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Sample records for neuro2a cells cotransfected

  1. Protective effects of osmolytes in cryopreserving adherent neuroblastoma (Neuro-2a) cells.

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    Bailey, Trisha L; Wang, Mian; Solocinski, Jason; Nathan, Britto P; Chakraborty, Nilay; Menze, Michael A

    2015-12-01

    A simple method to cryopreserve adherent monolayers of neuronal cells is currently not available, but the development of this technique could facilitate numerous applications in the field of biomedical engineering, cell line development, and drug screening. However, complex tissues of some exceptional animals survive freezing in nature. These animals are known to accumulate several small molecular weight solutes prior to freezing. Following a similar strategy, we investigated the effects of osmolytes such as trehalose, proline, and sucrose as additives to the traditional cryoprotectant dimethyl sulfoxide (Me2SO) in modulating the cryopreservation outcome of mouse neuroblastoma (Neuro-2a) cells. Neuro-2a cells adhered to cell culture plates were incubated for 24 h at varying concentrations of trehalose, proline, sucrose and combinations of these compounds. Cells were cryopreserved for 24 h and cell viability post-freezing and thawing was quantified by trypan blue exclusion assay. On average, only 13.5% of adherent cells survived freezing in the presence of 10% Me2SO alone (control). Pre-incubation of cells with medium containing both trehalose and proline severely decreased cell proliferation, but increased cell recovery to about 53% of control. Furthermore, characterization using Raman microspectroscopy revealed that the addition of both trehalose and proline to 10% Me2SO substantially increased the size, and altered the nature, of ice crystals formed during freezing. Our results suggest that pre-incubation of Neuro-2a cells with trehalose and proline in combination provides cell protection along with alterations of ice structure in order to increase cell survival post-freezing. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. β-Hydroxy-β-Methylbutyrate (HMB Promotes Neurite Outgrowth in Neuro2a Cells.

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    Rafael Salto

    Full Text Available β-Hydroxy-β-methylbutyrate (HMB has been shown to enhance cell survival, differentiation and protein turnover in muscle, mainly activating phosphoinositide-3-kinase/protein kinase B (PI3K/Akt and mitogen-activated protein kinases/ extracellular-signal-regulated kinases (MAPK/ERK signaling pathways. Since these two pathways are related to neuronal survival and differentiation, in this study, we have investigated the neurotrophic effects of HMB in mouse neuroblastoma Neuro2a cells. In Neuro2a cells, HMB promotes differentiation to neurites independent from any effects on proliferation. These effects are mediated by activation of both the PI3K/Akt and the extracellular-signal-regulated kinases (ERK1/2 signaling as demonstrated by the use of specific inhibitors of these two pathways. As myocyte-enhancer factor 2 (MEF2 family of transcription factors are involved in neuronal survival and plasticity, the transcriptional activity and protein levels of MEF2 were also evaluated. HMB promoted MEF2-dependent transcriptional activity mediated by the activation of Akt and ERK1/2 pathways. Furthermore, HMB increases the expression of brain glucose transporters 1 (GLUT1 and 3 (GLUT3, and mTOR phosphorylation, which translates in a higher protein synthesis in Neuro2a cells. Furthermore, Torin1 and rapamycin effects on MEF2 transcriptional activity and HMB-dependent neurite outgrowth support that HMB acts through mTORC2. Together, these findings provide clear evidence to support an important role of HMB in neurite outgrowth.

  3. The role of apoptosis in Listeria monocytogenes neural infection: listeriolysin O interaction with neuroblastoma Neuro-2a cells.

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    Parra, Maria C; Baquero, Fernando; Perez-Diaz, Jose C

    2008-01-01

    Listeria monocytogenes is the etiological agent of meningitis that affects individuals at high risk such as pregnant women, neonates, the elderly and immunocompromised individuals. Infection by this intracellular pathogen can be lethal if not diagnosed and treated. Mouse neuroblastoma Neuro-2a cells, a neuron-like cell line, were infected with L. monocytogenes. In this study apoptotic changes of neuroblastoma Neuro-2a cells infected with strains of Listeria producing different listeriolysin O levels are investigated by cytotoxicity assay, cellular viability assay, DAPI staining, intranucleosomal DNA fragmentation test, and monoclonal antibodies against ss-DNA. Results show that after internalization, the bacteria induced morphological, functional and genetic changes in the cells characteristic of apoptosis, which was dose-and time-dependent on listeriolysin O. Neuroblastoma Neuro-2a cells represent an interesting model cell line to further the understanding of Listeria pathogenesis within the central nervous system.

  4. Comparative analysis of the effect of low-dimensional alumina structures on cell lines L929 and Neuro-2a

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    Fomenko, A. N., E-mail: alserova@ispms.tsc.ru; Korovin, M. S., E-mail: msk@ispms.tsc.ru [National Research Tomsk Polytechnic University, Lenin Avenue 30, Tomsk, 634050 (Russian Federation)

    2016-08-02

    The paper presents the toxicity evaluation of nanostructures on the basis of alumina of different shape (nanofibers, nanoplates, nanosheets, nanosheet agglomerates) and with similar physical and chemical properties (particle size, specific surface area, phase composition, and zeta potential). The nanostructures were examined by transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD), low-temperature nitrogen adsorption, and dynamic light scattering (DLS). The cytotoxicity of nanostructures was estimated using L929 fibroblast cells and Neuro-2a tumor cells. It has been found that the L929 cells are less subject to the influence of alumina nanoparticles than the Neuro-2a tumor cells. Probably, the differences in the proliferation activity of normal and tumor cells in contact with the synthesized nanostructures are due to a change in the pH of the cell microenvironment.

  5. Magnetic nanoparticles trigger cell proliferation arrest of neuro-2a cells and ROS-mediated endoplasmic reticulum stress response

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    Wang, Pingping; Chen, Chuanfang; Zeng, Kun; Pan, Weidong; Song, Tao

    2014-11-01

    Magnetic nanoparticles (MNPs) have been increasingly applied in various areas, such as the biomedical and electronic industries. The unique properties of MNPs are beneficial to their applications, but concerns about their safety to human health along with the growing applications and production also arise. In this study, the cytotoxicity of superparamagnetic MNPs, with an average diameter of 10 nm and typical diameter range between 5 and 30 nm, was investigated using neuro-2a cells. The MNPs internalized into the cytoplasm of neuro-2a cells and inhibited the cell viability in a dose-dependent manner at concentrations ranging from 100 to 500 μg/mL. The cell growth inhibition would be partly attributed to the MNP-induced cell cycle arrest in the G0/G1 phase. MNPs triggered the endoplasmic reticulum (ER) stress response, as indicated by the up-regulated expression of the classical ER stress genes, binding immunoglobulin protein, activating transcription factor 6, and CCAAT-enhancer-binding protein homologous protein (CHOP). The induced production of cellular reactive oxygen species (ROS) and increased expression of heme oxygenase 1 and nuclear factor erythroid two-related factor two genes demonstrated that oxidative stress was also induced. Furthermore, the clearance of ROS by free radical scavenger N-acetylcysteine reduced the up-regulation of MNP-induced CHOP mRNA expressions, thereby suggesting that ROS was involved in the process of ER stress response induced by MNPs.

  6. Nimodipine but Not Nifedipine Promotes Expression of Fatty Acid 2-Hydroxylase in a Surgical Stress Model Based on Neuro2a Cells

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    Eva Herzfeld

    2017-05-01

    Full Text Available Nimodipine is well characterized for the management of aneurysmal subarachnoid hemorrhage and has been shown to promote a better outcome and less delayed ischemic neurological deficits. Animal and clinical trials show neuroprotective efficacy following nerve injuries. We showed a neuroprotective effect on Neuro2a cells. Subsequent microarray analysis revealed—among others—fatty acid 2-hydroxylase (FA2H upregulated by nimodipine in vitro, which is a component of myelin synthesis. Differentiated Neuro2a cells were analyzed for nimodipine-mediated survival considering stress treatment in comparison to nifedipine-treatment. Cell survival was determined by measurement of LDH activity in the culture medium. Nimodipine decreased surgery-like stress-induced cell death of differentiated Neuro2a cells. Neuro2a cell culture was analyzed for changes in FA2H expression induced by nimodipine or nifedipine in surgery-like stress conditions. We analyzed expression levels of FA2H mRNA and protein by qPCR using fa2h specific primers or a FA2H-specific antibody in nimodipine or nifedipine non- and pre-treated Neuro2a cell culture, respectively. Nimodipine but not nifedipine increases FA2H protein levels and also significantly increases mRNA levels of FA2H in both undifferentiated and differentiated Neuro2a cells. Our findings indicate that higher expression of FA2H induced by nimodipine may cause higher survival of Neuro2a cells stressed with surgery-like stressors.

