NS-1 Cells Research Articles

Activin A, a Novel Chemokine, Induces Mouse NK Cell Migration via AKT and Calcium Signaling.

Natural killer (NK) cells can migrate quickly to the tumor site to exert cytotoxic effects on tumors, and some chemokines, including CXCL8, CXCL10 or and CXCL12, can regulate the migration of NK cells. Activin A, a member of the transforming growth factor β (TGF-β) superfamily, is highly expressed in tumor tissues and involved in tumor development and immune cell activation. In this study, we focus on the effects of activin A on NK cell migration. In vitro, activin A induced NK cell migration and invasion, promoted cell polarization and inhibited cell adhesion. Moreover, activin A increased Ca2+, p-SMAD3 and p-AKT levels in NK cells. An AKT inhibitor and Ca2+ chelator partially blocked activin A-induced NK cell migration. In vivo, exogenous activin A increased tumor-infiltrating NK cells in NS-1 cell solid tumors and inhibited tumor growth, and blocking endogenous activin A with anti-activin A antibody reduced tumor-infiltrating NK cells in 4T-1 cell solid tumors. These results suggest that activin A induces NK cell migration through AKT signaling and calcium signaling and may enhance the antitumor effect of NK cells by increasing tumor-infiltrating NK cells.

Fungal Duel between Penicillium brasilianum and Aspergillus nomius Results in Dual Induction of Miktospiromide A and Kitrinomycin A.

Cocultivation of the fungi Penicillium brasilianum MST-FP1927 and Aspergillus nomius MST-FP2004 resulted in the reciprocal induction of two new compounds, miktospiromide A (1) from A. nomius and kitrinomycin A (2) from P. brasilianum. A third new compound, kitrinomycin B (3), was also identified from an axenic culture of P. brasilianum, along with the previously reported compounds austalide K (4), 17S-dihydroaustalide K (5), verruculogen (6), and fumitremorgin B (7). The structures of 1-3 were elucidated by detailed spectroscopic analysis and DFT calculations, while 4-7 were identified by comparison to authentic standards. The genome of A. nomius MST-FP2004 was sequenced, and a putative biosynthetic gene cluster for 1 was identified. Compound 2 showed activity against murine melanoma NS-1 cells (LD99 7.8 μM) and the bovine parasite Tritrichomonas foetus (LD99 4.8 μM).

ERK-dependent induction of the immediate-early gene Egr1 and the late gene Gpr50 contribute to two distinct phases of PACAP Gs-GPCR signaling for neuritogenesis.

Gs-coupled GPCR-stimulated neuritogenesis in PC12 and NS-1 - cells depends on activation of the MAP kinase ERK. Here, we examine changes in ERK activation (phosphorylation), and the time course of ERK-dependent gene induction, to seek transcriptional determinants for this process. Quenching of ERK activation by inhibition of MEK with U0126 at any time point for at least 24 h following addition of PACAP resulted in arrest of neurite formation. Changes in the transcriptome profile throughout this time period revealed at least two phases of gene induction: an early phase dominated by induction of immediate-early genes, and a later phase of gene induction after 4-6h of exposure to PACAP with persistent elevation of phospho-ERK levels. Genes induced by PACAP in both phases consisted in those whose induction was dependent on ERK (i.e., blocked by U0126), and some whose induction was blocked by the protein kinase A inhibitor H89. ERK-dependent "late gene" transcripts included Gpr50, implicated earlier in facilitation of NGF-induced neurite formation in NS-1 cells. Gpr50 induction by PACAP, but not NGF, was dependent on the guanine nucleotide exchange factor RapGEF2, which has been shown to be required for PACAP-induced neuritogenesis in NS-1 cells. Expression of a Gpr50-directed shRNA lowered basal levels of Gpr50 mRNA and attenuated Gpr50 mRNA and GPR50 protein induction by PACAP, with a corresponding attenuation of PACAP-induced neuritogenesis. Gs-GPCR-stimulated neuritogenesis first requires immediate-early gene induction, including that of Egr1 (Zif268/NGF1A/Krox24) as previously reported. This early phase of gene induction, however, is insufficient to maintain the neuritogenic process without ERK-dependent induction of additional late genes, including Gpr50,upon continuous exposure to neurotrophic neuropeptide. Early (Egr1) and late (Gpr50) gene induction by NGF, like that for PACAP, was inhibited by U0126, but was independent of RapGEF2, confirming distinct modes of ERK activation by Gs-coupled GPCRs and neurotrophic tyrosine receptor kinases, converging on a final common ERK-dependent signaling pathway for neuritogenesis.

