Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Modification of liposome surface with polyethylene glycol was used to improve oligodeoxyribonucleotide (ODN) loading, stability of the resulting complexes, and specificity of cellular delivery of ODN by cationic liposomes. Liposomes composed of a cationic lipid (DOTAP, DOGS, DDAB), a neutral lipid (DOPE), and a phospholipid derivative of polyethylene glycol (PEG-PE) formed a complex with 18-mer phosphorothioate up to ODN/lipid molar ratio of 0.25. The complexes showed intact vesicular structures similar to original liposomes and their size (100-130 nm) was unchanged after several weeks of storage, whereas complexes lacking PEG-PE showed progressive aggregation and/or precipitation. After exposure to human plasma, PEG-modified cationic liposomes retained over 60% of the originally bound ODN. PEG-coated complexes resulted in 4-13-fold enhancement of the ODN uptake by human breast cancer cells in serum-supplemented growth medium, relative to free ODN. Complexes containing conjugated anti-HER2 F(ab') fragments at the distal termini of PEG chains efficiently delivered ODN primarily into the cytoplasm and nuclei of HER2 overexpressing cancer cells and greatly enhanced the biological activity of antisense ODN. The development of PEG-modified cationic liposomes may lead to improved ODN potency in vivo.
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PMID:Cationic liposomes coated with polyethylene glycol as carriers for oligonucleotides. 962 54

The c-Mer receptor tyrosine kinase (RTK) is most closely related to chicken c-Eyk and belongs to the Axl RTK subfamily. Although not detected in normal lymphocytes, c-Mer is expressed in B- and T-cell leukemia cell lines, suggesting an association with lymphoid malignancies. To gain an understanding of the role of this receptor in lymphoid cells, we expressed in murine interleukin-3 (IL-3)-dependent Ba/F3 pro-B-lymphocyte cells a constitutively active receptor, CDMer, formed from the CD8 extracellular domain and the c-Mer intracellular domain. Cells transfected with a plasmid encoding the CDMer receptor became IL-3 independent. When tyrosine (Y)-to-phenylalanine (F) mutations were introduced into c-Mer, only the Y867 change significantly reduced the IL-3-independent cell proliferation. The Y867 residue in the CDMer receptor mediated the binding of Grb2, which recruited the p85 phosphatidylinositol 3-kinase (PI 3-kinase). Despite the difference in promotion of proliferation, both the CDMer and mutant F867 receptors activated Erk in transfected cells. On the other hand, we found that both transcriptional activation of NF-kappaB and activation of PI 3-kinase were significantly suppressed with the F867 mutant receptor, suggesting that the activation of antiapoptotic pathways is the major mechanism for the observed phenotypic difference. Consistent with this notion, apoptosis induced by IL-3 withdrawal was strongly prevented by CDMer but not by the F867 mutant receptor.
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PMID:Biological effects of c-Mer receptor tyrosine kinase in hematopoietic cells depend on the Grb2 binding site in the receptor and activation of NF-kappaB. 989 Oct 51

ARH-77 human myeloma cells invade into type I collagen gels but become non-invasive when engineered to express syndecan-1, a heparan sulphate proteoglycan that promotes cell adhesion to collagen. To determine if syndecan-1 expression influences the activity of proteases that may facilitate invasion, we analysed media harvested from syndecan-1 expressing and non-expressing cells. High levels of a 92 kD gelatinase accumulated in serum-free growth medium of both parental and control-transfected ARH-77, but much less 92 kD gelatinase accumulated in the medium of ARH-77 transfectants expressing syndecan-1. The gelatinase was identified as matrix metalloproteinase (MMP)-9 because its activity was immunoprecipitated with a MMP-9-specific monoclonal antibody. Gelatinase activity and Western blot analyses revealed 2-3-fold less MMP-9 in medium from syndecan-1 transfected cells than in medium from parental cells. Decreased MMP-9 was not due to increased association of MMP-9 with cells expressing syndecan-1. An inverse correlation between the syndecan 1 level and the level of MMP-9 accumulation in the media was observed using a panel of ARH-77 transfectants expressing syndecan-1. Investigation of six unrelated human myeloma cell lines confirmed that high gelatinase levels were recovered from conditioned media of those that did not express syndecan-1 (ARH-77, Mer and Col) and one line that expressed a low level of syndecan-1 (RPMI-8226), but low gelatinase levels were recovered from media of lines that expressed high levels of syndecan-1 (ARK and clone 2+). Therefore syndecan-1 may play a dual role in inhibiting the metastasis of tumour cells by promoting cell adhesion to the extracellular matrix and suppressing the proteolytic activity needed for invasion.
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PMID:Syndecan-1 expression suppresses the level of myeloma matrix metalloproteinase-9. 1005 Jul 21