  7. Nimodipine but Not Nifedipine Promotes Expression of Fatty Acid 2-Hydroxylase in a Surgical Stress Model Based on Neuro2a Cells

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    Herzfeld, Eva; Speh, Lea; Strauss, Christian; Scheller, Christian

    2017-01-01

    Nimodipine is well characterized for the management of aneurysmal subarachnoid hemorrhage and has been shown to promote a better outcome and less delayed ischemic neurological deficits. Animal and clinical trials show neuroprotective efficacy following nerve injuries. We showed a neuroprotective effect on Neuro2a cells. Subsequent microarray analysis revealed—among others—fatty acid 2-hydroxylase (FA2H) upregulated by nimodipine in vitro, which is a component of myelin synthesis. Differentiated Neuro2a cells were analyzed for nimodipine-mediated survival considering stress treatment in comparison to nifedipine-treatment. Cell survival was determined by measurement of LDH activity in the culture medium. Nimodipine decreased surgery-like stress-induced cell death of differentiated Neuro2a cells. Neuro2a cell culture was analyzed for changes in FA2H expression induced by nimodipine or nifedipine in surgery-like stress conditions. We analyzed expression levels of FA2H mRNA and protein by qPCR using fa2h specific primers or a FA2H-specific antibody in nimodipine or nifedipine non- and pre-treated Neuro2a cell culture, respectively. Nimodipine but not nifedipine increases FA2H protein levels and also significantly increases mRNA levels of FA2H in both undifferentiated and differentiated Neuro2a cells. Our findings indicate that higher expression of FA2H induced by nimodipine may cause higher survival of Neuro2a cells stressed with surgery-like stressors. PMID:28467360

  8. Protection of neuroblastoma Neuro2A cells from hypoxia-induced apoptosis by cyclic phosphatidic acid (cPA.

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    Mari Gotoh

    Full Text Available Cyclic phosphatidic acid (cPA is a naturally occurring phospholipid mediator with a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. We have previously shown that cPA significantly suppresses ischemia-induced delayed neuronal death and the accumulation of glial fibrillary acidic protein in the CA1 region of the rat hippocampus. These results indicated that the systemic administration of cPA can protect hippocampal neurons against ischemia-induced delayed neuronal cell death. In the current study, we investigated the effects of cPA on neuronal cell death caused by hypoxia in vitro and the molecular mechanisms underlying these effects. We used cobalt chloride (CoCl(2 to expose cells to hypoxic conditions in vitro. Treating mouse neuroblastoma (Neuro2A cells with CoCl(2 induced nuclear DNA condensation and phosphatidylserine exposure. However, adding cPA led to the suppression of CoCl(2-induced apoptosis in a cPA dose-dependent manner and attenuated the increase in the Bax/Bcl-2 ratio caused by CoCl(2. Quantitative PCR analysis showed that Neuro2A cells strongly express the LPA(1, LPA(2, and LPA(6, which are G-protein coupled receptors that can be activated by cPA. To date, LPA(1 and LPA(2 have been reported to exhibit antiapoptotic activity. Therefore, to assess the roles of LPA(1 and LPA(2 on cPA-induced neuroprotective functions, Ki16425, a selective LPA(1 and LPA(3 antagonist, was adopted to know the LPA(1 function and siRNA was used to knockdown the expression of LPA(2. On the basis of our results, we propose that cPA-induced protection of Neuro2A cells from CoCl(2-induced hypoxia damage is mediated via LPA(2.

  9. Oxygen-glucose deprivation regulates BACE1 expression through induction of autophagy in Neuro-2a/APP695 cells

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    Rong-fu Chen

    2015-01-01

    Full Text Available Our previous findings have demonstrated that autophagy regulation can alleviate the decline of learning and memory by eliminating deposition of extracellular beta-amyloid peptide (Aβ in the brain after stroke, but the exact mechanism is unclear. It is presumed that the regulation of beta-site APP-cleaving enzyme 1 (BACE1, the rate-limiting enzyme in metabolism of Aβ, would be a key site. Neuro-2a/amyloid precursor protein 695 (APP695 cell models of cerebral ischemia were established by oxygen-glucose deprivation to investigate the effects of Rapamycin (an autophagy inducer or 3-methyladenine (an autophagy inhibitor on the expression of BACE1. Either oxygen-glucose deprivation or Rapamycin down-regulated the expression of BACE1 while 3-methyladenine up-regulated BACE1 expression. These results confirm that oxygen-glucose deprivation down-regulates BACE1 expression in Neuro-2a/APP695 cells through the introduction of autophagy.

  10. Bovine herpesvirus 1 can efficiently infect the human (SH-SY5Y) but not the mouse neuroblastoma cell line (Neuro-2A).

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    Thunuguntla, Prasanth; El-Mayet, Fouad S; Jones, Clinton

    2017-03-15

    Bovine herpesvirus 1 (BoHV-1) is a significant bovine pathogen that establishes a life-long latent infection in sensory neurons. Previous attempts to develop immortalized bovine neuronal cells were unsuccessful. Consequently, our understanding of the BoHV-1 latency-reactivation cycle has relied on studying complex virus-host interactions in calves. In this study, we tested whether BoHV-1 can infect human (SH-SY5Y) or mouse (Neuro-2A) neuroblastoma cells. We provide new evidence that BoHV-1 efficiently infects SH-SY5Y cells and yields virus titers approximately 100 fold less than bovine kidney cells. Conversely, virus titers from productively infected Neuro-2A cells were approximately 10,000 fold less than bovine kidney cells. Using a β-Gal expressing virus (gC-Blue), we demonstrate that infection of Neuro-2A cells (actively dividing or differentiated) does not result in efficient virus spread, unlike bovine kidney or SH-SY5Y cells. Additional studies demonstrated that lytic cycle viral gene expression (bICP4 and gE) was readily detected in SH-SY5Y cells: conversely bICP4 was not readily detected in productively infected Neuro-2A cells. Finally, infection of SH-SY5Y and bovine kidney cells, but not Neuro-2A cells, led to rapid activation of the Akt protein kinase. These studies suggest that the Neuro-2A cell line may be a novel cell culture model to identify factors that regulate BoHV-1 productive infection in neuronal cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Piperine-like alkamides from Piper nigrum induce BDNF promoter and promote neurite outgrowth in Neuro-2a cells.

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    Yun, Young Sook; Noda, Sachie; Takahashi, Shigeru; Takahashi, Yuji; Inoue, Hideshi

    2018-01-01

    Black pepper (Piper nigrum) contains a variety of alkamides. Among them, piperine has been reported to have antidepressant-like effects in chronically stressed mice, but little is known about the biological activity of other alkamides. In this study, we investigated the effects of alkamides from white pepper (P. nigrum) on neuronal cells. Twelve alkamides were isolated from white pepper MeOH extracts, and their chemical structures were identified by NMR and MS analyses. The compounds were subjected to assays using the luciferase-reporter gene under the control of the BDNF promoter or cAMP response element in mouse neuroblastoma Neuro-2a cells. In both assays, marked reporter-inducing activity was observed for piperine (1), piperettine (2) and piperylin (7), all of which have in common an (E)-5-(buta-1,3-dien-1-yl)benzo[d] [1, 3] dioxole moiety. Piperettine (2) and piperylin (7) tended to increase endogenous BDNF protein levels. Furthermore, piperylin (7) promoted retinoic acid-induced neurite outgrowth. These results suggest that piperylin (7), or analogues thereof, may have a beneficial effect on disorders associated with dysregulation of BDNF expression, such as depression.

  12. Insulin inhibits extracellular regulated kinase 1/2 phosphorylation in a phosphatidylinositol 3-kinase (PI3) kinase-dependent manner in Neuro2a cells

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    Gispen, W.H.; Heide, L.P. van der; Hoekman, M.F.; Biessels, G.J.

    2003-01-01

    Insulin signalling is well studied in peripheral tissue, but not in neuronal tissue. To gain more insight into neuronal insulin signalling we examined protein kinase B (PKB) and extracellular regulated kinase 1 and 2 (ERK1/2) regulation in serum-deprived Neuro2a cells. Insulin phosphorylated PKB in

  13. 8-oxoG DNA Glycosylase-1 Inhibition Sensitizes Neuro-2a Cells to Oxidative DNA Base Damage Induced by 900 MHz Radiofrequency Electromagnetic Radiation

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    Xiaoya Wang

    2015-09-01

    Full Text Available Background/Aims: The purpose of this study was to explore the in vitro putative genotoxicity during exposure of Neuro-2a cells to radiofrequency electromagnetic fields (RF-EMFs with or without silencing of 8-oxoG DNA glycosylase-1 (OGG1. Methods: Neuro-2a cells treated with or without OGG1 siRNA were exposed to 900 MHz Global System for Mobile Communication (GSM Talk signals continuously at a specific absorption rate (SAR of 0, 0.5, 1 or 2 W/kg for 24 h. DNA strand breakage and DNA base damage were measured by the alkaline comet assay and a modified comet assay using formamidopyrimidine DNA glycosylase (FPG, respectively. Reactive oxygen species (ROS levels and cell viability were monitored using the non-fluorescent probe 2, 7-dichlorofluorescein diacetate (DCFH-DA and CCK-8 assay. Results: Exposure to 900 MHz RF-EMFs with insufficient energy could induce oxidative DNA base damage in Neuro-2a cells. These increases were concomitant with similar increases in the generation of reactive oxygen species (ROS. Without OGG1 siRNA, 2 W/kg RF-EMFs induced oxidative DNA base damage in Neuro-2a cells. Interestingly, with OGG1 siRNA, RF-EMFs could cause DNA base damage in Neuro-2a cells as low as 1 W/kg. However, neither DNA strand breakage nor altered cell viability was observed. Conclusion: Even if further studies remain conducted we support the hypothesis that OGG1 is involved in the process of DNA base repair and may play a pivotal role in protecting DNA bases from RF-EMF induced oxidative damage.

