TE671 Cells Research Articles

Actions of piperidine alkaloid teratogens at fetal nicotinic acetylcholine receptors

Teratogenic alkaloids are found in many species of plants including Conium maculatum L., Nicotiana glauca, Nicotiana tabaccum, and multiple Lupinus spp. Fetal musculoskeletal defects produced by alkaloids from these plants include arthrogyropisis, scoliosis, torticollis, kyposis, lordosis, and cleft palate. A pharmacodynamic comparison of the alkaloids ammodendrine, anabasine, anabaseine, anagyrine, and coniine in SH-SY5Y cells and TE-671 cells was made. These alkaloids and their enantiomers were more effective in depolarizing TE-671 cells which express the human fetal-muscle type nicotinic acetylcholine receptor (nAChR) relative to SH-SY5Y cells which predominately express autonomic nAChRs. The rank order of potency in TE-671 cells was: anabaseine > (+)-anabasine > (−)-anabasine > (±)-anabasine > anagyrine > (−)-coniine > (±)-coniine > (+)-coniine > (±)-ammodendrine > (+)-ammodendrine. The rank order potency in SH-SY5Y cells was: anabaseine > (+)-anabasine > (−)-coniine > (+)-coniine > (+)-ammodendrine > anagyrine > (−)-anabasine > (±)-coniine > (±)-anabasine > (−)-ammodendrine. The actions of these alkaloids at nAChRs in both cell lines could be distinguished by their maximum effects in depolarizing cell membrane potential. The teratogenic action of these compounds may be related to their ability to activate and subsequently desensitize nAChRs.

Reactivity of Zn-, Cd-, and Apo-Metallothionein with Nitric Oxide Compounds: In Vitro and Cellular Comparison

The reactivity of Zn(7)- and Cd(7)-metallothionein (MT) with S-nitrosopenicillamine (SNAP), S-nitrosoglutathione (GSNO), and 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA/NO) was investigated to explore the hypothesis that metallothionein is a signficant site of cellular reaction of nitric oxide or NO compounds. Zn(7)-MT reacted with SNAP or GSNO only under aerobic conditions and in the presence of light, which stimulates the decomposition of S-nitrosothiolates to NO. Zn(2+) is released, and protein thiols are modified. DEA/NO, which degrades spontaneously to release NO, also reacted with Zn(7)-MT only when oxygen was present. Anaerobically, DEA/NO reacted with Zn(7)-MT in the presence of 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, which converts NO to NO(2). Glutathione competed effectively with Zn(7)-MT for reactive nitrogen oxide species in reaction mixtures. Reaction of Cd(7)-MT with SNAP also required oxygen and light to react. In this case, only a fraction of the Cd(2+) bound to Cd(7)-MT was displaced by SNAP. Apo-metallothionein was much more reactive with SNAP and DEA-NO than Zn(7)- or Cd(7)-MT. TE671 and LLC-PK(1) cell lines were incubated with DEA/NO to examine the role that MT might play in the cellular reactions of this NO donor compound. Incubation of cells with 0-80 microM Zn(2+) for 24 h resulted in progressively increasing concentrations of Zn-unsaturated MT. One hour of cellular exposure to a range of DEA/NO concentrations followed by 24 h of incubation caused no evident acute toxicity at less than 0.45 mM. Preinduction of MT did not alter this response. The effects of DEA/NO on proteomic, metallothionein, and low molecular weight (LMW) thiol pools, including glutathione (GSH), were measured. Substantial fractions of the proteomic and LMW thiol pools underwent reaction with little dislocation of Zn(2+). In addition, one-third of the MT thiol pool reacted without labilizing any of the bound Zn(2+). These results demonstrated that it was free thiols associated with MT that reacted with DEA/NO not those bound to Zn(2+). Moreover, under the conditions of the experiments, DEA/NO reacted with the spectrum of cellular thiols in proportion to their fraction in the cytosol and did not preferentially react with MT sulfhydryl groups.