1. Carrier mediated uptake (uptake-1) transport of norepinephrine (NE) plays a key role in the regulation of sympathetic neurotransmission. Recent investigations indicate that nitric oxide (NO) may modulate uptake-1 activity, possibly in a cyclic GMP independent manner. 2. Carrier mediated transport of [(3)H-NE] and [(3)H-dopamine, DA] was examined in CHO cells transfected with cDNA for the NE and DA transporters (NET, DAT) respectively. 3. While exposure to the NO donor S-nitroso-N-acetylpenicillamine (100 microM, SNAP) significantly reduced [(3)H-NE] uptake (P<0.001), no effect on [(3)H-DA] transport was apparent. 4. Comparison of the amino acid sequences for NET and DAT identified cysteine residue 351 in NET, which was not present in DAT. Site-directed mutagenesis of Cys 351 to Ser produced a functional NET that was resistant to the inhibitory effects of SNAP. 5. The presence of SNAP mediated nitrosylation of the cysteine residue in an 8-mer model peptide based around Cys 351 in NET was confirmed by both biochemical and mass spectroscopic means. 6. These data indicate the potential regulatory role for NO in modulating sympathetic neurotransmission, and further confirm the importance of non-cyclic GMP dependent mechanisms in mediating the actions of NO.
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PMID:Nitric oxide mediated modulation of norepinephrine transport: identification of a potential target for S-nitrosylation. 1088 90

Pluripotential hematopoietic stem cells grow in close association with bone marrow stromal cells, which play a critical role in sustaining hematopoiesis in long-term bone marrow cultures. The mechanisms through which stromal cells act to support pluripotential hematopoietic stem cells are largely unknown. This study demonstrates that growth arrest-specific gene-6 (GAS6) plays an important role in this process. GAS6 is a ligand for the Axl (Ufo/Ark), Sky (Dtk/Tyro3/Rse/Brt/Tif), and Mer (Eyk) family of tyrosine kinase receptors and binds to these receptors via tandem G domains at its C terminus. After translation, GAS6 moves to the lumen of the endoplasmic reticulum, where it is extensively gamma-carboxylated. The carboxylation process is vitamin K dependent, and current evidence suggests that GAS6 must be gamma-carboxylated to bind and activate any of the cognate tyrosine kinase receptors. Here, we show that expression of GAS6 is highly correlated with the capacity of bone marrow stromal cells to support hematopoiesis in culture. Nonsupportive stromal cell lines express little to no GAS6, whereas supportive cell lines express high levels of GAS6. Transfection of the cDNA encoding GAS6 into 3T3 fibroblasts is sufficient to render this previously nonsupportive cell line capable of supporting long-term hematopoietic cultures. 3T3 cells, genetically engineered to stably express GAS6 (GAS6-3T3), produce a stromal layer that supports the generation of colony-forming units in culture (CFU-c) for up to 6 wk. Hematopoietic support by genetically engineered 3T3 is not vitamin K dependent, and soluble recombinant GAS6 does not substitute for coculturing the hematopoietic progenitors with genetically modified 3T3 cells.
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PMID:Hematopoietic progenitor cells grow on 3T3 fibroblast monolayers that overexpress growth arrest-specific gene-6 (GAS6). 1105 Feb 45