Gs‐coupled GPCR‐mediated Activation of Rapgef2‐ and ERK‐dependent Gene Regulation and Neuritogenesis in NS‐1 Cells

Gs‐coupled GPCRs signal through the second messenger cAMP. In the neuroendocrine NS‐1 cell line, cAMP acts through three parcellated signal pathways using distinct cAMP sensors as third messenger: protein kinase A (PKA) is linked to neuroendocrine‐specific gene expression, the exchange protein (EPAC) to cell proliferation arrest, and the neuritogenic cAMP sensor NCS‐Rapgef2 to neuritogenesis (Xu et al., J. Neuroendocrinol. 33: e12974). NCS‐RapGEF2‐mediated neuritogenesis occurs via the Rap‐Braf‐MEK‐ERK pathway, which is activated by a variety of Gs‐coupled GPCRs, including the endogenously expressed PAC1 receptor, and receptors for GLP‐1 (GLP1R) and VIP (VIPR1, VIPR2) when exogenously expressed in NS‐1 cells, followed by exposure to exendin‐4 or VIP (Xu et al., ibid) or by ADRB2, after exposure to isoproterenol. We have noted previously that other Gs‐coupled GPCRs expressed in NS‐1 cells mediate measurable cAMP elevation, which however is apparently below the threshold required for triggering and maintaining neuritogenesis (Xu et al., ibid). Here, we identify changes in the NS‐1 cell transcriptome associated with PACAP activation of PAC1 receptor and the role of ERK phosphorylation in sustaining gene expression potentially associated with cAMP‐dependent neuritogenesis. Continuous ERK phosphorylation is required for neuritogenic signaling: inhibition of ERK phosphorylation with U0126 for up to 24 hours after PACAP treatment arrests neuritogenesis, while inhibition of PKA with H89 has no effect on neuritogenesis.NS‐1 cells were treated for 24 hours with PACAP in the presence and absence of either the MEK inhibitor U0126 or the PKA inhibitor H89, and subjected to transcriptomic analysis. We identified 25 genes significantly upregulated by PACAP but downregulated (P<0.001, >2 fold, n=3) by co‐treatment with U0126. DCLK1 is a serine/threonine protein kinase important for neuronal development. Dclk1 mRNA is upregulated ~six‐fold at 8 hr and four‐fold at 24 hr after PACAP treatment, and this was reduced to <two‐fold by U0126. Dlg2 encodes a postsynaptic scaffolding protein, PSD‐93, and Dlg2 mRNA is also upregulated significantly at 8 and 24 hr after PACAP (P<0.01, >2 fold compared to no treat) and its upregulation by PACAP is also blocked by U0126. The gene encoding the orphan GPCR, GPR50, is upregulated more than 100‐fold by 8 hours after PACAP, and induction of GPR50 mRNA is blocked more than 97% by U0126, with partial blockade (80%) by H89. We are currently investigating the impact of GPR50 on neuritogenesis using GPR50 knockdown with shRNA in NS‐1 cells. Forty‐five genes upregulated by PACAP and blocked by H89, but not U0126, include Slit2 and Gas1. Regulation of these genes by Gs‐coupled GPCR signaling through PKA may mediate NS‐1 cell responses separate from activation of neuritogenesis.Cyclic AMP elevation associated with Gs‐coupled GPCR signaling in NS‐1 cells results in upregulation of both RapGEF2/ERK‐dependent and PKA‐dependent gene induction. A threshold for cAMP elevation, either in amount or duration of elevation, or both, appears to be a requirement for Gs‐coupled GPCR‐mediated neuritogenesis. NS‐1 cells provide a model for investigating the functional importance of RapGEF2‐ and PKA‐dependent gene regulation initiated by cAMP in neuroendocrine cells.