Chemical Stimulation of Adherent Cells by Localized Application of Acetylcholine from a Microfluidic System

Chemical stimulation of cells is inherently cell type selective in contrast to electro-stimulation. The availability of a system for localized application of minute amounts of chemical stimulants could be useful for dose related response studies to test new compounds. It could also bring forward the development of a novel type of neuroprostheses. In an experimental setup microdroplets of an acetylcholine solution were ejected from a fluidic microsystem and applied to the bottom of a nanoporous membrane. The solution traveled through the pores to the top of the membrane on which TE671 cells were cultivated. Calcium imaging was used to visualize cellular response with temporal and spatial resolution. Experimental demonstration of chemical stimulation for both threshold gated stimulation as well as accumulated dose–response was achieved by either employing acetylcholine as chemical stimulant or applying calcein uptake, respectively. Numerical modeling and simulation of transport mechanisms involved were employed to gain a theoretical understanding of the influence of pore size, concentration of stimulant and droplet volume on the spatial-temporal distribution of stimulant and on the cellular response. Diffusion, pressure driven flow and evaporation effects were taken into account. Fast stimulation kinetic is achieved with pores of 0.82 μm diameter, whereas sustained substance delivery is obtained with nanoporous membranes. In all cases threshold concentrations ranging from 0.01 to 0.015 μM acetylcholine independent of pore size were determined.

GD3 Accumulation in Cell Surface Lipid Rafts Prior to Mitochondrial Targeting Contributes to Amyloid-β-induced Apoptosis

Neuronal apoptosis induced by amyloid β-peptide (Aβ) plays an important role in the pathophysiology of Alzheimer's disease (AD). However, the molecular mechanism underlying Aβ-induced apoptosis remains undetermined. The disialoganglioside GD3 involves ceramide-, Fas- and TNF-α-mediated apoptosis in lymphoid cells and hepatocytes. Although the implication of GD3 has been suggested, the precise role of GD3 in Aβ-induced apoptosis is still unclear. Here, we investsigated the changes of GD3 metabolism and characterized the distribution and trafficking of GD3 during Aβ-induced apoptosis using human brain-derived TE671 cells. Extracellular Aβ-induced apoptosis in a mitochondrial-dependent manner. GD3 level was negligible in the basal condition. However, in response to extracellular Aβ, both the expression of GD3 synthase mRNA and the intracellular GD3 level were dramatically increased. Neosynthesized GD3 rapidly accumulated in cell surface lipid microdomains, and was then translocated to mitochondria to execute the apoptosis. Disruption of membrane lipid microdomains with methyl-β-cyclodextrin significantly prevented both GD3 accumulation in cell surface and Aβ-induced apoptosis. Our data suggest that rapidly accumulated GD3 in plasma membrane lipid microdomains prior to mitochondrial translocation is one of the key events in Aβ-induced apoptosis.

Antitumor activity and pharmacokinetics of oral gimatecan on pediatric cancer xenografts

This study compared the antitumor activity and the pharmacological profile of gimatecan given orally and irinotecan (CPT-11) on pediatric tumor xenografts. Gimatecan was tested in two neuroblastoma cell lines (SK-N-DZ and SK-N-(BE)2c) and on TE-671 rhabdomyosarcoma cells using two different schedules. We characterized its pharmacokinetic profile in nude mice bearing human SK-N-DZ and TE-671 cell lines. Gimatecan appears to have high plasma disposition. The drug was present in plasma almost completely as the intact lactone form and showed substantial activity in all tumor models. Prolonged daily treatment with low doses of gimatecan produced significant tumor regression in all tumor xenografts. The antitumor activity and the promising pharmacological profile indicate gimatecan as an excellent candidate for clinical treatment of pediatric tumors.

The human urocortin 2 gene is regulated by hypoxia: identification of a hypoxia-responsive element in the 3′-flanking region