We have constructed a series of 22 phosphorothioate 20-mer antisense oligonucleotides directed against different regions of the human (EGFR) mRNA. Treatment with EGFR antisense oligonucleotides showed a dose-dependent inhibition of human GEO colon cancer cell growth in soft agar. Western blot analysis demonstrated a significant reduction in EGFR expression after treatment with each EGFR antisense oligonucleotide. The ability to inhibit GEO anchorage-independent growth, however, varied among the EGFR antisense sequences with an IC(50) ranging between 0.5 and 3.5 microM. Two of these antisense oligonucleotides targeting the regions between 2457-2476 and 614-4633 bases of the human EGFR mRNA have been modified as hybrid DNA/RNA mixed backbone oligonucleotides (MBO) to examine their anticancer properties in vivo. The 2 EGFR antisense MBOs retained the same biological properties of the fully phosphorothioate EGFR antisense oligonucleotides targeting the same EGFR mRNA sequences, such as blocking EGFR synthesis, inhibiting cell growth and enhancing programmed cell death in human cancer cell lines that express functional EGFRs. Furthermore, a potentiation in the growth inhibitory effect on GEO cancer cells was observed after treatment with these EGFR antisense MBOs in combination with cytotoxic drugs, including cisplatin, doxorubicin, paclitaxel, or topotecan. These results show the antiproliferative activity of specific EGFR antisense oligonucleotides and allow to identify novel EGFR antisense MBOs that deserve further evaluation as potential selective anticancer agents alone or in combination with cytotoxic drugs in human carcinomas that express functional EGFRs.
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PMID:Antisense oligonucleotides targeting the epidermal growth factor receptor inhibit proliferation, induce apoptosis, and cooperate with cytotoxic drugs in human cancer cell lines. 1141 Aug 62

Recognition of the essential role of dendritic cells (DCs) as professional antigen-presenting cells has prompted investigators to search for methods to use DCs as natural adjuvants in immunotherapy. A number of antigenic oligopeptides, recognized by CD8(+) cytotoxic T lymphocytes (CTLs) specific for cancer cells, have been applied in clinical trials using DCs. Such a monovalent vaccine with a single epitope for a particular type of HLA class 1 molecule would be effective. However, a polyvalent vaccine might be more potent. We designed a novel protein delivery system consisting of hydrophobized polysaccharides complexed with target proteins. The truncated HER2 protein encompassing 147 N-terminal amino acids, including the 9-mer HER2p63-71 peptide (HER2p63), TYLPTNASL, the human homologue of an antigenic murine tumor rejection peptide, was prepared. We report here that HLA-A2402(+) DCs could incorporate hydrophobized polysaccharide-truncated HER2 protein complexes and process the protein to present major histocompatibility complex class 1-binding HER2p63 peptide. The complexes enter DCs by phagocytosis, and then the truncated protein is processed through a pathway similar to that for endogenous proteins. DCs sensitized by these complexes primed and boosted HER2p63-specific CD8(+) T cells in the context of HLA-A2402. Vaccination with DCs incorporating these complexes completely suppressed lung metastases in a HER2-expressing murine tumor model. We also generated 3 CD4(+) clones reactive with different HER2- derived 25-mer peptides from lymph node cells in mice treated with CHP/HER2-147. Thus, hydrophobized polysaccharide-protein complexes are promising candidates for the construction of polyvalent vaccines.
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PMID:Presentation of a major histocompatibility complex class 1-binding peptide by monocyte-derived dendritic cells incorporating hydrophobized polysaccharide-truncated HER2 protein complex: implications for a polyvalent immuno-cell therapy. 1198 28

