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1.
Metabolism ; 65(1): 16-25, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26683793

ABSTRACT

OBJECTIVE: Although peroxisome proliferator-activated receptor (PPAR) δ agonists have been shown to improve the serum lipoprotein profiles in humans, the impact of the changes in these lipoprotein profiles on atherosclerosis remains to be elucidated. The aim of this study was to investigate the relationship between the selective PPARδ agonist-induced alterations of serum lipoprotein profiles and the development of atherosclerosis in human apolipoprotein B100 and cholesterol ester transfer protein double transgenic (hApoB100/hCETP-dTg) mice with human-like hypercholesterolemic dyslipidemia. METHODS: hApoB100/hCETP-dTg mice fed an atherogenic diet received a novel PPARδ agonist (PYPEP) or vehicle for 18 weeks, followed by evaluation of atherosclerosis. Serum samples were collected during the treatment period at least at 3-week intervals to determine the lipoprotein levels and the levels of an inflammatory marker, macrophage chemotactic protein-1 (MCP-1), and to analyze the lipoprotein profile by fast protein liquid chromatography. The cholesterol efflux capacity of high-density lipoprotein (HDL) was examined using [(3)H]-cholesterol labeled macrophages. RESULTS: Compared with vehicle treatment, PYPEP treatment caused increases in the serum levels of HDL cholesterol and apolipoprotein A-I (ApoA-I), as well as reductions in the serum non-HDL cholesterol and MCP-1 levels. The HDL fraction from the PYPEP-treated group maintained its cholesterol efflux capacity and showed an increased population of smaller HDL particles. PYPEP substantially suppressed atherosclerotic lesion progression, and the lesion areas had significant correlations with non-HDL cholesterol, HDL cholesterol, ApoA-I and MCP-1 by Pearson's correlation analysis. A multiple regression analysis revealed that non-HDL cholesterol and ApoA-I were significantly associated with the atherosclerotic lesion area. CONCLUSION: A novel PPARδ agonist, PYPEP, suppressed atherosclerotic lesion progression by improving the serum lipoprotein profiles, including increased levels of ApoA-I and functional HDL particles, as well as a reduced non-HDL cholesterol level, in hApoB100/hCETP-dTg mice with human-like hypercholesterolemic dyslipidemia.


Subject(s)
Apolipoprotein B-100/genetics , Atherosclerosis/prevention & control , Cholesterol Ester Transfer Proteins/genetics , PPAR delta/agonists , Piperidines/pharmacology , Pyrrolidines/pharmacology , Animals , Apolipoprotein A-I/blood , Atherosclerosis/blood , Chemokine CCL2/blood , Female , Humans , Lipoproteins, HDL/blood , Mice , Mice, Transgenic
2.
Anal Biochem ; 380(2): 249-56, 2008 Sep 15.
Article in English | MEDLINE | ID: mdl-18570884

ABSTRACT

The expression of recombinant proteins is a well-accepted technology, but their detection and purification often require time-consuming and complicated processes. This paper describes the development of a novel double epitope tag (GEPGDDGPSGAEGPPGPQG) for rapid and accurate quantification of recombinant protein by a homogeneous immunoassay based on fluorescence resonance energy transfer. In our double epitope tagging system, recombinant proteins can be simply measured on a microtiter plate by addition of a pair of fluorophore-labeled monoclonal antibodies (their epitopes; GEPGDDGPS and GPPGPQG). The sensitivity of the immunoassay with an incubation time of only 5 min is almost equal to that of labor-intensive Western blotting. In addition, culture media and extracts of host cells generally used for protein expression have little effect on this immunoassay. To investigate the utility of our proposed tag for protein production, several different proteins containing this tag were practically expressed and purified. The data presented demonstrate that the double epitope tag is a reliable tool that can alleviate the laborious and troublesome processes of protein production.