Ucn2 (urocortin 2) has been shown to exert potent beneficial effects in the cardiovascular system, including inhibition of apoptosis, improvement of cardiomyocyte contractility and decrease of oxidative stress. The mechanisms that contribute to the regulation of hUcn2 (human Ucn2) expression in cardiovascular pathologies are not known. In the present study, we analysed the mechanism by which hypoxia, a major stimulus in ischaemic heart disease, regulates Ucn2 gene expression. Hypoxia and CPX (ciclopirox olamine), which prevents proteolytic degradation of HIF (hypoxia-inducible factor), significantly increased hUcn2 mRNA levels in TE-671 cells. Gene silencing of endogenous HIF1alpha abolishes this increase. Hypoxia and CPX activated a luciferase-linked fragment of the 3'FLR (3'-flanking region) of the hUcn2 gene containing two putative HREs (hypoxia-response elements), HRE1 and HRE2. Site-directed mutagenesis experiments demonstrated that HRE1 is required for HIF1alpha-dependent luciferase activation. This activation was conserved in constructs with the 3'FLR fragment placed upstream of the luciferase gene, indicating an enhancer function for HRE1. Competition assays revealed direct binding between HRE1 and HIF1alpha. Regulation of Ucn2 by hypoxia was confirmed in rat neonatal cardiomyocytes and in cardiac-derived H9c2 cells transfected with constructs of the 3'FLR of the hUcn2 gene. In conclusion, our study demonstrates that hypoxia induces hUcn2 expression via a specific HRE in the 3'FLR of the hUcn2 gene, which interacts with the transcription factor HIF1alpha. Hypoxia-mediated stimulation of cardioprotective Ucn2 may help to preserve cardiac function and prevent apoptosis in ischaemic conditions in the heart.

Skeletal muscle cell MHC I expression: Implications for statin-induced myopathy

Statins can induce necrotizing or inflammatory myopathies in some patients. Increased major histocompatibility complex class I (MHC I) expression has been shown in muscle biopsies of statin-induced myopathy. Therefore, we investigated the effect of statins on the expression of MHC I in muscle cells. Using flow cytometry and polymerase chain reaction (PCR), the rhabdomyosarcoma cell line TE671 and primary cultured skeletal muscle cells (SKMC) were investigated for MHC I expression after incubation with different statins and/or interferon-gamma (IFN-gamma). TE671 and SKMC express MHC I in the untreated condition. Statins alone reduced the expression of MHC I in SKMC and had no effect on MHC I in TE671 cells. Statins potentiated the MHC I-inducing effect of IFN-gamma in TE671, but not in SKMC, neither at the protein level nor at the mRNA level. The increased muscle MHC I expression in statin-induced myopathy might not be induced directly by statins themselves.

Expression of glutamate receptor subunits in human cancers

Emerging evidence suggests a role for glutamate and its receptors in the biology of cancer. This study was designed to systematically analyze the expression of ionotropic and metabotropic glutamate receptor subunits in various human cancer cell lines, compare expression levels to those in human brain tissue and, using electrophysiological techniques, explore whether cancer cells respond to glutamate receptor agonists and antagonists. Expression analysis of glutamate receptor subunits NR1-NR3B, GluR1-GluR7, KA1, KA2 and mGluR1-mGluR8 was performed by means of RT-PCR in human rhabdomyosarcoma/medulloblastoma (TE671), neuroblastoma (SK-NA-S), thyroid carcinoma (FTC 238), lung carcinoma (SK-LU-1), astrocytoma (MOGGCCM), multiple myeloma (RPMI 8226), glioma (U87-MG and U343), lung carcinoma (A549), colon adenocarcinoma (HT 29), T cell leukemia cells (Jurkat E6.1), breast carcinoma (T47D) and colon adenocarcinoma (LS180). Analysis revealed that all glutamate receptor subunits were differentially expressed in the tumor cell lines. For the majority of tumors, expression levels of NR2B, GluR4, GluR6 and KA2 were lower compared to human brain tissue. Confocal imaging revealed that selected glutamate receptor subunit proteins were expressed in tumor cells. By means of patch-clamp analysis, it was shown that A549 and TE671 cells depolarized in response to application of glutamate agonists and that this effect was reversed by glutamate receptor antagonists. This study reveals that glutamate receptor subunits are differentially expressed in human tumor cell lines at the mRNA and the protein level, and that their expression is associated with the formation of functional channels. The potential role of glutamate receptor antagonists in cancer therapy is a feasible goal to be explored in clinical trials.