We isolated a cDNA encoding the Xenopus member of Sky/Axl/Mer receptor tyrosine kinase family (referred as Sky family), termed Xksy. The predicted Xksy protein has conserved structural characteristics of the Sky family: an unique extracellular domain of two immunoglobulin (Ig)-like repeats, two fibronectin type III (FNIII)-like repeats and an intracellular tyrosine kinase. Homology analysis of Xksy showed the highest identity to mammalian Sky protein. In contrast to the predominant expression of sky mRNA in the adult mammalian nervous system, Northern blot analysis showed ubiquitous expression of a single 5.2-kb Xksy mRNA in tissues of the adult Xenopus. RNase protection assays revealed that, during development, Xksy mRNA is expressed from mid neurulation stage. Levels increase through the tadpole stage and become restricted to the head region in embryos by stage 40. Whole-mount in situ hybridization analyses revealed that expression of Xksy is localized to the nervous system of the tadpole stage, including origins of sensory organs and branchial arches. When a chimeric receptor (EGFR-Xksy), composed of the extracellular region of epidermal growth factor (EGF) receptor and the transmembrane/intracellular regions of Xksy, was expressed in a doxycycline repressive manner in HEK 293 cells, EGF-stimulus without doxycycline induced tyrosine phosphorylation of the chimeric receptor and evoke morphological changes. EGF treatment also induced growth modifications of EGFR-Xksy cells. And doxycycline pre-treatment eliminated these activities. These findings suggest that Xksy may play an important role in growth, differentiation and the accurate migration of cells during embryogenesis and early neural development.
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PMID:Molecular cloning, expression and partial characterization of Xksy, Xenopus member of the Sky family of receptor tyrosine kinases. 1203 91

The phagocytosis of photoreceptor outer segments by retinal pigment epithelial (RPE) cells plays a critical role in preserving normal retinal function. Recently the receptor protein tyrosine kinase (RTK) Mer, has been shown to be necessary for this cellular process to take place. Gas6, the ligand for the Mer RTK, can specifically and selectively stimulate the phagocytosis of photoreceptor outer segments (OS) by normal cultured rat RPE cells, as we have previously reported. The Gas6 protein has been shown to associate with plasma membrane phosphatidylserine by its amino-terminal portion, while its carboxyl-terminal portion can bind and activate Mer and its related RTKs, Axl and Tyro-3. Given the capability of Gas6 to interact with more than one molecule, we have performed a series of experiments to further dissect the interactions of Gas6 with the OS and RPE and to determine the specific calcium requirements necessary for Gas6 to exert its stimulatory effect on phagocytosis. These experiments show that Gas6 must bind to OS before the stimulation of OS ingestion can occur and that this binding requires a Ca(2+) concentration of 500-600 microM. The same Ca(2+) concentration is required for the Gas6 mediated stimulation of OS ingestion. We further demonstrate that in order to bind to OS and to stimulate OS phagocytosis, Gas6 requires gamma-carboxylation in a vitamin K-dependent reaction. By analogy with other systems, we propose that Ca(2+) mediates the linkage between the gamma-carboxyglutamic acid (Gla)-rich N-terminal region of Gas6 with phospholipids, presumably phosphatidylserine, in the plasma membrane of the OS. Only after this binding has occurred can Gas6 interact with receptor molecule(s) on the surface of the RPE, and activate RPE cell signaling pathways leading to OS ingestion. These studies further underscore the importance of Gas6 in the phagocytic function of the RPE and open new avenues of investigation to understand the molecular events mediated and triggered by Gas6, and its interaction with the OS and RPE.
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PMID:Gas6 binding to photoreceptor outer segments requires gamma-carboxyglutamic acid (Gla) and Ca(2+) and is required for OS phagocytosis by RPE cells in vitro. 1238 86

Inappropriate expression of fibroblast growth factors (FGFs) or activation of FGF receptors (FGFRs) could contribute to several human angiogenic pathologies. In an attempt to design antagonists of FGF, we developed a screening procedure for identifying peptide ligands binding to FGFR1. To retain the natural conformation of FGFR1 during screening, we expressed recombinant FGFR1 on the surface of Sf9 insect cells. A 6-mer phage display peptide library was then screened on the cell surface and a group of hydrophobic peptide sequences were identified. Further experiments demonstrated that the phages displaying these sequences can specifically bind to FGFR1. The docking analysis suggests that the peptide ValTyrMetSerProPhe can specifically bind to the hydrophobic surface of FGFR1. The synthetic peptide Ac-ValTyrMetSerProPhe-NH2 can inhibit mitogenic activity of aFGF and has the potential to become a therapeutic agent as an aFGF antagonist.
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PMID:Selection of peptide ligands binding to fibroblast growth factor receptor 1. 1244 May 21


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