  14. 8-oxoG DNA glycosylase-1 inhibition sensitizes Neuro-2a cells to oxidative DNA base damage induced by 900 MHz radiofrequency electromagnetic radiation.

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    Wang, Xiaoya; Liu, Chuan; Ma, Qinglong; Feng, Wei; Yang, Lingling; Lu, Yonghui; Zhou, Zhou; Yu, Zhengping; Li, Wei; Zhang, Lei

    2015-01-01

    The purpose of this study was to explore the in vitro putative genotoxicity during exposure of Neuro-2a cells to radiofrequency electromagnetic fields (RF-EMFs) with or without silencing of 8-oxoG DNA glycosylase-1 (OGG1). Neuro-2a cells treated with or without OGG1 siRNA were exposed to 900 MHz Global System for Mobile Communication (GSM) Talk signals continuously at a specific absorption rate (SAR) of 0, 0.5, 1 or 2 W/kg for 24 h. DNA strand breakage and DNA base damage were measured by the alkaline comet assay and a modified comet assay using formamidopyrimidine DNA glycosylase (FPG), respectively. Reactive oxygen species (ROS) levels and cell viability were monitored using the non-fluorescent probe 2, 7-dichlorofluorescein diacetate (DCFH-DA) and CCK-8 assay. Exposure to 900 MHz RF-EMFs with insufficient energy could induce oxidative DNA base damage in Neuro-2a cells. These increases were concomitant with similar increases in the generation of reactive oxygen species (ROS). Without OGG1 siRNA, 2 W/kg RF-EMFs induced oxidative DNA base damage in Neuro-2a cells. Interestingly, with OGG1 siRNA, RF-EMFs could cause DNA base damage in Neuro-2a cells as low as 1 W/kg. However, neither DNA strand breakage nor altered cell viability was observed. Even if further studies remain conducted we support the hypothesis that OGG1 is involved in the process of DNA base repair and may play a pivotal role in protecting DNA bases from RF-EMF induced oxidative damage. © 2015 The Author(s) Published by S. Karger AG, Basel.

  15. Investigation of the Neuroprotective Impact of Nimodipine on Neuro2a Cells by Means of a Surgery-Like Stress Model

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    Eva Herzfeld

    2014-10-01

    Full Text Available Nimodipine is well characterized for the management of SAH (subarachnoid hemorrhage and has been shown to promote a better outcome and less DIND (delayed ischemic neurological deficits. In rat experiments, enhanced axonal sprouting and higher survival of motoneurons was demonstrated after cutting or crushing the facial nerve by nimodipine. These results were confirmed in clinical trials following vestibular Schwannoma surgery. The mechanism of the protective competence of nimodipine is unknown. Therefore, in this study, we established an in vitro model to examine the survival of Neuro2a cells after different stress stimuli occurring during surgery with or without nimodipine. Nimodipine significantly decreased ethanol-induced cell death of cells up to approximately 9% in all tested concentrations. Heat-induced cell death was diminished by approximately 2.5% by nimodipine. Cell death induced by mechanical treatment was reduced up to 15% by nimodipine. Our findings indicate that nimodipine rescues Neuro2a cells faintly, but significantly, from ethanol-, heat- and mechanically-induced cell death to different extents in a dosage-dependent manner. This model seems suitable for further investigation of the molecular mechanisms involved in the neuroprotective signal pathways influenced by nimodipine.

  16. Investigation of the neuroprotective impact of nimodipine on Neuro2a cells by means of a surgery-like stress model.

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    Herzfeld, Eva; Strauss, Christian; Simmermacher, Sebastian; Bork, Kaya; Horstkorte, Rüdiger; Dehghani, Faramarz; Scheller, Christian

    2014-10-14

    Nimodipine is well characterized for the management of SAH (subarachnoid hemorrhage) and has been shown to promote a better outcome and less DIND (delayed ischemic neurological deficits). In rat experiments, enhanced axonal sprouting and higher survival of motoneurons was demonstrated after cutting or crushing the facial nerve by nimodipine. These results were confirmed in clinical trials following vestibular Schwannoma surgery. The mechanism of the protective competence of nimodipine is unknown. Therefore, in this study, we established an in vitro model to examine the survival of Neuro2a cells after different stress stimuli occurring during surgery with or without nimodipine. Nimodipine significantly decreased ethanol-induced cell death of cells up to approximately 9% in all tested concentrations. Heat-induced cell death was diminished by approximately 2.5% by nimodipine. Cell death induced by mechanical treatment was reduced up to 15% by nimodipine. Our findings indicate that nimodipine rescues Neuro2a cells faintly, but significantly, from ethanol-, heat- and mechanically-induced cell death to different extents in a dosage-dependent manner. This model seems suitable for further investigation of the molecular mechanisms involved in the neuroprotective signal pathways influenced by nimodipine.

  17. Potentiated Osteoinductivity via Cotransfection with BMP-2 and VEGF Genes in Microencapsulated C2C12 Cells

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    Yang Shen

    2015-01-01

    Full Text Available Microcapsules with entrapped cells hold great promise for repairing bone defects. Unfortunately, the osteoinductivity of microcapsules has been restricted by many factors, among which the deficiency of functional proteins is a significant priority. We potentiated the osteoinductivity of microencapsulated cells via cotransfection with BMP-2 and VEGF genes. Various tissue-derived mesenchymal stem cells and cell lines were compared for BMP-2 and VEGF cotransfection. Ethidium bromide (EB/Calcein AM staining revealed that all of the cell categories could survive for 4 weeks after microencapsulation. An ELISA assay indicated that all microencapsulated BMP-2 or VEGF transfected cells could secrete gene products constitutively for 1 month. Particularly, the recombinant microencapsulated C2C12 cells released the most desirable level of BMP-2 and VEGF. Further experiments demonstrated that microencapsulated BMP-2 and VEGF cotransfected C2C12 cells generated both BMP-2 and VEGF for 4 weeks. Additionally, the cotransfection of BMP-2 and VEGF in microencapsulated C2C12 cells showed a stronger osteogenic induction against BMSCs than individual BMP-2-transfected microencapsulated C2C12 cells. These results demonstrated that the cotransfection of BMP-2 and VEGF into microencapsulated C2C12 cells is of potent utility for the potentiation of bone regeneration, which would provide a promising clinical strategy for cellular therapy in bone defects.

  18. Novel of core-shell AlOOH/Cu nanostructures: Synthesis, characterization, antimicrobial activity and in vitro toxicity in Neuro-2a cells

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    Bakina, O. V., E-mail: ovbakina@ispms.tsc.ru; Fomenko, A. N., E-mail: alserova@ispms.tsc.ru; Korovin, M. S., E-mail: msk@ispms.tsc.ru; Glazkova, E. A., E-mail: eagl@ispms.tsc.ru; Svarovskaya, N. V., E-mail: nvsv@ispms.tsc.ru [Institute of Strength Physics and Materials Sciences SB RAS, Akademicheskii Pr. 2/4, Tomsk, 634055 (Russian Federation); National Research Tomsk Polytechnic University, Lenin Avenue 30, Tomsk, 634050 (Russian Federation)

    2016-08-02

    Core-shell micro/nanostructures were fabricated by the reaction of Al/Cu bimetallic nanoparticles with water. Al/Cu nanoparticles have been obtained using the method of simultaneous electrical explosion of a pair of the corresponding metal wires in an argon atmosphere. The nanoparticles are chemically active and interact with water at 60°C to form core-shell micro/nanostructures. The obtained products were characterized by means of X-ray diffraction, scanning electron microscopy, transmission electron microscopy and dynamic light scattering and the nitrogen adsorption method. The antibacterial activity of the synthesized structures was investigated against E. coli and St. aureus. The toxic effect of these nanostructures against the Neuro-2a neuroblastoma cell line was investigated. AlOOH/Cu nanostructures are shown to inhibit cell proliferation. The AlOOH/Cu nanostructures are good candidates for medical applications.

  19. The all-trans retinoic acid (atRA)-regulated gene Calmin (Clmn) regulates cell cycle exit and neurite outgrowth in murine neuroblastoma (Neuro2a) cells

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    Marzinke, Mark A. [Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706-1544 (United States); Clagett-Dame, Margaret, E-mail: dame@biochem.wisc.edu [Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706-1544 (United States); Pharmaceutical Science Division, University of Wisconsin-Madison, Madison, WI 53705-2222 (United States)

    2012-01-01

    The vitamin A metabolite all-trans retinoic acid (atRA) functions in nervous system development and regulates cell proliferation and differentiation. Neuroblastoma cells (SH-SY5Y and Neuro2a or N2A) exposed to atRA undergo growth inhibition and neuronal differentiation, both of which are preceded by an increase in Clmn mRNA. Treatment of N2A cells with atRA produces a reduction in phosphohistone 3 immunostaining and BrdU incorporation, both indicators of a reduction in cell proliferation. These effects are nearly eliminated in atRA-treated shClmn knockdown cells. Loss of Clmn in the mouse N2A cell line also results in a significant reduction of atRA-mediated neurite outgrowth, a response that can be rescued by reintroduction of the Clmn sequence. In contrast, ectopic overexpression of Clmn produces an increase in the cyclin dependent kinase inhibitor, p21{sup Cip1}, a decrease in cyclin D1 protein and an increase in hypophosphorylated Rb, showing that Clmn participates in G{sub 1}/S arrest. Clmn overexpression alone is sufficient to inhibit N2A cell proliferation, whereas both Clmn and atRA must be present to induce neurite outgrowth. This study shows that the atRA-responsive gene Clmn promotes exit from the cell cycle, a requisite event for neuronal differentiation. -- Highlights: Black-Right-Pointing-Pointer Calmin is a retinoic acid-responsive gene. Black-Right-Pointing-Pointer Calmin promotes cell cycle exit in N2A cells. Black-Right-Pointing-Pointer Calmin overexpression increases p21Cip1 and decreases cyclin D1. Black-Right-Pointing-Pointer Calmin is required for RA-induced growth inhibition and neurite outgrowth.