Subject(s)
Epitopes/chemistry , Fluorescence Resonance Energy Transfer/methods , Peptides/chemistry , Recombinant Proteins/analysis , Amino Acid Sequence , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Blotting, Western , Collagen Type II/immunology , Epitopes/analysis , Humans , Immunoassay/methods , Immunoglobulin Fragments/analysis , Immunoglobulin Fragments/biosynthesis , Immunoglobulin Fragments/chemistry , Immunoglobulin Fragments/genetics , Molecular Sequence Data , Organometallic Compounds/chemistry , Peptides/immunology , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Spermidine Synthase/biosynthesis , Spermidine Synthase/chemistry , Spermidine Synthase/genetics
3.
Neuropharmacology ; 51(4): 727-36, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16806303

ABSTRACT

During the course of neuronal development or regeneration, the axonal growth cone protein growth-associated protein 43 (GAP-43) is expressed in a great majority of differentiating neurons, suggesting that the regulation of this gene is tied to important differentiation signals common to many neurons. In order to discover non-peptide molecules capable of mimicking the effects of NGF, we developed a reporter gene assay system based on measurement of light production in PC12 cells stably transfected with the luciferase reporter gene, the expression of which depends on the transcriptional activation of GAP-43. High throughput screening of the proprietary compound collection using this system revealed (E,E)-1-[5-(3,4-dihydroxyphenyl)-1-oxo-2,4-pentadienyl]piperidine (HU0622), a piperine derivative, to be an activator of GAP-43 transcription. HU0622 strongly induced neurite outgrowth and extension in PC12 and sensory neuronal cultures of chick dorsal root ganglia. The compound induced sustained extracellular signal-regulated kinase (ERK) activation that is crucial for neurite outgrowth activity without activating NGF receptor, TrkA. Furthermore, HU0622 as well as NGF promoted PC12 survival under serum-free conditions and activated Akt/protein kinase B downstream from phosphatidylinositol 3-kinase (PI3K). HU0622 also promoted survival of rat dorsal root ganglion neurons deprived of NGF. HU0622, a small non-peptidyl molecule, may be a novel promising lead compound for the stimulation of nerve regeneration.


Subject(s)
Alkaloids/pharmacology , GAP-43 Protein/metabolism , Nerve Growth Factors/pharmacology , Neurons/drug effects , Piperidines/pharmacology , Alkaloids/chemistry , Animals , Benzodioxoles , Cell Survival/drug effects , Cells, Cultured , Chick Embryo , Dose-Response Relationship, Drug , Drug Interactions , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Extracellular Signal-Regulated MAP Kinases/metabolism , Flavonoids/pharmacology , GAP-43 Protein/genetics , Ganglia, Spinal/cytology , Immunohistochemistry/methods , Luciferases/metabolism , Neurites/drug effects , Neurons/cytology , Neurons/metabolism , Piperidines/chemistry , Polyunsaturated Alkamides , Rats , Transfection/methods , Tubulin/metabolism
4.
J Biol Chem ; 280(40): 34268-77, 2005 Oct 07.
Article in English | MEDLINE | ID: mdl-16055446

ABSTRACT

We found that a monokine induced by interferon-gamma (Mig, CXCL9), which belongs to the CXC chemokine subfamily, acts as a neurotrophic factor on PC12 cells and rat primary sympathetic neurons. PC12 cells were shown to express a single class of high affinity binding sites for Mig (670 receptors/cell, Kd = 2.9 nm). Mig induced neurite outgrowth in PC12 cells in a dose-dependent manner. Comparison of extracellular signal-regulated kinase signaling pathways between Mig and nerve growth factor (NGF) revealed that these pathways are crucial for Mig action as well as NGF. K252a, an inhibitor of tyrosine autophosphorylation of tyrosine kinase receptors (Trks) did not inhibit the action of Mig, suggesting that Mig action occurs via a different receptor from that of NGF. Furthermore, Mig as well as NGF promoted PC12 survival under serum-free conditions and activated Akt/protein kinase B downstream from phosphatidylinositol 3-kinase (PI3K). Because the PI3K inhibitor LY294002 prevented the Mig- and NGF-induced survival effect, this effect is probably mediated by the PI3K signaling pathway. Mig also promoted survival of rat primary sympathetic neurons that die when deprived of NGF. These results suggest that chemokines, including Mig (CXCL9) have neurotrophic effects on the nervous system.


Subject(s)
Chemokines, CXC/physiology , Interferon-gamma/physiology , Nerve Growth Factors/physiology , Animals , Antibodies , Blotting, Western , Cell Differentiation , Cell Survival , Chemokine CXCL9 , Dose-Response Relationship, Drug , Humans , Microscopy, Confocal , Neurites , Neurons , PC12 Cells , Phosphatidylinositol 3-Kinases/metabolism , Rats , Signal Transduction , Sympathetic Nervous System
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