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.

Epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells is one of the causative mechanisms of kidney fibrosis. In our study, we screened lipophilic compounds using a lipid library including approximately 200 lipids to identify those that suppressed EMT induced by a transforming growth factor (TGF)-β1 stimulus. Initial screening was performed with the immortalized HK-2 renal tubule epithelial cell line. The most promising compounds were further tested in RPTEC primary renal tubule epithelial cells. We found that the synthetic lipid AM251 suppressed two hallmark events associated with EMT, the upregulation of collagen 1A1 (COL1A1) and downregulation of E-cadherin. Though AM251 is known to act as an antagonist for the cannabinoid receptor type 1 (CB1) and an agonist for the G protein-coupled receptor 55 (GRP55), the suppression of EMT by AM251 was not mediated through either receptor. Microarray analyses revealed that AM251 inhibited induction of several EMT transcription factors such as SNAIL1, which is the key inducer of EMT, and the AP-1 transcription factors FOSB and JUNB. Activation of SMAD2/3 and p38 mitogen-activated protein kinase (MAPK) was inhibited by AM251, with greater inhibition of the latter, indicating that AM251 acted upstream of SMAD/p38 MAPK in the TGF-β signaling pathway. Our findings regarding the effects of AM251 on the TGF-β signaling pathway may inform development of a novel therapeutic agent suppressing EMT, thus preventing kidney fibrosis.

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.

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.

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, K-d = 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.

Differential hybridization screening of the cDNA libraries derived from the rat facial nucleus was performed, and a number of cDNA clones were isolated and found to be upregulated after facial nerve axotomy. One of the isolated cDNA clones encoded the A‐chain of C1q (C1q‐A) of the rat, whose cloning has not been reported in the literature. Sequence analysis showed that C1q‐A is well conserved in the rat, mouse and human on the DNA and protein levels. In situ hybridization demonstrated that there were strong signals for C1q‐A mRNA within the rat facial nucleus damaged by axotomy, and the signals were localized in activated microglia. Immunohistochemistry showed strong immunoreactivity for C1q protein in the activated microglia of the rat facial nucleus damaged by axotomy. C1q‐immunoreactivity was also found in the extracellular space of the damaged facial nucleus. No significant C1q‐A mRNA signals and C1q‐immunoreactivity were observed in the normal brain tissue. These results suggest that microglia markedly up‐regulate the C1q‐A gene to produce and secrete C1q in response to the neuronal damage caused by axotomy.

In fetal mammalian heart, constitutive adenylyl cyclase/cyclic AMP-dependent protein kinase A (cAMP-PKA)-mediated phosphorylation, independent of β-adrenergic receptor stimulation, could under such circumstances play an important role in sustaining the L-type calcium channel current (ICa,L) and regulating other PKA dependent phosphorylation targets. In this study, we investigated the regulation of L-type Ca2+ channel (LTCC) in murine embryonic ventricles. The data indicated a higher phosphorylation state of LTCC at early developmental stage (EDS, E9.5–E11.5) than late developmental stage (LDS, E16.5–E18.5). An intrinsic adenylyl cyclase (AC) activity, PKA activity and basal cAMP concentration were obviously higher at EDS than LDS. The cAMP increase in the presence of isobutylmethylxanthine (IBMX, nonselective phosphodiesterase inhibitor) was further augmented at LDS but not at EDS by chelation of intracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA)-acetoxymethyl ester (BAPTA-AM). Furthermore, ICa,L increased with time after patch rupture in LDS cardiomyocytes dialyzed with pipette solution containing BAPTA whereas not at EDS. Thus we conclude that the high basal level of LTCC phosphorylation is due to the high intrinsic PKA activity and the high intrinsic AC activity at EDS. The latter is possibly owing to the little or no effect of Ca2+ influx via LTCCs on AC activity, leading to the inability to inhibit AC.

