Search results for "COS Cells"
showing 10 items of 111 documents
Phosphorylation of the DNA repair protein APE/REF-1 by CKII affects redox regulation of AP-1
1999
The DNA repair protein apurinic endonuclease (APE/Ref-1) exerts several physiological functions such as cleavage of apurinic/apyrimidinic sites and redox regulation of the transcription factor AP-1, whose activation is part of the cellular response to DNA damaging treatments. Here we demonstrate that APE/Ref-1 is phosphorylated by casein kinase II (CKII). This was shown for both the recombinant APE/Ref-1 protein (Km=0.55 mM) and for APE/Ref-1 expressed in COS cells. Phosphorylation of APE/Ref-1 did not alter the repair activity of the enzyme, whereas it stimulated its redox capability towards AP-1, thus promoting DNA binding activity of AP-1. Inhibition of CKII mediated phosphorylation of A…
Overlapping peptides of melanocyte differentiation antigen melan-A/MART-1 recognized by autologous cytolytic T lymphocytes in association with HLA-B4…
1998
From the peripheral blood lymphocytes (PBLs) of melanoma patient SK29(AV) we have previously isolated 2 independent cytolytic T lymphocyte (CTL) clones (CTL7/147 and CTL13/211), which lysed autologous tumor cells in association with HLA-B45.1. As demonstrated here, both CTL clones were directed against melanocyte differentiation antigen Melan-A/MART-1, which also was recognized by HLA-A2.1-restricted CTLs from the same patient. By generating and transfecting 3'-deletion mutants of Melan-A/MART-1 cDNA, we localized its peptide-coding regions. The HLA-B45.1-presented peptides were derived from a hydrophobic region of the protein and largely overlapped the peptides recognized by CTLs from the …
High frequency of a non-functional TAP1/LMP2 promoter polymorphism in human tumors
2002
The Tap1 and Tap2 genes encoding for a heterodimeric peptide transporter play a key role in antigen processing and presentation. The TAP complex mediates the transport of peptides generated by the IFN-gamma-inducible proteasome subunits LMP2, 7 and 10 from the cytosol into the endoplasmic reticulum (ER), where they bind to MHC class I molecules. In contrast to the frequent polymorphisms within the rat Tap genes which exert functional differences, polymorphic regions within the human Tap genes have been demonstrated, but not systematically analyzed in terms of their functional significance. Both the Tap1 and Lmp2 genes are transcribed from a bidirectional intergenic promoter which is regulat…
Immunoadhesins of interleukin-6 and the IL-6/soluble IL-6R fusion protein hyper-IL-6.
1999
Signal transduction in response to interleukin-6 (IL-6) results from homodimerization of gp130. This dimerization occurs after binding of IL-6 to its surface receptor (IL-6R) and can also be triggered by the complex of soluble IL-6R and IL-6. We fused IL-6 to the constant region of a human IgG1 heavy chain (Fc). IL-6Fc was expressed in COS-7 cells and purified via Protein A Sepharose. Using three different assays we found that the biological activity of this dimeric IL-6 protein is comparable with monomeric IL-6. Recently, we described the designer cytokine Hyper-IL-6 (H-IL-6) in which soluble IL-6R and IL-6 are connected via a flexible peptide linker. This molecule turned out to be 100-100…
Interaction of Neuronal Calcium Sensor-1 (NCS-1) with Phosphatidylinositol 4-Kinase β Stimulates Lipid Kinase Activity and Affects Membrane Trafficki…
2001
Phosphatidylinositol 4-kinases (PI4K) catalyze the first step in the synthesis of phosphatidylinositol 4,5-bisphosphate, an important lipid regulator of several cellular functions. Here we show that the Ca(2+)-binding protein, neuronal calcium sensor-1 (NCS-1), can physically associate with the type III PI4Kbeta with functional consequences affecting the kinase. Recombinant PI4Kbeta, but not its glutathione S-transferase-fused form, showed enhanced PI kinase activity when incubated with recombinant NCS-1, but only if the latter was myristoylated. Similarly, in vitro translated NCS-1, but not its myristoylation-defective mutant, was found associated with recombinant- or in vitro translated P…
Cell fate specification in an in vitro model of neural development
1998
We have studied in an in vitro model of neural development the effect of neighboring cells on the fate of single fluorescently labeled precursor cells. In one line of experiments, PCC7-Mz1 embryonal carcinoma cells were transiently transfected with "green fluorescent protein" (GFP) and, following incubation with 0.1 microM all-trans retinoic acid (RA), the number and morphology of derivatives (neuronal or non-neuronal) was determined that form groups of GFP-expressing cells in a surrounding of unlabeled cells. Because single PCC7-Mz1 cells can produce single-lineage and mixed-lineage derivatives, they are individually pluripotent. In another line of experiments, we have analyzed the fate of…
Subcellular targeting of multiligand-binding protein gC1qR.
1999
Abstract gC1q receptor, a protein originally described as the cell surface receptor for the globular heads of complement factor C1q, has been found to bind human H-kininogen with high affinity and specificity. Therefore, gC1qR has been considered candidate kininogen docking site on the surfaces of platelets, neutrophils and endothelial cells. Recent work demonstrating that gC1qR is an intracellular protein that is tightly associated with mitochondria rather than targeted to the cell surface has challenged this view. To further probe cellular trafficking routes of gC1qR, we overexpressed human gC1qR in a mammalian cell and monitored cell surface exposure of recombinant gC1qR by virtue of its…
Comm Sorts Robo to Control Axon Guidance at the Drosophila Midline
2002
AbstractAxon growth across the Drosophila midline requires Comm to downregulate Robo, the receptor for the midline repellent Slit. We show here that comm is required in neurons, not in midline cells as previously thought, and that it is expressed specifically and transiently in commissural neurons. Comm acts as a sorting receptor for Robo, diverting it from the synthetic to the late endocytic pathway. A conserved cytoplasmic LPSY motif is required for endosomal sorting of Comm in vitro and for Comm to downregulate Robo and promote midline crossing in vivo. Axon traffic at the CNS midline is thus controlled by the intracellular trafficking of the Robo guidance receptor, which in turn depends…
The Process-inducing Activity of Transmembrane Agrin Requires Follistatin-like Domains
2009
Clustering or overexpression of the transmembrane form of the extracellular matrix proteoglycan agrin in neurons results in the formation of numerous highly motile filopodia-like processes extending from axons and dendrites. Here we show that similar processes can be induced by overexpression of transmembrane-agrin in several non-neuronal cell lines. Mapping of the process-inducing activity in neurons and non-neuronal cells demonstrates that the cytoplasmic part of transmembrane agrin is dispensable and that the extracellular region is necessary for process formation. Site-directed mutagenesis reveals an essential role for the loop between beta-sheets 3 and 4 within the Kazal subdomain of t…
RPGR ORF15 isoform co-localizes with RPGRIP1 at centrioles and basal bodies and interacts with nucleophosmin
2005
The ORF15 isoform of RPGR (RPGR(ORF15)) and RPGR interacting protein 1 (RPGRIP1) are mutated in a variety of retinal dystrophies but their functions are poorly understood. Here, we show that in cultured mammalian cells both RPGR(ORF15) and RPGRIP1 localize to centrioles. These localizations are resistant to the microtubule destabilizing drug nocodazole and persist throughout the cell cycle. RPGR and RPGRIP1 also co-localize at basal bodies in cells with primary cilia. The C-terminal (C2) domain of RPGR(ORF15) (ORF15(C2)) is highly conserved across 13 mammalian species, suggesting that it is a functionally important domain. Using matrix-assisted laser desorption ionization time-of-flight mas…