  20. Porcine Hemagglutinating Encephalomyelitis Virus Enters Neuro-2a Cells via Clathrin-Mediated Endocytosis in a Rab5-, Cholesterol-, and pH-Dependent Manner.

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    Li, Zi; Zhao, Kui; Lan, Yungang; Lv, Xiaoling; Hu, Shiyu; Guan, Jiyu; Lu, Huijun; Zhang, Jing; Shi, Junchao; Yang, Yawen; Song, Deguang; Gao, Feng; He, Wenqi

    2017-12-01

    Porcine hemagglutinating encephalomyelitis virus (PHEV) is a highly neurovirulent coronavirus that invades the central nervous system (CNS) in piglets. Although important progress has been made toward understanding the biology of PHEV, many aspects of its life cycle remain obscure. Here we dissected the molecular mechanism underlying cellular entry and intracellular trafficking of PHEV in mouse neuroblastoma (Neuro-2a) cells. We first performed a thin-section transmission electron microscopy (TEM) assay to characterize the kinetics of PHEV, and we found that viral entry and transfer occur via membranous coating-mediated endo- and exocytosis. To verify the roles of distinct endocytic pathways, systematic approaches were used, including pharmacological inhibition, RNA interference, confocal microscopy analysis, use of fluorescently labeled virus particles, and overexpression of a dominant negative (DN) mutant. Quantification of infected cells showed that PHEV enters cells by clathrin-mediated endocytosis (CME) and that low pH, dynamin, cholesterol, and Eps15 are indispensably involved in this process. Intriguingly, PHEV invasion leads to rapid actin rearrangement, suggesting that the intactness and dynamics of the actin cytoskeleton are positively correlated with viral endocytosis. We next investigated the trafficking of internalized PHEV and found that Rab5- and Rab7-dependent pathways are required for the initiation of a productive infection. Furthermore, a GTPase activation assay suggested that endogenous Rab5 is activated by PHEV and is crucial for viral progression. Our findings demonstrate that PHEV hijacks the CME and endosomal system of the host to enter and traffic within neural cells, providing new insights into PHEV pathogenesis and guidance for antiviral drug design. IMPORTANCE Porcine hemagglutinating encephalomyelitis virus (PHEV), a nonsegmented, positive-sense, single-stranded RNA coronavirus, invades the central nervous system (CNS) and causes

  1. Down-regulation of the mitochondrial aspartate-glutamate carrier isoform 1 AGC1 inhibits proliferation and N-acetylaspartate synthesis in Neuro2A cells.

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    Profilo, Emanuela; Peña-Altamira, Luis Emiliano; Corricelli, Mariangela; Castegna, Alessandra; Danese, Alberto; Agrimi, Gennaro; Petralla, Sabrina; Giannuzzi, Giulia; Porcelli, Vito; Sbano, Luigi; Viscomi, Carlo; Massenzio, Francesca; Palmieri, Erika Mariana; Giorgi, Carlotta; Fiermonte, Giuseppe; Virgili, Marco; Palmieri, Luigi; Zeviani, Massimo; Pinton, Paolo; Monti, Barbara; Palmieri, Ferdinando; Lasorsa, Francesco Massimo

    2017-06-01

    The mitochondrial aspartate-glutamate carrier isoform 1 (AGC1) catalyzes a Ca 2+ -stimulated export of aspartate to the cytosol in exchange for glutamate, and is a key component of the malate-aspartate shuttle which transfers NADH reducing equivalents from the cytosol to mitochondria. By sustaining the complete glucose oxidation, AGC1 is thought to be important in providing energy for cells, in particular in the CNS and muscle where this protein is mainly expressed. Defects in the AGC1 gene cause AGC1 deficiency, an infantile encephalopathy with delayed myelination and reduced brain N-acetylaspartate (NAA) levels, the precursor of myelin synthesis in the CNS. Here, we show that undifferentiated Neuro2A cells with down-regulated AGC1 display a significant proliferation deficit associated with reduced mitochondrial respiration, and are unable to synthesize NAA properly. In the presence of high glutamine oxidation, cells with reduced AGC1 restore cell proliferation, although oxidative stress increases and NAA synthesis deficit persists. Our data suggest that the cellular energetic deficit due to AGC1 impairment is associated with inappropriate aspartate levels to support neuronal proliferation when glutamine is not used as metabolic substrate, and we propose that delayed myelination in AGC1 deficiency patients could be attributable, at least in part, to neuronal loss combined with lack of NAA synthesis occurring during the nervous system development. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Dopamine induces apoptosis in APPswe-expressing Neuro2A cells following Pepstatin-sensitive proteolysis of APP in acid compartments.

    Science.gov (United States)

    Cagnin, Monica; Ozzano, Matteo; Bellio, Natascia; Fiorentino, Ilaria; Follo, Carlo; Isidoro, Ciro

    2012-08-30

    A pathological hallmark of Alzheimer's disease (AD) is the presence within neurons and the interneuronal space of aggregates of β-amyloid (Aβ) peptides that originate from an abnormal proteolytic processing of the amyloid precursor protein (APP). The aspartyl proteases that initiate this processing act in the Golgi and endosomal compartments. Here, we show that the neurotransmitter dopamine stimulates the rapid endocytosis and processing of APP and induces apoptosis in neuroblastoma Neuro2A cells over-expressing transgenic human APP (Swedish mutant). Apoptosis could be prevented by impairing Pepstatin-sensitive and acid-dependent proteolysis of APP within endosomal-lysosomal compartments. The γ-secretase inhibitor L685,458 and the α-secretase stimulator phorbol ester elicited protection from dopamine-induced proteolysis of APP and cell toxicity. Our data shed lights on the mechanistic link between dopamine excitotoxicity, processing of APP and neuronal cell death. Since AD often associates with parkinsonian symptoms, which is suggestive of dopaminergic neurodegeneration, the present data provide the rationale for the therapeutic use of lysosomal activity inhibitors such as chloroquine or Pepstatin A to alleviate the progression of AD leading to onset of parkinsonism. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. Characterization of intracellular dynamics of inoculated PrP-res and newly generated PrPSc during early stage prion infection in Neuro2a cells

    Science.gov (United States)

    Yamasaki, Takeshi; Baron, Gerald S; Suzuki, Akio; Hasebe, Rie; Horiuchi, Motohiro

    2014-01-01

    Summary To clarify the cellular mechanisms for the establishment of prion infection, we analyzed the intracellular dynamics of inoculated and newly generated abnormal isoform of prion protein (PrPSc) in Neuro2a cells. Within 24 h after inoculation, the newly generated PrPSc was evident at the plasma membrane, in early endosomes, and in late endosomes, but this PrPSc was barely evident in lysosomes; in contrast, the majority of the inoculated PrPSc was evident in late endosomes and lysosomes. However, during the subsequent 48 h, the newly generated PrPSc increased remarkably in early endosomes and recycling endosomes. Overexpression of wild-type and mutant Rab proteins showed that membrane trafficking along not only the endocytic-recycling pathway but also the endo-lysosomal pathway is involved in de novo PrPSc generation. These results suggest that the trafficking of exogenously introduced PrPSc from the endo-lysosomal pathway to the endocytic-recycling pathway is important for the establishment of prion infection. PMID:24503096

  4. Enhanced transient recombinant protein production in CHO cells through the co-transfection of the product gene with Bcl-xL

    Science.gov (United States)

    Zustiak, Matthew P.; Jose, Lisa; Xie, Yueqing; Zhu, Jianwei; Betenbaugh, Micheal J.

    2014-01-01

    Transient gene expression is gaining popularity as a method to rapidly produce recombinant proteins in mammalian cells. Although significant improvements have been made, in terms of expression, more improvements are needed to compete with the yields achievable in stable gene expression. Much progress has come from optimization of transfection media and parameters, as well as altering culturing conditions to enhance productivity. Recent studies have included using cell lines engineered for apoptosis resistance through the constitutive expression of an anti-apoptotic protein, Bcl-xL. In this study we examine an alternative method of using the benefits of anti-apoptotic gene expression to enhance the transient expression of biotherapeutics, namely, through the co-transfection of bcl-xL and the product-coding gene. CHO-S cells were co-transfected with the product-coding gene and a vector containing Bcl-xL using polyethylenimine. Cells co-transfected with Bcl-xL showed reduced levels of apoptosis, increased specific productivity, and an overall increase in product yield of approximately 100%. Similar results were produced by employing another anti-apoptotic protein, Bcl-2 delta in CHO cells, or through the co-transfection with bcl-xL using HEK-293E cells. This work provides an alternative method for increasing yields of therapeutic proteins in TGE applications without generating a prior stable cell line and subsequent screening which are both time and resource consuming. PMID:24604826

  5. JNK1 and JNK3 play a significant role in both neuronal apoptosis and necrosis. Evaluation based on in vitro approach using tert-butylhydroperoxide induced oxidative stress in neuro-2A cells and perturbation through 3-aminobenzamide.

