare Messungen sind nach Isolierung der Lymphozyten mit Optiprep© möglich. Der Zeitfaktor ist bei LFCM-Messungen vernachlĂ€ssigbar. Es gibt keine signifikanten MeĂabweichungen direkt nach Beschickung der KĂŒvette oder nach 10 min. Die vertikale Anordnung des MeĂpunktes hat keinen EinfluĂ auf das Ergebnis. Bei der horizontalen Positionierung muĂ die Base line des LFCM nach Herstellervorgabe angeordnet werden. Die MeĂwerte der ZellzĂ€hlung verringern sich je weiter das MeĂfenster in die Probe gelegt wird. Das LFCM ermöglicht die LymphozytenzĂ€hlung von 0-500 Z/l, darĂŒber findet eine SĂ€ttigung im MeĂfenster statt
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