The Autophagosomes Containing Dengue Virus Proteins and Full-Length Genomic RNA Are Infectious.

Autophagic machinery is involved in selective and non-selective recruitment as well as degradation or exocytosis of cargoes, including pathogens. Dengue virus (DENV) infection induces autophagy that enhances virus replication and vesicle release to evade immune system surveillance. This study reveals that DENV2 induces autophagy in lung and liver cancer cells and showed that DENV2 capsid, envelope, NS1, NS3, NS4B and host cell proinflammatory high mobility group box 1 (HMGB1) proteins associated with autophagosomes which were purified by gradient centrifugation. Capsid, NS1 and NS3 proteins showing high colocalization with LC3 protein in the cytoplasm of the infected cells were detected in the purified double-membrane autophagosome by immunogold labeling under transmission electron microscopy. In DENV infected cells, the levels of capsid, envelope, NS1 and HMGB1 proteins are not significantly changed compared to the dramatic accumulation of LC3-II and p62/SQSTM1 proteins when autophagic degradation was blocked by chloroquine, indicating that these proteins are not regulated by autophagic degradation machinery. We further demonstrated that purified autophagosomes were infectious when co-cultured with uninfected cells. Notably, these infectious autophagosomes contain DENV2 proteins, negative-strand and full-length genomic RNAs, but no viral particles. It is possible that the infectivity of the autophagosome originates from the full-length DENV RNA. Moreover, we reveal that DENV2 promotes HMGB1 exocytosis partially through secretory autophagy. In conclusion, we are the first to report that DENV2-induced double-membrane autophagosomes containing viral proteins and full-length RNAs are infectious and not undergoing autophagic degradation. Our novel finding warrants further validation of whether these intracellular vesicles undergo exocytosis to become infectious autophagic vesicles.

Cyclic AMP‐dependent Activation of ERK Via GLP‐1 Receptor Signaling Requires the Neuroendocrine Cell‐Selective Guanine Nucleotide Exchanger NCS‐RapGEF2

G-protein coupled receptors activate various intracellular processes upon ligand engagement depending on coupling to adenylyl cyclase (Gs, Gi/o), phospholipase C (Gq), or RhoGEFs (G12/13). Cyclic AMP activation of the Rap-Braf-MEK-ERK pathway after signaling initiated by the neuropeptide PACAP, via the Gs-protein coupled receptor (GsPCR) PAC1, occurs uniquely through the neuritogenic cAMP sensor Rap guanine nucleotide exchange factor 2 (NCS-RapGEF2) in NS-1 neuroendocrine cells. We examined the expression of other Family B GsPCRs in this cell line and assessed cAMP elevation and neuritogenesis after treatment with their cognate peptide ligands. Exposure of NS-1 cells to the VIPR1/2 agonist VIP, or the GLP1R agonist exendin-4, did not induce neuritogenesis, or elevation of cAMP, presumably due to insufficient receptor protein expression. VIP and exendin-4 did induce neuritogenesis after transduction of human VIPR1, VIPR2 and GLP1R into NS-1 cells. Exendin-4/GLP1R-stimulated neuritogenesis was MEK-ERK- dependent (blocked by U0126), indicating its use of the cAMP->RapGEF2->ERK neuritogenic signaling pathway previously identified for PACAP/PAC1 signaling in NS-1 cells. NCS- RapGEF2 is expressed in the beta cell tumor-derived cell lines MIN6 and INS-1, and in human pancreatic islets. As in NS-1 cells, exendin-4 caused ERK phosphorylation in INS-1 cells. Reduction in RapGEF2 expression after its specific shRNA treatment reduced exendin-4-induced ERK phosphorylation. Transcriptome analysis of INS-1 cells after one hour exposure to exendin- 4 revealed an immediate early-gene response that was composed of both ERK-dependent and ERK-independent signaling targets. ERK-dependent targets, and presumably involving NCS-RapGEF2 signaling, included Nr4a2, Btg1, Skil, Plkl2, Per1 and Dyrk3, while ERK-independent targets and presumably involving PKA or Epac signaling, included Nr4a1, Nr4a3, Crem, Fosl2, Sik1 and Rgs2. We propose that cAMP signaling initiated by GLP-1 in pancreatic beta cells causes parallel activation of multiple cAMP effectors, including NCS- RapGEF2, Epac, and protein kinase A, to separately control various facets of GLP-1 action, including insulin secretion and transcriptional modulation. Identification of coupling of GsPCRs to ERK activation via NCS-RapGEF2 provides a novel signaling mode for GLP-1 in pancreatic beta cells and potentially brain and elsewhere. RapGEF2-dependent ERK activation is likely to be a feature of additional nervous and endocrine targets of family B receptor ligands including PACAP, VIP, CRH, GIP, secretin, glucagon, PTH, calcitonin, and GHRH. Whether or not ligands for these receptors, including GLP-1, might activate the NCS-RapGEF2, PKA, or Epac cAMP-dependent pathways in a biased fashion, with differential physiological effects, has yet to be investigated. This abstract is dedicated to the memory of Andrew C. Emery.