    Science.gov (United States)

    Muthaiah, Vijaya Prakash Krishnan; Michael, Felicia Mary; Palaniappan, Tamilselvi; Rajan, Sridhar Skylab; Chandrasekar, Kirubhanand; Venkatachalam, Sankar

    2017-06-01

    In spinal cord injury (SCI), oxidative stress in the penumbra of the injury site is a characteristic feature. The predominance of necrosis over apoptosis in the ensuing delayed cell death results in progressive waves of necrosis affecting neighboring cells and thus exaggerates the severity of the lesion. Necrosis has been classified into subtypes based on the active molecular players and parthanatos is one among them, which is characterized by the over activation of PARP1 as the pre-mitochondrial event that triggers necrosis. Parthanatos being the necrosis mode reported in SCI, we intended to study the molecular players in the elusive pre-mitochondrial events of PARP1 over activation using an in vitro model. tert-Butylhydroperoxide (tBuOOH) was reported to induce oxidative stress in various cell types including Neuro-2A cells. Using a tailored protocol, a predominantly PARP1 mediated necrotic mode of cell death was obtained in Neuro-2A cells using tBuOOH. By perturbing the progress of necrosis using 3-amniobenzamide, a known PARP1 inhibitor, it was found that JNK1 and JNK3 but not JNK2 were involved in pre-mitochondrial stages of PARP1 mediated cell death. Given that JNK1 and JNK3 play a role in apoptosis also, they may serve as common targets to counter both apoptosis and necrosis. The in vitro model used in the present study may be useful in delineating molecular mechanisms in necrosis. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Co-transfection of dendritic cells with AFP and IL-2 genes enhances the induction of tumor antigen-specific antitumor immunity.

    Science.gov (United States)

    Yang, Jing-Yue; Li, Xiao; Gao, Li; Teng, Zeng-Hui; Liu, Wen-Chao

    2012-10-01

    Dendritic cells (DCs) are highly efficient, specialized antigen-presenting cells and DCs transfected with tumor-related antigens are regarded as promising vaccines in cancer immunotherapy. The aim of the present study was to investigate whether DCs co-transfected with the α-fetoprotein (AFP) and human interleukin-2 (IL-2) genes were able to induce stronger therapeutic antitumor immunity in transfected DCs. In this study, DCs from hepatocellular carcinoma (HCC) patients were co-transfected with the IL-2 gene and/or the AFP gene. The reverse transcription-PCR (RT-PCR) data revealed that the DCs transfected with the adenovirus AdAFP/IL-2 expressed AFP and IL-2. The DCs co-transfected with IL-2 and AFP (AFP/IL-2-DCs) enhanced the cytotoxicities of cytotoxic T lymphocytes (CTLs) and increased the production of IL-2 and interferon-γ significantly compared with their AFP-DC, green fluorescent protein (GFP)-DC, DC or phosphate-buffered saline (PBS) counterparts. In vivo data suggested that immunization with AFP-DCs enhances antigen-specific antitumor efficacy more potently than immunization with IL-2-DCs or AFP-DCs. These findings provide a potential strategy to improve the efficacy of DC-based tumor vaccines.

  7. [RECOMBINANT ADENOVIRUS-MEDIATED BONE MORPHOGENETIC PROTEIN 9 AND ERYTHROPOIETIN GENES CO-TRANSFECTION IN PROMOTING OSTEOGENIC DIFFERENTIATION OF ADIPOSE-DERIVED STEM CELLS IN VITRO].

    Science.gov (United States)

    Zhang, Guangde; Su, Chengshuai; Jin, Xia; Yang, Shimao; Fang, Dianji; Guo, Yanwei

    2016-03-01

    To investigate the effect of recombinant adenovirus-mediated bone morphogenetic protein 9 (BMP-9) and erythropoietin (EPO) genes co-transfection on osteogenic differentiation of adipose-derived stem cells (ADSCs) in vitro. The inguinal adipose tissue was harvested from 4-month-old New Zealand rabbits, ADSCs were isolated with enzyme digestion and adherence method, and multipotent differentiation capacity was identified. The 3rd generation ADSCs were divided into 5 groups: normal cells (group A), empty plasmid control group (group B), BMP-9 or EPO recombinant adenovirus transfected cells (groups C and D), BMP-9 and EPO recombinant adenovirus co-transfected cells (group E). The inverted phase contrast microscope was used to observe the cell growth at 7 days; the expression of cell fluorescence was observed under a fluorescence microscope at 14 days, and viral transfection efficiency was calculated at 48 hours; Western blot was used to detect the expressions of BMP-9 and EPO proteins at 14 days. The expression of alkaline phosphatase (ALP) activity was detected at 3, 7, and 14 days after osteogenic induction, and alizarin red staining was used to detect calcium nodules formation and real-time fluorescence quantitative PCR to detect the expressions of osteopontin (OPN) and osteocalcin (OCN) at 3 weeks. At 7 days after transfected, some cells showed oval, round, and irregular shape under the inverted phase contrast microscope in groups A and B; a few fusiform cells were observed in groups C and D; oval cells increased obviously, and there were only few round cells in group E. The fluorescence microscope observation showed that BMP-9 and EPO, BMP-9/EPO recombinant adenovirus could stably transfected ADSCs, with transfection efficiency of 80%-93%. The expressions of BMP-9 and EPO proteins significantly higher in group E than the other groups by Western blot (P transfect ADSCs, which can stably express in ADSCs, BMP-9/EPO genes co-transfection can more promote the

  8. File list: ALL.Neu.20.AllAg.Neuro-2a [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Neu.20.AllAg.Neuro-2a mm9 All antigens Neural Neuro-2a SRX814799,SRX367977,SRX3...X691794,SRX691798,SRX691797,SRX691795 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Neu.20.AllAg.Neuro-2a.bed ...

  9. File list: ALL.Neu.05.AllAg.Neuro-2a [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Neu.05.AllAg.Neuro-2a mm9 All antigens Neural Neuro-2a SRX367977,SRX814799,SRX3...X706576,SRX691799,SRX691797,SRX691795 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Neu.05.AllAg.Neuro-2a.bed ...

  10. File list: Unc.Neu.20.AllAg.Neuro-2a [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Neu.20.AllAg.Neuro-2a mm9 Unclassified Neural Neuro-2a SRX814799,SRX814802,SRX8...14803 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Unc.Neu.20.AllAg.Neuro-2a.bed ...

  11. File list: Oth.Neu.50.AllAg.Neuro-2a [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Neu.50.AllAg.Neuro-2a mm9 TFs and others Neural Neuro-2a SRX706575,SRX691800,SR...X691796,SRX706573,SRX691799,SRX691794,SRX691798,SRX691797,SRX691795 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Neu.50.AllAg.Neuro-2a.bed ...

  12. Cytotoxic Effects of Serum from Equine Grass Sickness Cases on Neuro-2a and PC12 Tet-Off Cell Lines: Implication for Using In Vitro Methods as Antemortem Diagnostic Tools

    NARCIS (Netherlands)

    Malekinejad, H.|info:eu-repo/dai/nl/314000461; Bull, S.; Rahmani, F.; Fink-Gremmels, J.|info:eu-repo/dai/nl/119949997

    2012-01-01

    A wide variety of clinical and paraclinical methods have been used for diagnosis of equine grass sickness (EGS), but none of them could absolutely confirm the diagnosis, and postmortem pathologic examination is still considered the final step in precise diagnosis of EGS. Use of in vitro cell

  13. File list: InP.Neu.20.AllAg.Neuro-2a [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Neu.20.AllAg.Neuro-2a mm9 Input control Neural Neuro-2a SRX691801,SRX706576,SRX...706574 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Neu.20.AllAg.Neuro-2a.bed ...

  14. Neuroprotective effect of olfactory ensheathing cells co-transfected with Nurr1 and Ngn2 in both in vitro and in vivo models of Parkinson's disease.