Characterization of an alternative packaging system derived from the cat RD114 retrovirus for gene delivery

Retroviral vectors derived from the Moloney murine leukemia virus (MLV) are widely used in gene therapy. Pseudotyping of these vectors with the cat RD114 retrovirus envelope increases their potential for delivering genes into human hematopoietic cells. In the present study, we have further investigated the potential of the RD114 retrovirus in gene therapy. We describe and characterize an alternative retroviral packaging system derived from the RD114 retrovirus. RD114-derived recombinant retroviruses were produced transiently by transfection of 293T cells, and viral titers were assessed on TE671 cells by measuring the percentage of infected green fluorescent protein (GFP) positive cells by fluorescence-activated cell sorter (FACS) analysis. Purified human hematopoietic cells (lymphocytes and CD34(+) cells) were activated and transduced on retronectin-coated plates. Two days later, the percentage of GFP positive cells was evaluated by FACS analysis. We demonstrate that RD114 viral particles could package MLV transfer vectors, and that, in addition to its natural envelope, RD114 cores could be efficiently pseudotyped by the Gibbon ape leukemia, the MLV-amphotropic and the vesicular stomatitis virus G protein envelopes. Furthermore, we found that RD114 viral particles were highly efficient to transduce human lymphocytes and CD34(+) cells. This is the first demonstration that replication-defective RD114 viral particles can be generated and used for efficient gene delivery into human hematopoietic cells. We conclude that RD114-derived vectors could be useful in the field of gene therapy.

Japanese encephalitis virus NS2B-NS3 protease induces caspase 3 activation and mitochondria-mediated apoptosis in human medulloblastoma cells

Japanese encephalitis virus (JEV) causes severe neurological diseases with a high fatality rate. Clinical, neurophysiological and radiological features of Japanese encephalitis JE patients showed that JEV infection resulted in widespread involvement of the nervous system, including thalamus, basal ganglia, brainstem, cerebellum, cerebral cortex and spinal cord. In this study, we characterized the apoptotic effect of JEV infection and its viral proteins on the TE671 human medulloblastoma cells. JEV replicated in TE671 cells, inducing caspase 3-mediated apoptosis in MOI- and time-dependent manners. Of viral proteins, co-expression of JEV NS3 protease with NS2B cofactor significantly induced higher degrees of apoptosis and triggered higher caspase 3 activities than single expression of E, NS1, NS2B or NS3 protease in human medulloblastoma cells. Moreover, JEV NS2B-NS3 protease induced reduction of mitochondrial membrane potential and release of mitochondrial cytochrome C, which were responsible for the mitochondria-mediated apoptosis. In addition, the production of reactive oxygen species production and activation of ASK1-p38 MAPK signaling pathway might be associated with JEV NS2B-NS3 protease-induced mitochondria-mediated apoptosis. The results demonstrated that the JEV infection and the co-expression of JEV NS3 protease with NS2B cofactor induced caspase 3 activation and mitochondria-mediated apoptosis in human medulloblastoma cells, being valuable insight for cellular and molecular levels of JEV pathogenesis.

Biochemical, Functional, and Pharmacological Characterization of AT-56, an Orally Active and Selective Inhibitor of Lipocalin-type Prostaglandin D Synthase

We report here that 4-dibenzo[a,d]cyclohepten-5-ylidene-1-[4-(2H-tetrazol-5-yl)-butyl]-piperidine (AT-56) is an orally active and selective inhibitor of lipocalin-type prostaglandin (PG) D synthase (L-PGDS). AT-56 inhibited human and mouse L-PGDSs in a concentration (3–250 μm)-dependent manner but did not affect the activities of hematopoietic PGD synthase (H-PGDS), cyclooxygenase-1 and -2, and microsomal PGE synthase-1. AT-56 inhibited the L-PGDS activity in a competitive manner against the substrate PGH2 (Km = 14 μm) with a Ki value of 75 μm but did not inhibit the binding of 13-cis-retinoic acid, a nonsubstrate lipophilic ligand, to L-PGDS. NMR titration analysis revealed that AT-56 occupied the catalytic pocket, but not the retinoid-binding pocket, of L-PGDS. AT-56 inhibited the production of PGD2 by L-PGDS-expressing human TE-671 cells after stimulation with Ca2+ ionophore (5 μm A23187) with an IC50 value of about 3 μm without affecting their production of PGE2 and PGF2α but had no effect on the PGD2 production by H-PGDS-expressing human megakaryocytes. Orally administered AT-56 (<30 mg/kg body weight) decreased the PGD2 production to 40% in the brain of H-PGDS-deficient mice after a stab wound injury in a dose-dependent manner without affecting the production of PGE2 and PGF2α and also suppressed the accumulation of eosinophils and monocytes in the bronco-alveolar lavage fluid from the antigen-induced lung inflammation model of human L-PGDS-transgenic mice.