Total Synthesis of the Antitumor-Antitubercular 2,6'-Bijuglone Natural Product Diospyrin and Its 3,6'-Isomer.

The 2,6'-bijuglone natural product diospyrin and its unnatural 3,6'-isomer idospyrin have been synthesized in seven steps each from N,N-diethylsenecioamide in overall yields of 12% and 13%, respectively. The syntheses diverge from ramentaceone (7-methyljuglone) and include a key Suzuki-Miyaura cross-coupling. Diospyrin, idospyrin, and several synthetic precursors exhibit potent and selective cytotoxicity to the murine myeloma NS-1 cell line over neonatal foreskin cells.

Synthesis of keratine, silver, and flavonols nanocomposites to inhibit oxidative stress in pancreatic beta-cell (INS-1) and reduce intracellular reactive oxygen species production

Flavonols (FLA) fromVaccinium macrocarpon (V. macrocarpon) were identified using high-performance liquid chromatography coupled with mass spectrometry detection. Nanoparticles were prepared using highly crosslinked keratin (KER) from human hair andsilver and entrapped with flavonols [KER + FLA + AgNPs].Nanocomposites were characterized using UV–Vis spectroscopy, transmission electron microscopy (TEM), X-ray diffraction,zeta potential, and dynamic light scattering, andrelease profiles.The interactions between the capping agent and the silver core have been investigated using FTIR spectroscopy·H2O2 is a source of Reactive Oxygen Species (ROS) and acts as an activator of oxidative stress affecting NS-1 cells, and the protective effect of the nanocomposites were evaluated against H2O2-induced pancreatic β-cell damage.LC-MS/MS and HPLC analyses revealed the presence of 12 flavonols inV. macrocarponplant extract.The cell apoptosis and proliferation, were evaluated by Hoechst 33342 staining, flow cytometry and Cell Counting Kit-8 respectively. Preincubation of the NS-1 cells with 250 µg/mL of H2O2induced oxidative stress conditions that showpancreatic β-cell dysfunction, includingROS, cell death, mitochondrial function, antioxidant enzymes, and lipid peroxidation. Nevertheless, pretreatment with FLA and [KER + FLA + AgNPs] prevented mitochondria disruption, maintained cellular ATP levels, inhibited LDH release, intracellular ROS production, decreased lipid peroxidation, increased expression of antioxidant enzymes (CAT, SOD, and GPx) and GSH levels. These results indicate that nanocomposites couldprotect rat INS-1 pancreaticβ-cell from H2O2-induced oxidative damage, apoptosis and proliferation by reducing the production of intracellular reactive oxygen species.

A Novel Anti-PD-L1 Antibody Exhibits Antitumor Effects on Multiple Myeloma in Murine Models via Antibody-Dependent Cellular Cytotoxicity.