A MAb 9F6 was capable of staining HPV16 E7 in a human cervical carcinoma line, CaSki, and rat 3Y1 cells stably expressing HPV16 E7 gene. Contrary to the current understanding of E7 as a nuclear protein, the site of staining was clearly cytoplasmic. The subcellular localization of E7 was further studied by using the beta-galactosidase (beta-gal) receptor method. A fusion protein composed of E7 and beta-gal was stably expressed in rat 3Y1 cells. The beta-gal activity in these cells was detected mostly in the nucleus, even though 9F6 still stained the cytoplasm of these cells. The fusion protein was also found to be oncogenic since transfected 3Y1 cells acquired transformed phenotypes such as increased saturation density and anchorage-independent growth. These results indicate that biologically active E7 exists mostly in the nucleus, but nuclear E7 is masked from 9F6. A series of deletion mutants of E7 further demonstrated that the amino acid sequence from 16 to 41 was enough to transport beta-gal into the nucleus. A mutation either at amino acid 24 or 26 which is known to disrupt the binding of E7 to RB, the retinoblastoma gene product, did not strongly affect the nuclear localization of the fusion protein, suggesting that the nuclear transportation of E7 is mostly independent of RB binding.

C33 Ag (CD82) is a member of the transmembrane 4 superfamily (TM4SF) and an activation Ag of T-cells. Recent studies have shown that CD82 associates with CD4 or CD8 and delivers costimulatory signals for the TCR/CD3 pathway. We have isolated cDNA and genomic clones of mouse CD82. Mouse CD82 has 266 amino acid residues with 76% identity to human CD82. The mouse CD82 gene consists of nine exons and spans more than 20 kb of genomic DNA. The genomic organization of CD82 is quite similar to that of three other TM4SF members whose genomic structures were described, i.e., Tapa-1 (CD81), CD53, and CD63. By mapping the 5′ end of CD82 transcripts, we found a single major transcription initiation site 144 bp upstream of the ATG initiation codon. We also determined the sequence of the 5′ flanking region of CD82 gene for about 2 kb. The 5′ flanking sequence has a housekeeping promoter with potential binding motifs for various transcriptional factors. Northern blot analysis showed quite variable expression of the CD82 gene among different organs. The highest expression was seen in the spleen and the kidney. The expression was low in skeletal muscle and hardly detectable in the heart. Northern blot analysis was also carried out for CD81, CD53, and CD63. The expression of the CD81 gene was ubiquitous and similar among different organs, while that of CD53 was seen only in the spleen. The expression of the CD63 gene was ubiquitous, with the highest expression in the kidney. These results together with the comparison of the structures of 5′ flanking sequences of these genes indicate distinct regulations of gene expression for these four members of TM4SF.

To identify the cellular receptors and other cell surface molecules playing essential roles in the transmission of human T-cell leukemia virus type 1 (HTLV-1), we have been isolating monoclonal antibodies (mAbs) that are capable of inhibiting HTLV-1-induced syncytium formation. In the present study, we isolated two mAbs, H11 (IgM) and H14 (IgG1), inhibitory to syncytium formation in the coculture of TOM-1 or C91/PL (both HTLV-1-positive human T-cell lines) and MOLT-4/8 (HTLV-1-negative human T-cell line) by immunizing the membrane fraction of human osteosarcoma line HOS. By immunoprecipitation and immunoblotting, H11 and H14 were found to be specific for MHC class I heavy chain and beta 2-microglobulin (beta 2 M), respectively. Among the four commercially obtained mAbs, two mAbs for MHC class I antigen and two mAbs to beta 2 M, one mAb to MHC class I antigen and one mAb to beta 2 M were also found to be inhibitory to the syncytium formation. The functional comparison of these mAbs revealed that the syncytium-inhibitory mAbs induced strong homotypic cell adhesion particularly in the HTLV-1-positive T-cell lines. This cell adhesion was dependent on temperature, energy metabolism, and microfilament function but not on the activity of protein kinase C or divalent cations. These results suggest a novel type of LFA-1-independent cell adhesion induced by signal transduction via MHC class I antigen.