Simple Magnetic Cell Patterning Using Streptavidin Paramagnetic Particles

A simple methodology for cell patterning has been developed that can potentially be used to position different types of mammalian cells with high precision. In this method, cell membrane proteins were first biotinylated and then bound to streptavidin paramagnetic particles. The magnetically labeled cells were then seeded onto culture dishes and patterned using low magnetic fields. Highly defined cell patterns were achieved using HeLa, TE671 cells and human monocytes. HeLa and TE671 cells were also sequentially patterned and successfully co-cultured on the same plate using this technique. Cell viability studies proved that this magnetic labeling method was not toxic to cells. Transmission electron microscopy showed that the magnetically labeled HeLa and TE671 cells internalized some of the paramagnetic particles after two days of culture, while the labeled human monocytes did the same after only one hour. Uptake of these particles did not affect the cell patterning and cell viability. This magnetic labeling process is fast, as it involves affinity-based attachment of paramagnetic particles and does not rely on cellular uptake of magnetic materials. It may be adaptable and scalable for various applications.

Human muscle cells express the costimulatory molecule B7‐H3, which modulates muscle–immune interactions

Interactions between the family of B7 ligands and their receptors are increasingly recognized as crucial for stimulation and/or inhibition of immune responses. The present study was undertaken to examine the expression and functional relevance of B7 homolog 3 (B7-H3), a novel B7 homolog attributed significant immunoregulatory functions, in human muscle cells in vivo and in vitro. Thirty-five muscle biopsy specimens obtained from patients with polymyositis, dermatomyositis, inclusion body myositis, or noninflammatory myopathies and normal controls were analyzed by immunohistochemistry for B7-H3 expression. The expression of B7-H3 protein on primary human myoblasts and TE671 muscle rhabdomyosarcoma cells was studied by flow cytometry and Western blot analysis. B7-H3 small interfering RNA (siRNA) was used to study the impact of knockdown of B7-H3 on CD8+ cell-mediated lysis in skeletal muscle cells. B7-H3 was not detectable on normal muscle fibers. In contrast, its expression was markedly increased on muscle fibers from patients with inflammatory myopathies. Cell-surface staining was most prominent in the contact areas between muscle fibers and inflammatory cells. B7-H3 protein was detected on myoblasts cultured from control and myositis patient muscle tissue as well as in TE671 muscle rhabdomyosarcoma cells. Knockdown of B7-H3 by siRNA in TE671 cells enhanced CD8+ T cell-specific lysis, indicating a functional role of B7-H3 in the protection of skeletal muscle from immune-mediated lysis. Our results demonstrate that human muscle cells express B7-H3, a functional coinhibitory molecule of the B7 family. B7-H3 may play an important role in muscle-immune interactions, providing further evidence of the active role of muscle cells in local immunoregulatory processes.

The EphA3 receptor is expressed in a subset of rhabdomyosarcoma cell lines and suppresses cell adhesion and migration

Elevated expression of the Eph receptor tyrosine kinase EphA3 is associated with lymphocytic leukaemia, but little is known about its expression or function in solid tumours. Out of a panel of cancer cell lines, we found that EphA3 was expressed only on two rhabdomyosarcoma (RMS) cell lines of the embryonal histological subtype and on one of the alveolar RMS subtype, whereas it was not detected on two other cell lines of the alveolar subtype. Other EphA receptors (1-7) were, either not expressed in any, or expressed in all five RMS cell lines. Stimulation of EphA3-expressing TE671 and RD RMS cells with ephrinA5 resulted in loss of adhesion to fibronectin, decreased migration towards the stromal cell-derived growth factor-I (SDF-I), increased EphA3 phosphorylation, and increased Rho GTPase activity. In contrast, ectopic expression of EphA3 in the EphA3 negative CRL2061 cell line resulted in decreased cell adhesion. Finally, suppression of EphA3 expression by siRNA in RD cells results in increased SDF-I-mediated motility. These data indicate that EphA3 expression may define subsets of RMS tumours, and that EphA3 suppresses motility through regulation of Rho GTPases in RMS cells.