Multiple myeloma is a malignant cancer of plasma cells. Despite recent progress with immunomodulatory drugs and proteasome inhibitors, it remains an incurable disease that requires other strategies to overcome its recurrence and non-response. Based on the high expression levels of programmed death-ligand 1 (PD-L1) in human multiple myeloma isolated from bone marrow and the murine myeloma cell lines, NS-1 and MOPC-315, we propose PD-L1 molecule as a target of anti-multiple myeloma therapy. We developed a novel anti-PD-L1 antibody containing a murine immunoglobulin G subclass 2a (IgG2a) fragment crystallizable (Fc) domain that can induce antibody-dependent cellular cytotoxicity. The newly developed anti-PD-L1 antibody showed significant antitumor effects against multiple myeloma in mice subcutaneously, intraperitoneally, or intravenously inoculated with NS-1 and MOPC-315 cells. The anti-PD-L1 effects on multiple myeloma may be related to a decrease in the immunosuppressive myeloid-derived suppressor cells (MDSCs), but there were no changes in the splenic MDSCs after combined treatment with lenalidomide and the anti-PD-L1 antibody. Interestingly, the newly developed anti-PD-L1 antibody can induce antibody-dependent cellular cytotoxicity in the myeloma cells, which differs from the existing anti-PD-L1 antibodies. Collectively, we have developed a new anti-PD-L1 antibody that binds to mouse and human PD-L1 and demonstrated the antitumor effects of the antibody in several syngeneic murine myeloma models. Thus, PD-L1 is a promising target to treat multiple myeloma, and the novel anti-PD-L1 antibody may be an effective anti-myeloma drug via antibody-dependent cellular cytotoxicity effects.

Contributions of Chondroitin Sulfate, Keratan Sulfate and N-linked Oligosaccharides to Inhibition of Neurite Outgrowth by Aggrecan.

The role of proteoglycans in the central nervous system (CNS) is a rapidly evolving field and has major implications in the field of CNS injury. Chondroitin sulfate proteoglycans (CSPGs) increase in abundance following damage to the spinal cord and inhibit neurite outgrowth. Major advances in understanding the interaction between outgrowing neurites and CSPGs has created a need for more robust and quantitative analyses to further our understanding of this interaction. We report the use of a high-throughput assay to determine the effect of various post-translational modifications of aggrecan upon neurite outgrowth from NS-1 cells (a PC12 cell line derivative). Aggrecan contains chondroitin sulfate, keratan sulfate, and N-linked oligosaccharides (N-glycans), each susceptible to removal through different enzymatic digestions. Using a sequential digestion approach, we found that chondroitin sulfate and N-glycans, but not keratan sulfate, contribute to inhibition of neurite outgrowth by substrate-bound aggrecan. For the first time, we have shown that N-linked oligosaccharides on aggrecan contribute to its inhibition of neuritogenesis.

Involvement of CHOP in activin A‑induced myeloma NS‑1 cell apoptosis.

Activin A, a multifunctional cytokine, is a member of transforming growth factor-β (TGF-β) superfamily. It is associated with a variety of pathophysiological processes, including inflammation, fibrosis, and tumorigenesis. Chronic or prolonged endoplasmic reticulum (ER) stress can lead to cells apoptosis. However, whether ER stress-related proteins, such as CHOP, GADD34 are involved in activin A-induced myeloma cell apoptosis remains unknown. In the present study, it was revealed that activin A inhibited the proliferation of myeloma cell line NS-1 cells and induced NS-1 cell apoptosis. Activin A upregulated the expression of CHOP, GADD34, caspase-3, and caspase-12. Moreover, both Smad3 and p-Smad3 levels were increased with treatment of activin A. Further studies revealed that the overexpression of activin signaling protein Smad3 in NS-1 cells increased the levels of CHOP, caspase-3, and p-Smad3. These data indicated that the CHOP protein of the ER stress pathway may be involved in activin A-induced NS-1 cell apoptosis, and also indicated the potential therapy of activin A-induced apoptosis via CHOP signaling for multiple myeloma.