    Science.gov (United States)

    Liu, Qingqing; Qin, Qi; Sun, Hongxue; Zhong, Di; An, Ran; Tian, Yushuang; Chen, Hongping; Jin, Jing; Wang, Haining; Li, Guozhong

    2018-02-01

    The aim of the study is to evaluate the neuroprotective effects of olfactory ensheathing cells (OECs) with the overexpression of nuclear receptor-related factor 1 (Nurr1) and neurogenin 2 (Ngn2) in experimental models of Parkinson's disease (PD) and to elucidate the potential mechanism underlying the neuroprotective effects of OECs-Nurr1-Ngn2. In vitro study, OECs-Nurr1-Ngn2 conditioned medium (CM) was added to MPP + -treated PC12 cells for 24h, and then the viability of PC12 cells, oxidative stress and apoptosis were detected. In vivo study, 48 male Sprague-Dawley (SD) rats were randomly divided into four groups. OECs/VMCs and OECs-Nurr1-Ngn2/VMCs groups were transplanted with 2×10 5 cells each of OECs or OECs-Nurr1-Ngn2 and VMCs into the right striatum one week after a unilateral 6-OHDA lesion. Control and PD groups were injected with 0.9% NaCl and 0.2% ascorbic acid into the same region. Rotational behavior was determined at 2, 4, 6 and 8weeks after injection or implantation in all groups. Neuronal differentiation markers, oxidative stress- and apoptosis-related indicators were detected at 8weeks post-grafting. OECs-Nurr1-Ngn2 increased the viability of PC12 cells, inhibited oxidative stress and apoptosis, and these effects could be reversed by pre-treatment of k252a, a TrkB receptor inhibitor. The behavioral deficits of PD rat were ameliorated by the transplantation of OECs-Nurr1-Ngn2/VMCs. These results suggest that OECs-Nurr1-Ngn2 exhibits substantial neuroprotective, anti-oxidant, and anti-apoptotic effects against PD via the up-regulation of the neurotrophic factor-TrkB pathway. Copyright © 2018 Elsevier Inc. All rights reserved.

  15. HBV DNA genome co-transfection procedure for the evaluation of relative fitness.

    Directory of Open Access Journals (Sweden)

    Ina Sevic

    Full Text Available Hepatitis B virus (HBV has a high mutation rate and exists as a mixture of genetically different but closely related variants. We present a HBV DNA co-transfection fitness assay and use it to evaluate the relative fitness of different HBV variants in two scenarios: seroconversion process and occupation of an ecological niche. In the seroconversion experiment, subgenotype D1 (sgtD1 deletion (1763-1770 had significantly lower fitness comparing with both sgtD1 wild type and sgtD1mut G1896A, while, in the case of occupation of ecological niche experiment, the results showed the same relative fitness between all of the genotype combinations, except F1b-F4. In this case sgtF1b clearly overgrow sgtF4, which is in accordance with the observation that F1b is the most prevalent in the new infections in Argentina. In summary, we present a method aimed to evaluate HBV viral fitness which improve the analysis of the relative frequency of viral variants during the HBV infection process.

  16. A beta-lactone related to lactacystin induces neurite outgrowth in a neuroblastoma cell line and inhibits cell cycle progression in an osteosarcoma cell line.

    OpenAIRE

    Fenteany, G; Standaert, R F; Reichard, G A; Corey, E J; Schreiber, S L

    1994-01-01

    Lactacystin, a microbial natural product, induces neurite outgrowth in Neuro 2A mouse neuroblastoma cells and inhibits progression of synchronized Neuro 2A cells and MG-63 human osteosarcoma cells beyond the G1 phase of the cell cycle. A related beta-lactone, clasto-lactacystin beta-lactone, formally the product of elimination of N-acetylcysteine from lactacystin, is also active, whereas the corresponding clastolactacystin dihydroxy acid is completely inactive. Structural analogs of lactacyst...

  17. Pim-1 kinase inhibits the activation of reporter gene expression in Elk-1 and c-Fos reporting systems but not the endogenous gene expression: an artifact of the reporter gene assay by transient co-transfection

    Directory of Open Access Journals (Sweden)

    Yan B.

    2006-01-01

    Full Text Available We have studied the molecular mechanism and signal transduction of pim-1, an oncogene encoding a serine-threonine kinase. This is a true oncogene which prolongs survival and inhibits apoptosis of hematopoietic cells. In order to determine whether the effects of Pim-1 occur by regulation of the mitogen-activated protein kinase pathway, we used a transcriptional reporter assay by transient co-transfection as a screening method. In this study, we found that Pim-1 inhibited the Elk-1 and NFkappaB transcriptional activities induced by activation of the mitogen-activated protein kinase cascade in reporter gene assays. However, Western blots showed that the induction of Elk-1-regulated expression of endogenous c-Fos was not affected by Pim-1. The phosphorylation and activation of neither Erk1/2 nor Elk-1 was influenced by Pim-1. Also, in the gel shift assay, the pattern of endogenous NFkappaB binding to its probe was not changed in any manner by Pim-1. These data indicate that Pim-1 does not regulate the activation of Erk1/2, Elk-1 or NFkappaB. These contrasting results suggest a pitfall of the transient co-transfection reporter assay in analyzing the regulation of transcription factors outside of the chromosome context. It ensures that results from reporter gene expression assay should be verified by study of endogenous gene expression.

  18. NGF and TERT co-transfected BMSCs improve the restoration of cognitive impairment in vascular dementia rats.

    Directory of Open Access Journals (Sweden)

    Fei Wang

    Full Text Available Vascular dementia (VaD is a mental disorder caused by brain damage due to cerebrovascular disease, and incidence of VaD is rising. To date, there is no known effective cure for VaD, so effort in developing an effective treatment for VaD is of great importance. The differentiation plasticity of BMSCs, in conjunction with its weak immunogenicity, makes manipulated BMSCs an attractive strategy for disease treatment. However, BMSCs often display disabled differentiation, premature aging, and unstable proliferation, reducing their neuroprotective function. These problems may be caused by the lack of telomerase activity in BMSCs. Our results show that NGF-TERT co-transfected BMSCs have a better therapeutic effect than BMSCs lacking NGF and TERT expression, demonstrated by significant improvements in learning and memory in VaD rats. The underlying mechanism might be increased expression of NGF, TrkA and SYN in the hippocampal CA1 area, which has potential implication in advancing therapeutics for VaD.

  19. Neurite outgrowth stimulatory effects of culinary-medicinal mushrooms and their toxicity assessment using differentiating Neuro-2a and embryonic fibroblast BALB/3T3.

    Science.gov (United States)

    Phan, Chia-Wei; David, Pamela; Naidu, Murali; Wong, Kah-Hui; Sabaratnam, Vikineswary

    2013-10-11

    Mushrooms are not only regarded as gourmet cuisine but also as therapeutic agent to promote cognition health. However, little toxicological information is available regarding their safety. Therefore, the aim of this study was to screen selected ethno-pharmacologically important mushrooms for stimulatory effects on neurite outgrowth and to test for any cytotoxicity. The stimulatory effect of mushrooms on neurite outgrowth was assessed in differentiating mouse neuroblastoma (N2a) cells. Neurite length was measured using Image-Pro Insight processor system. Neuritogenesis activity was further validated by fluorescence immunocytochemical staining of neurofilaments. In vitro cytotoxicity was investigated by using mouse embryonic fibroblast (BALB/3T3) and N2a cells for any embryo- and neuro-toxic effects; respectively. Aqueous extracts of Ganoderma lucidum, Lignosus rhinocerotis, Pleurotus giganteus and Grifola frondosa; as well as an ethanol extract of Cordyceps militaris significantly (p < 0.05) promoted the neurite outgrowth in N2a cells by 38.4 ± 4.2%, 38.1 ± 2.6%, 33.4 ± 4.6%, 33.7 ± 1.5%, and 35.8 ± 3.4%; respectively. The IC50 values obtained from tetrazolium (MTT), neutral red uptake (NRU) and lactate dehydrogenase (LDH) release assays showed no toxic effects following 24 h exposure of N2a and 3T3 cells to mushroom extracts. Our results indicate that G. lucidum, L. rhinocerotis, P. giganteus, G. frondosa and C. militaris may be developed as safe and healthy dietary supplements for brain and cognitive health.

  20. Neurite outgrowth stimulatory effects of culinary-medicinal mushrooms and their toxicity assessment using differentiating Neuro-2a and embryonic fibroblast BALB/3T3

    Science.gov (United States)

    2013-01-01

    Background Mushrooms are not only regarded as gourmet cuisine but also as therapeutic agent to promote cognition health. However, little toxicological information is available regarding their safety. Therefore, the aim of this study was to screen selected ethno-pharmacologically important mushrooms for stimulatory effects on neurite outgrowth and to test for any cytotoxicity. Methods The stimulatory effect of mushrooms on neurite outgrowth was assessed in differentiating mouse neuroblastoma (N2a) cells. Neurite length was measured using Image-Pro Insight processor system. Neuritogenesis activity was further validated by fluorescence immunocytochemical staining of neurofilaments. In vitro cytotoxicity was investigated by using mouse embryonic fibroblast (BALB/3T3) and N2a cells for any embryo- and neuro-toxic effects; respectively. Results Aqueous extracts of Ganoderma lucidum, Lignosus rhinocerotis, Pleurotus giganteus and Grifola frondosa; as well as an ethanol extract of Cordyceps militaris significantly (p < 0.05) promoted the neurite outgrowth in N2a cells by 38.4 ± 4.2%, 38.1 ± 2.6%, 33.4 ± 4.6%, 33.7 ± 1.5%, and 35.8 ± 3.4%; respectively. The IC50 values obtained from tetrazolium (MTT), neutral red uptake (NRU) and lactate dehydrogenase (LDH) release assays showed no toxic effects following 24 h exposure of N2a and 3T3 cells to mushroom extracts. Conclusion Our results indicate that G. lucidum, L. rhinocerotis, P. giganteus, G. frondosa and C. militaris may be developed as safe and healthy dietary supplements for brain and cognitive health. PMID:24119256

  1. Sialidase NEU4 hydrolyzes polysialic acids of neural cell adhesion molecules and negatively regulates neurite formation by hippocampal neurons.