Enhancement of prostaglandin D 2 production through cyclooxygenase-2 and lipocalin-type prostaglandin D synthase by upstream stimulatory factor 1 in human brain-derived TE671 cells under serum starvation

We found that prostaglandin (PG) D 2 production was induced through transcriptional activation of cyclooxygenase (COX)-2 and lipocalin-type PGD synthase (L-PGDS) genes under serum-starved conditions in human brain-derived TE671 cells. Analysis of promoter and intron regions of the human L-PGDS gene demonstrated that an atypical E-box within intron 4 mediated serum starvation-induced up-regulation of L-PGDS gene expression. The results of electrophoretic mobility shift assay and chromatin immunoprecipitation assay showed that upstream stimulatory factor (USF) 1 bound to this atypical E-box. USF1 gene expression was also enhanced during serum starvation in TE671 cells through activation of p38 mitogen activated protein kinase, and the efficiency of the binding of USF1 to the atypical E-box was clearly increased by serum starvation. Administration of USF1 siRNA suppressed both L-PGDS and COX-2 gene expression and PGD 2 production. Moreover, NS-398, a COX-2 inhibitor and AT-56, an L-PGDS inhibitor, suppressed PGD 2 production in TE671 cells cultured under the serum-starved condition. These results indicate that PGD 2 production stimulated by serum starvation is mediated by both COX-2 and L-PGDS through enhancement of USF1 in TE671 cells.

Stereoselective Potencies and Relative Toxicities of Coniine Enantiomers

Coniine, one of the major toxic alkaloids present in poison hemlock ( Conium maculatum), occurs in two optically active forms. A comparison of the relative potencies of (+)- and (-)-coniine enantiomers has not been previously reported. In this study, we separated the enantiomers of coniine and determined the biological activity of each enantiomer in vitro and in vivo. The relative potencies of these enantiomers on TE-671 cells expressing human fetal nicotinic neuromuscular receptors had the rank order of (-)-coniine > (+/-)-coniine > (+)-coniine. A mouse bioassay was used to determine the relative lethalities of (-)-, (+/-)-, and (+)-coniine in vivo. The LD 50 values of the coniine enantiomers were 7.0, 7.7, and 12.1 mg/kg for the (-)-, (+/-)-, and (+)- forms of coniine, respectively. The results from this study demonstrate that there is a stereoselective difference in the in vitro potencies of the enantiomers of coniine that directly correlates with the relative toxicities of the enantiomers in vivo.

Viral Vector–mediated Delivery of Estrogen Receptor-α to the Hippocampus Improves Spatial Learning in Estrogen Receptor-α Knockout Mice

Estrogen, which influences both classical genomic and rapid membrane-associated signaling cascades, has been implicated in the regulation of hippocampal function, including spatial learning. Gene mutation studies suggest that estrogen effects are mediated by estrogen receptor-α (ER-α); however, because gonadal steroids influence the organization of the hippocampus during development, it has been difficult to distinguish developmental effects from those specific to adults. In this study we show that lentiviral delivery of the gene encoding ER-α to the hippocampus of adult ER-α-knockout (ER-αKO) mice restores hippocampal responsiveness to estrogen and rescues spatial learning. We propose that constitutive estrogen receptor activity is important for maintaining hippocampus-dependent memory function in adults.

Aloe-emodin is an interferon-inducing agent with antiviral activity against Japanese encephalitis virus and enterovirus 71