Establishment of stable cell line expressing human bocavirus type 1 non-structural protein NS1 and its trans-transcriptional activation

Human bocavirus 1 (HBoV1) non-structural protein NS1 is a multifunctional protein important for virus replication and induction of apoptosis in host cell. To better understand the function of the NS1 protein, it is urgent to address reducing the toxicity of NS1 to host cells. In the present study, we established a stable cell line that regulates expression of NS1 of HBoV1. The recombinant lentivirus plasmid containing a regulatable promoter fused with ns1 gene was constructed and transfected into HEK 293T cells using transfection reagent. The HEK 293T cell lines stably expressing NS1-100 and NS1-70 proteins were established by screening resistant cells with puromycin and inducing NS1 expression with doxycycline. The expression of NS1 protein was determined by fluorescent labeling protein and Western blotting. HBoV1 promoter was transfected into stably expressing NS1 cell line and its trans-transcriptional activity was analyzed. The results showed that NS1 protein was expressed stably in the established cell lines and had a strong activation activity on the HBoV1 promoter driving luciferase gene. Taken together, this study provides a solid basis for further research on the function of NS1 and the pathogenesis of human bocavirus.

Evaluation of different transfection methodologies to achieve efficient expression of the NS1 dengue protein in HepG2 cells

HepG2, a human hepatocarcinoma cell line, has been used as a model to study infection by several pathogens including dengue virus. However, this cell line is notoriously difficult to be transfected with plasmid DNAs by traditional methods, which is a limitation for some studies involving heterologous gene expression. In the present work, we analyzed different protocols for transfection of HepG2 with the plasmid pcENS1, which encodes the dengue NS1 protein, in order to evaluate the best methodology for achieving high cell viability and transfection efficiency. We analyzed two transfection approaches using lipid-based methods (Lipofectamine and FuGENE 6) or electroporation by nucleofection. Expression of the recombinant NS1 protein was evaluated by immunofluorescence and flow cytometry. Transfection with either of the two lipid-based methods led to very low number of HepG2 cells expressing NS1 (3.9% and 6.8% with Lipofectamine and FuGene, respectively) and high cell death rates. On the other hand, the efficiency of cell transfection was remarkable higher with nucleofection when compared to these other methods, achieving 63% of cells expressing NS1 protein and more than 60% of viability in the optimized condition.

A stress-enhanced model for discovery of disease-modifying gene: Ece1-suppresses the toxicity of α-synuclein A30P

Parkinson's disease (PD) is a progressive motor neurodegenerative disorder, characterized by a selective loss of dopaminergic neurons in the substantia nigra. The complexity of disease etiology includes both genetic and environmental factors. No effective drug that can modify disease progression and protect dopamine neurons from degeneration is presently available. Human α-Synuclein A30P (A30P) is a mutant gene identified in early onset PD and showed to result selective dopamine neuron loss in transgenic A30P flies and mice. Paraquat (PQ) is an herbicide and an oxidative stress generator, linked to sporadic PD. We hypothesized that vital PD modifier genes are conserved across species and would show unique transcriptional changes to oxidative stress in animals expressing a PD-associated gene, such as A30P. We also hypothesized that manipulation of PD modifier genes would provide neuroprotection across species. To identify disease modifier genes, we performed two independently-duplicated experiments of microarray, capturing genome-wide transcriptional changes in A30P flies, chronically fed with PQ-contaminated food. We hypothesized that the best time point of identifying a disease modifier gene is at time when flies showed maximal combined toxicity of A30P transgene and PQ treatment during an early stage of disease and that effective disease modifiers gene are those showing transcriptional changes to oxidative stress in A30P expressing and not in wild type animals. Fly Neprilysin3 (Nep3) is one identified gene that is highly conserved. Its mouse and human homolog is endothelin-converting enzyme-1 (Ece1). To investigate the neuroprotective effect of Ece1, we used NS1 cells and mouse midbrain neurons expressing A30P, treated with or without PQ. We found that ECE1 expression protected against A30P toxicity on cell viability, on neurite outgrowth and ameliorated A30P accumulation in vitro. Expression of ECE1 in vivo suppressed dopamine neuron loss and alleviated the corresponding motor deficits in mice with A30P-expression. Our study leverages a new approach to identify disease modifier genes using a stress-enhanced PD animal model.

Evaluating vacquinol-1 in rats carrying glioblastoma models RG2 and NS1.