    Science.gov (United States)

    Takahashi, Kohta; Mitoma, Junya; Hosono, Masahiro; Shiozaki, Kazuhiro; Sato, Chihiro; Yamaguchi, Kazunori; Kitajima, Ken; Higashi, Hideyoshi; Nitta, Kazuo; Shima, Hiroshi; Miyagi, Taeko

    2012-04-27

    Modulation of levels of polysialic acid (polySia), a sialic acid polymer, predominantly associated with the neural cell adhesion molecule (NCAM), influences neural functions, including synaptic plasticity, neurite growth, and cell migration. Biosynthesis of polySia depends on two polysialyltransferases ST8SiaII and ST8SiaIV in vertebrate. However, the enzyme involved in degradation of polySia in its physiological turnover remains uncertain. In the present study, we identified and characterized a murine sialidase NEU4 that catalytically degrades polySia. Murine NEU4, dominantly expressed in the brain, was found to efficiently hydrolyze oligoSia and polySia chains as substrates in sialidase in vitro assays, and also NCAM-Fc chimera as well as endogenous NCAM in tissue homogenates of postnatal mouse brain as assessed by immunoblotting with anti-polySia antibodies. Degradation of polySia by NEU4 was also evident in neuroblastoma Neuro2a cells that were co-transfected with Neu4 and ST8SiaIV genes. Furthermore, in mouse embryonic hippocampal primary neurons, the endogenously expressed NEU4 was found to decrease during the neuronal differentiation. Interestingly, GFP- or FLAG-tagged NEU4 was partially co-localized with polySia in neurites and significantly suppressed their outgrowth, whereas silencing of NEU4 showed the acceleration together with an increase in polySia expression. These results suggest that NEU4 is involved in regulation of neuronal function by polySia degradation in mammals.

  2. Innate immune mechanisms in Japanese encephalitis virus infection: effect on transcription of pattern recognition receptors in mouse neuronal cells and brain tissue.

    Science.gov (United States)

    Fadnis, Prachi Rahul; Ravi, Vasanthapuram; Desai, Anita; Turtle, Lance; Solomon, Tom

    2013-12-01

    Very little information is available on the role of innate immune mechanisms in Japanese encephalitis virus (JEV) infection. This study was designed to investigate the role of all Pattern Recognition Receptors (PRRs) in JEV infection in a mouse neuronal cell line in comparison to events that occur in vivo, using JEV infected suckling and adult mice. Analysis of mRNA expression was carried out using RT-PCR for detection of PRR genes and their downstream pathway genes, while a PCR array technique was used to examine the complete transcription analysis. Amongst the various innate immune receptors, TLR3 gene exhibited differential expression in JEV-infected Neuro2a, in suckling mice and adult mouse brain cells but not in uninfected control cells. The downstream events of TLR3 were confirmed by increased mRNA expression of IRF3 and interferon-β in JEV-infected Neuro2a cells and suckling mice brain tissue. To confirm the functional significance of this observation, TLR3 gene silencing experiments were carried using specific siRNA in Neuro2a cells. The results revealed a significant enhancement of JEV replication in TLR3 gene silenced JEV-infected Neuro2a cells, thereby suggesting that TLR3 serves a protective role against JEV. The expression levels of other PRRs varied. JEV-infected adult mice showed significant upregulation of TLR2 and MDA5 as compared to JEV-infected suckling mice and Neuro2a cells. In addition, upregulation of Myd88 and IRF7 was also noted in adult mice. These observations, coupled with the fact that adult mice infected with JEV exhibited longer survival rates, suggests that the host antiviral TLR2 response seen in adult mice was eventually countered by the virus by using MDA5 receptor. Our findings suggest that different PRRs appear to be involved in JEV infection in Neuro2a cells and brains of suckling and adult mice.

  3. Basic helix-loop-helix proteins E2A and HEB induce immature T-cell receptor rearrangements in nonlymphoid cells

    NARCIS (Netherlands)

    A.W. Langerak (Anton); I.L. Wolvers-Tettero; E.J. van Gastel-Mol (Ellen); M.E. Oud; J.J.M. van Dongen (Jacques)

    2001-01-01

    textabstractT-cell receptor (TCR) gene rearrangements are mediated via V(D)J recombination, which is strictly regulated during lymphoid differentiation, most probably through the action of specific transcription factors. Investigated was whether cotransfection of RAG1 and

  4. PDGF-like growth factor induces EGF-potentiated phenotypic transformation of normal rat kidney cells in the absence of TGFβ

    NARCIS (Netherlands)

    Zoelen, E.J.J. van; Oostwaard, Th.M.J.; Laat, S.W. de

    1986-01-01

    Using a growth factor defined assay for anchorage-independent growth (van Zoelen, E.J.J., van Oostwaard, Th.M.J., van der Saag, P.T. and de Laat, S.W. (1985) J. Cell. Physiol. 123, 151–160), we have studied the ability of polypeptide growth factors produced by Neuro-2A neuroblastoma cells to induce

  5. Mechanical response of single nerve cells estimated by femtosecond laser-induced impulsive force

    Science.gov (United States)

    Iino, Takanori; Furuno, Tadahide; Hagiyama, Man; Ito, Akihiko; Hosokawa, Yoichiroh

    2015-03-01

    Single nerve cell's mechanical response is an important issue for understanding function of nerve system, though, the response has been rarely clear. One of the factors is difficulty to stimulate the single cells by quantitative and controllable mechanical stress with subcellular spatial selectivity. As such mechanical stimulator, our group has focused on shock and stress waves generated by focusing the femtosecond laser under a microscope. When those waves impact on the biological cell, they act as an impulsive force. Although the impulsive force is available as a mechanical manipulator of the single cells, it was not confirmed that it could stimulate the nerve cells. Here we investigated the issue using neuro2a cells extending their neurite as an experimental model of nerve cell. Our results indicated that the impulsive force could be available as the stimulator to cause the mechanical response of the neuro2a cell.

  6. Polysulfide exerts a protective effect against cytotoxicity caused by t-buthylhydroperoxide through Nrf2 signaling in neuroblastoma cells.

    Science.gov (United States)

    Koike, Shin; Ogasawara, Yuki; Shibuya, Norihiro; Kimura, Hideo; Ishii, Kazuyuki

    2013-11-01

    Polysulfide is a bound sulfur species derived from endogenous H2S. When mouse neuroblastoma, Neuro2A cells were exposed to tert-butyl hydroperoxide after treatment with polysulfide, a significant decline in cell toxicity was observed. Rapid uptake of polysulfides induced translocation of Nrf2 into the nucleus, resulting in acceleration of GSH synthesis and HO-1 expression. We demonstrated that polysulfide reversibly modified Keap1 to form oxidized dimers and induced the translocation of Nrf2. Moreover, polysulfide treatment accelerated Akt phosphorylation, which is a known pathway of Nrf2 phosphorylation. Thus, polysulfide may mediate the activation of Nrf2 signaling, thereby exerting protective effects against oxidative damage in Neuro2A cells. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  7. Matrix effects on a cell-based assay used for the detection of paralytic shellfish toxins in bivalve shellfish samples.

    Science.gov (United States)

    Aballay-Gonzalez, Ambbar; Ulloa, Viviana; Rivera, Alejandra; Hernández, Víctor; Silva, Macarena; Caprile, Teresa; Delgado-Rivera, Lorena; Astuya, Allisson

    2016-05-01

    Detecting marine biotoxins such as paralytic shellfish toxins (PSTs) is essential to ensuring the safety of seafood. The mouse bioassay is the internationally accepted method for monitoring PSTs, but technical and ethical issues have led to a search for new detection methods. The mouse neuroblastoma cell-based assay (Neuro-2a CBA) using ouabain and veratridine (O/V) has proven useful for the detection of PSTs. However, CBAs are sensitive to shellfish-associated matrix interferences. As the extraction method highly influences matrix interferences, this study compared three extraction protocols: Association of Official Analytical Chemists (AOAC) 2005.06, AOAC 2011.02 and an alternative liquid-liquid method. These methods were used to assess the matrix effect of extracts from four commercially important bivalve species (Chilean mussel, Magellan mussel, clam and Pacific oyster) in Neuro-2a CBA. Extracts from all three protocols caused a toxic effect in Neuro-2a cells (without O/V) when tested at a concentration of 25 mg of tissue-equivalent (TE) ml(-1). The greatest toxicity was obtained through the AOAC 2011.02 protocol, especially for the Chilean mussel and Pacific oyster extracts. Similar toxicity levels (less than 15%) were observed in all extracts at 3.1 mg TE ml(-1). When assessed in Neuro-2a CBA, AOAC 2005.06 extracts presented the lowest matrix interferences, while the highest interferences were observed for AOAC 2011.02 in Magellan mussel and clam extracts. Finally, the AOAC 2005.06 and alternative protocols were compared using Chilean mussel samples fortified with 40 and 80 µg STX per 100 g meat. The AOAC 2005.06 method demonstrated better results. In conclusion, the AOAC 2005.06 extracts exhibited the fewest interferences in the Neuro-2a CBA. Therefore, this extraction method should be considered for the implementation of Neuro-2a CBA as a high-throughput screening methodology for PST detection.