In this study, aloe-emodin was identified as a potential interferon (IFN)-inducer by screening compounds from Chinese herbal medicine. Aloe-emodin showed low cytotoxicity to human HL-CZ promonocyte cells and TE-671 medulloblastoma cells and significantly activated interferon-stimulated response element (ISRE) and gamma-activated sequence (GAS)-driven cis-reporting systems. Moreover, aloe-emodin upregulated expression of IFN-stimulated genes such as dsRNA-activated protein kinase and 2′,5′-oligoisoadenylate synthase. Aloe-emodin resulted in significant activation of nitric oxide production. The antiviral activity of aloe-emodin against Japanese encephalitis virus (JEV) and enterovirus 71 (EV71) was evaluated using dose- and time-dependent plaque reduction assays in HL-CZ cells and TE-671 cells. The 50% inhibitory concentration (IC 50) of aloe-emodin ranged from 0.50 μg/mL to 1.51 μg/mL for JEV and from 0.14 μg/mL to 0.52 μg/mL for EV71. Aloe-emodin showed clearly potent virus inhibitory abilities and achieved high therapeutic indices, in particular for HL-CZ cells. Therefore, the study demonstrated dose- and time-dependent actions of aloe-emodin on the inhibition of JEV and EV71 replication via IFN signalling responses.

Interferon antagonist function of Japanese encephalitis virus NS4A and its interaction with DEAD-box RNA helicase DDX42

The interferon (IFN) antagonists of Japanese encephalitis virus (JEV) proteins contribute to the JE pathogenesis. Most flavivirus non-structural (NS) proteins correlate with virus-induced inflammation and immune escape. NS4A proteins of West Nile virus and dengue type 2 virus have been demonstrated to inhibit IFN signaling. In this study, JEV NS4A without the C-terminal 2K domain has been demonstrated to partially block activation of an IFN-stimulated response element (ISRE)-based cis-reporter by IFN-α/β. In addition, JEV NS4A significantly inhibited the phosphorylation levels of STAT1 and STAT2, but not TYK2 in the IFN-treated cells. Moreover, the N-terminus of a RNA helicase DDX42 protein identified using a phage display human brain cDNA library have been demonstrated to specifically bind to JEV NS4A in vitro using a co-immunoprecipitation assay. The interaction between JEV NS4A and RNA helicase DDX42 showed partial co-localization in human medulloblastoma TE-671 cells by confocal microscopy. Importantly, the expression of N-terminal DDX42 is able to overcome JEV-induced antagonism of IFN responses. Therefore, these results show that JEV NS4A without the C-terminal 2K domain is associated with modulation of the IFN response and the interaction of JEV NS4A with RNA helicase DDX42 could be important for JE pathogenesis.

Temporal Recruitment of Transcription Factors at the 3′,5′-Cyclic Adenosine 5′-Monophosphate-Response Element of the Human GnRH-II Promoter

GnRH-II is a potent GnRH subtype involved in modulating OVCAR-3 cell proliferation and the invasive properties of JEG-3 cells, and an atypical cAMP-response element (CRE) in the human GnRH-II promoter influences its activation. We demonstrated that the GnRH-II promoter is activated by 8-bromoadenosine-cAMP in several cell lines including alphaT3, TE671, JEG-3, and OVCAR-3 cells and that cAMP enhances GnRH-II mRNA levels in JEG-3 and OVCAR-3 cells. Moreover, 8-bromoadenosine-cAMP increases cAMP response element-binding protein (CREB) phosphorylation in JEG-3 and OVCAR-3 cells and augments CBP and CCAAT/enhancer-binding protein (C/EBP)-beta coimmunoprecipitation with phosphorylated CREB (p-CREB) in a temporally defined manner from nuclear extracts. When CREB, CBP, and C/EBPbeta levels were knocked down by small interfering RNA, reductions in any of these transcription factors reduced cAMP-enhanced GnRH-II promoter activity and GnRH-II mRNA levels in JEG-3 and OVCAR-3 cells. Importantly, chromatin immunoprecipitation assay showed that p-CREB bound the CRE within the endogenous GnRH-II promoter within 1 h and that p-CREB association with C/EBPbeta occurs within 2 h of cAMP stimulation, coincident with the first appearance of C/EBPbeta at the CRE. By contrast, maximum interactions between p-CREB and CBP do not occur until at least 4 h after cAMP stimulation, and this is reflected in the progressive loading of CBP at the CRE at 2-4 h, as demonstrated by chromatin immunoprecipitation. Taken together, these data suggest that p-CREB, C/EBPbeta, and CBP are recruited to the CRE of the GnRH-II promoter in a temporarily defined manner to enhance its transcription in JEG-3 and OVCAR-3 cells in response to cAMP.