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor, and available experimental and routine therapies result in limited survival benefits. A vulnerability of GBM cells to catastrophic vacuolization and cell death, a process termed methuosis, induced by Vacquinol-1 (VQ-1) has been described earlier. In the present study, we investigate the efficacy of VQ-1 treatment in two syngeneic rat GBM models, RG2 and NS1. VQ-1 treatment affected growth of both RG2 and NS1 cells in vitro. Intracranially, significant reduction in RG2 tumor size was observed, although no effect was seen on overall survival. No survival advantage or effect on tumor size was seen in animals carrying the NS1 models compared to untreated controls. Furthermore, immunological staining of FOXP3, CD4 and CD8 showed no marked difference in immune cell infiltrate in tumor environment following treatment. Taken together, a survival advantage of VQ-1 treatment alone could not be demonstrated here, even though some effect upon tumor size was seen. Staining for immune cell markers did not indicate that VQ-1 either reduced or increased host anti-tumor immune response.

Thyroid Hormone Enhances Neurite Outgrowth in Neuroscreen 1 Cells.

Objectives:Alzheimer’s disease (AD) is a neurodegenerative disorder that affects millions of individuals. Moreover, hypothyroidism has been identified as one of the risk factors that may contribute to the development of AD. Here, we investigated whether there was a correlation among expression levels of proteins involved in the formation of AD lesions, neurite outgrowth, and thyroid hormone levels.Methods:Cells were grown in media supplemented with different levels of 3,5,3’-triiodothyronine (T3) and then processed for neurite outgrowth and to prepare RNA samples. RNA samples were analysed using quantitative real-time PCR. Protein levels were measured using in cell-Western blotting analysis.Results:By using neurite outgrowth studies, it was demonstrated that T3 treatment enhanced neurite outgrowth in NS-1 cells in a time- and dose-dependent manner. Quantitative real-time PCR studies further confirmed that NS-1 cells expressed substantial levels of TRα and significantly less TRβ, either of which could be responsible for the T3-dependent effects on neurite outgrowth. Although the overall tau protein expression was not affected in response to T3 treatment, the splicing of tau exon 10 was impacted in the direction of producing more tau molecules that excluded the exon (tau 3R).Conclusion:The results of this study are critical not only to understand the probable link between hypothyroidism and AD but also in providing the basis for future prevention and treatment of AD in hypothyroid patients.

Recombinant Zika NS1 Protein Secreted from Vero Cells Is Efficient for Inducing Production of Immune Serum Directed against NS1 Dimer.

Zika virus (ZIKV) is a mosquito-borne flavivirus that recently emerged in the South Pacific, Americas, and Caribbean islands, where the larger epidemics were documented. ZIKV infection in humans is responsible for neurological disorders and microcephaly. Flavivirus NS1 is a non-structural glycoprotein that is expressed on the cell surface and secreted as a hexameric lipoprotein particle. Intracellular NS1 exists as a dimer that is required for viral replication, whereas the secreted NS1 hexamer interacts with host factors, leading to pathophysiological conditions. In an effort to dispose of specific anti-ZIKV NS1 immune serum, Vero cells were transduced with a lentiviral vector containing the NS1 gene from an epidemic strain of ZIKV. We showed that stably transduced Vero/ZIKV NS1 cell clone was efficient in the secretion of recombinant NS1 oligomer. Immunization of adult rat with purified extracellular NS1 developed anti-ZIKV antibodies that specifically react with the NS1 dimer produced in human cells infected with African and Asian strains of ZIKV. The rat antibody against ZIKV NS1 dimer is a reliable biological tool that enables the immunological detection of secreted NS1 from host-cells infected with ZIKV.

Activin A induces apoptosis of mouse myeloma cells via the mitochondrial pathway.

Activin A is a pleiotropic cytokine belonging to the transforming growth factor β superfamily. Abnormal expression of activin A is associated with tumorigenesis. Multiple myeloma is characterized by the development of osteolytic disease, which ultimately leads to cachexia. However, the involvement of activin A in myeloma cell viability and apoptosis remains to be fully elucidated. For this purpose, mouse myeloma NS-1 cells were treated with activin A, and subsequently subjected to 5-bromo-2'-deoxyuridine analysis, Hoechst 33342 staining, flow cytometry and western blot analysis. The results revealed that activin A significantly suppressed NS-1 cell viability, and induced NS-1 cell apoptosis. In addition, activin A-induced promotion of NS-1 cell apoptosis was accompanied by upregulated expression of BCL2 associated X, apoptosis regulator (Bax), but downregulated expression of B cell lymphoma-2 (Bcl-2), resulting in an increase of the Bax/Bcl-2 ratio. Furthermore, cytochrome c and caspase-3 protein expression also increased following treatment with activin A. These data suggest that activin A induces apoptosis in mouse myeloma NS-1 cells via the mitochondrial pathway, providing a novel insight into multiple myeloma treatment.

Transgenerational effects of early life trauma: Epigenetic mechanisms involving the germline

First messenger-dependent activation of MAP kinases in neuronal and endocrine cells is critical for cell differentiation and function and requires guanine nucleotide exchange factor (GEF)-mediated activation of downstream Ras family small GTPases, which ultimately lead to ERK, JNK, and p38 phosphorylation. Because there are numerous GEFs and also a host of Ras family small GTPases, it is important to know which specific GEF–small GTPase dyad functions in a given cellular process. Here we investigated the upstream activators and downstream effectors of signaling via the GEF Epac2 in the neuroendocrine NS-1 cell line. Three cAMP sensors, Epac2, PKA, and neuritogenic cAMP sensor–Rapgef2, mediate distinct cellular outputs: p38-dependent growth arrest, cAMP response element–binding protein–dependent cell survival, and ERK-dependent neuritogenesis, respectively, in these cells. Previously, we found that cAMP-induced growth arrest of PC12 and NS-1 cells requires Epac2-dependent activation of p38 MAP kinase, which posed the important question of how Epac2 engages p38 without simultaneously activating other MAP kinases in neuronal and endocrine cells. We now show that the small GTP-binding protein Rap2A is the obligate effector for, and GEF substrate of, Epac2 in mediating growth arrest through p38 activation in NS-1 cells. This new pathway is distinctly parcellated from the G protein—coupled receptor → Gs → adenylate cyclase → cAMP → PKA → cAMP response element–binding protein pathway mediating cell survival and the G protein—coupled receptor → Gs → adenylate cyclase → cAMP → neuritogenic cAMP sensor–Rapgef2 → B-Raf → MEK → ERK pathway mediating neuritogenesis in NS-1 cells.

Guanine nucleotide exchange factor Epac2–dependent activation of the GTP-binding protein Rap2A mediates cAMP-dependent growth arrest in neuroendocrine cells

First messenger-dependent activation of MAP kinases in neuronal and endocrine cells is critical for cell differentiation and function and requires guanine nucleotide exchange factor (GEF)-mediated activation of downstream Ras family small GTPases, which ultimately lead to ERK, JNK, and p38 phosphorylation. Because there are numerous GEFs and also a host of Ras family small GTPases, it is important to know which specific GEF-small GTPase dyad functions in a given cellular process. Here we investigated the upstream activators and downstream effectors of signaling via the GEF Epac2 in the neuroendocrine NS-1 cell line. Three cAMP sensors, Epac2, PKA, and neuritogenic cAMP sensor-Rapgef2, mediate distinct cellular outputs: p38-dependent growth arrest, cAMP response element-binding protein-dependent cell survival, and ERK-dependent neuritogenesis, respectively, in these cells. Previously, we found that cAMP-induced growth arrest of PC12 and NS-1 cells requires Epac2-dependent activation of p38 MAP kinase, which posed the important question of how Epac2 engages p38 without simultaneously activating other MAP kinases in neuronal and endocrine cells. We now show that the small GTP-binding protein Rap2A is the obligate effector for, and GEF substrate of, Epac2 in mediating growth arrest through p38 activation in NS-1 cells. This new pathway is distinctly parcellated from the G protein-coupled receptor → Gs → adenylate cyclase → cAMP → PKA → cAMP response element-binding protein pathway mediating cell survival and the G protein-coupled receptor → Gs → adenylate cyclase → cAMP → neuritogenic cAMP sensor-Rapgef2 → B-Raf → MEK → ERK pathway mediating neuritogenesis in NS-1 cells.