  8. Effect of Ultrasonic Vibration on Proliferation and Differentiation of Cells

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    Haruka Hino

    2016-12-01

    Full Text Available The effect of mechanical stimulation of vibration on proliferation and differentiation of cells has been studied in vitro. To apply the vibration on the cells, a piezoelectric element was attached on the outside surface of the bottom of the culture plate of six wells. The piezoelectric element was vibrated by sinusoidally alternating voltage at 1.0 MHz generated by a function generator. Five kinds of cells were used in the experiment: C2C12 (mouse myoblast cell, L929 (fibroblast connective tissue of mouse, Hepa1-6 (mouse hepatoma cell, HUVEC (human umbilical vein endothelial cell, and Neuro-2a (mouse neural crest-derived cell line. After the incubation for 24 hours, cells were exposed to the ultrasonic vibration intermittently for three days: for thirty minutes per day. At the end of the experiment, the number of cells was counted by colorimetric method with a microplate photometer. In the case of Neuro-2a, the total length of the neurite was calculated at the microscopic image. The experimental study shows following results. Cells are exfoliated by the strong vibration. Proliferation and differentiation of cells are accelerated with mild vibration. The optimum intensity of vibration depends on the kind of cells.

  9. DRAM1 Protects Neuroblastoma Cells from Oxygen-Glucose Deprivation/Reperfusion-Induced Injury via Autophagy

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    Mengqiang Yu

    2014-10-01

    Full Text Available DNA damage-regulated autophagy modulator protein 1 (DRAM1, a multi-pass membrane lysosomal protein, is reportedly a tumor protein p53 (TP53 target gene involved in autophagy. During cerebral ischemia/reperfusion (I/R injury, DRAM1 protein expression is increased, and autophagy is activated. However, the functional significance of DRAM1 and the relationship between DRAM1 and autophagy in brain I/R remains uncertain. The aim of this study is to investigate whether DRAM1 mediates autophagy activation in cerebral I/R injury and to explore its possible effects and mechanisms. We adopt the oxygen-glucose deprivation and reperfusion (OGD/R Neuro-2a cell model to mimic cerebral I/R conditions in vitro, and RNA interference is used to knock down DRAM1 expression in this model. Cell viability assay is performed using the LIVE/DEAD viability/cytotoxicity kit. Cell phenotypic changes are analyzed through Western blot assays. Autophagy flux is monitored through the tandem red fluorescent protein–Green fluorescent protein–microtubule associated protein 1 light chain 3 (RFP–GFP–LC3 construct. The expression levels of DRAM1 and microtubule associated protein 1 light chain 3II/I (LC3II/I are strongly up-regulated in Neuro-2a cells after OGD/R treatment and peaked at the 12 h reperfusion time point. The autophagy-specific inhibitor 3-Methyladenine (3-MA inhibits the expression of DRAM1 and LC3II/I and exacerbates OGD/R-induced cell injury. Furthermore, DRAM1 knockdown aggravates OGD/R-induced cell injury and significantly blocks autophagy through decreasing autophagosome-lysosome fusion. In conclusion, our data demonstrate that DRAM1 knockdown in Neuro-2a cells inhibits autophagy by blocking autophagosome-lysosome fusion and exacerbated OGD/R-induced cell injury. Thus, DRAM1 might constitute a new therapeutic target for I/R diseases.

  10. A beta-lactone related to lactacystin induces neurite outgrowth in a neuroblastoma cell line and inhibits cell cycle progression in an osteosarcoma cell line.

    Science.gov (United States)

    Fenteany, G; Standaert, R F; Reichard, G A; Corey, E J; Schreiber, S L

    1994-04-12

    Lactacystin, a microbial natural product, induces neurite outgrowth in Neuro 2A mouse neuroblastoma cells and inhibits progression of synchronized Neuro 2A cells and MG-63 human osteosarcoma cells beyond the G1 phase of the cell cycle. A related beta-lactone, clasto-lactacystin beta-lactone, formally the product of elimination of N-acetylcysteine from lactacystin, is also active, whereas the corresponding clastolactacystin dihydroxy acid is completely inactive. Structural analogs of lactacystin altered only in the N-acetylcysteine moiety are active, while structural or stereochemical modifications of the gamma-lactam ring or the hydroxyisobutyl group lead to partial or complete loss of activity. The inactive compounds do not antagonize the effects of lactacystin in either neurite outgrowth or cell cycle progression assays. The response to lactacystin involves induction of a predominantly bipolar morphology that is maximal 16-32 h after treatment and is distinct from the response to several other treatments that result in morphological differentiation. Neurite outgrowth in response to lactacystin appears to be dependent upon microtubule assembly, actin polymerization, and de novo protein synthesis. The observed structure-activity relationships suggest that lactacystin and its related beta-lactone may act via acylation of one or more relevant target molecule(s) in the cell.

  11. A G protein-coupled receptor (GPCR) in red: live cell imaging of the kappa opioid receptor-tdTomato fusion protein (KOPR-tdT) in neuronal cells

    Science.gov (United States)

    Huang, Peng; Chiu, Yi-Ting; Chen, Chongguang; Wang, Yujun; Liu-Chen, Lee-Yuan

    2013-01-01

    Introduction In contrast to green fluorescent protein and variants (GFPs), red fluorescent proteins (RFPs) have rarely been employed for generation of GPCR fusion proteins, likely because of formation of aggregates and cell toxicity of some RFPs. Among all the RFPs available, tdTomato (tdT), one of the non-aggregating RFP, has the highest brightness score (about 3 times that of eGFP) and unsurpassed photostability. Methods We fused tdT to the KOPR C-terminus. The KOPR-tdT cDNA construct was transfected into Neuro2A mouse neuroblastoma cell line (Neuro2A cells) and rat cortical primary neurons for characterization of pharmacological properties and imaging studies on KOPR trafficking. Results KOPR-tdT retained KOPR properties (cell surface expression, ligand binding, agonist-induced signaling and internalization) when expressed in Neuro2A cells and rat primary cortical neurons. Live cell imaging of KOPR-tdT enables visualization of time course of agonist-induced internalization of KOPR in real time for 60 min, without photobleaching and apparent cell toxicity. U50,488H-induced KOPR internalization occurred as early as 4 min and plateaued at about 30 min. A unique pattern of internalized KOPR in processes of primary neurons was induced by U50,488H. Discussion tdT is an alternative to, or even a better tool than, GFPs for fusing to GPCR for trafficking studies, because tdT has higher brightness and thus better resolution and less photobleaching problems due to reduced laser power used. It also has advantages associated with its longer-wavelength emission including spectral separation from autofluorescence and GFPs, reduced cell toxicity the laser may impose, and greater tissue penetration. These advantages of tdT over GPFs may be critical for live cell imaging studies of GPCRs in vitro and for studying GPCRs in vivo because of their low abundance. PMID:23856011

  12. THE MYC FAMILY OF ONCOGENES AND THEIR PRESENCE AND IMPORTANCE IN SMALL-CELL LUNG-CARCINOMA AND OTHER TUMOR TYPES

    NARCIS (Netherlands)

    DEVRIES, EGE; MULDER, NH

    1993-01-01

    The myc family of cellular oncogenes, c - myr, N - myc, encodes three highly related, cell cycle specific, nuclear phosphoproteins. All are able to transform primary rat embryo fibroblasts when cotransfected with the c - ras oncogene. Myc family genes am differentially expressed with respect to

  13. Co-expression of activin receptor-interacting protein 1 and 2 in mouse nerve cells.

    Science.gov (United States)

    Qi, Yan; Ge, Jing-Yan; Wang, Yi-Nan; Liu, Hai-Yan; Li, Yun-Man; Liu, Zhong-Hui; Cui, Xue-Ling

    2013-05-10

    Activin is a neurotrophic and neuroprotective factor in the central nervous system. Activin receptor-interacting protein 1 and 2 (ARIP1 and ARIP2) are identified as activin signal proteins in mouse brain. However, whether ARIP1 and ARIP2 are co-expressed in nerve cells and the differences of their biological activities are not well characterized. In the present study, we found that ARIP1 and ARIP2 mRNA expressions were detectable in mouse brain and their proteins were co-localized at the hypothalamus of cerebrum and granular layers in cerebellum, especially in Purkinje cells. Furthermore, ARIP1 and ARIP2 were co-expressed in mouse Neuro-2a cells, which is similar to the co-localization of ARIP1 and ARIP2 in hypothalamus neurons and Purkinje cells. Overexpression of ARIP1 in Neuro-2a cells inhibited activin signal transduction induced by activin A and Smad3, and activin A-induced voltage-gated Na(+) current (INa), while ARIP2 was only a negative regulator of signal transduction induced by activin A and did not alter activin A-induced INa. Taken together, these data demonstrate that ARIP1 and ARIP2 are co-expressed in some nerve cells and their biological activities are distinct. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  14. Polysulfide promotes neuroblastoma cell differentiation by accelerating calcium influx.

    Science.gov (United States)

    Koike, Shin; Shibuya, Norihiro; Kimura, Hideo; Ishii, Kazuyuki; Ogasawara, Yuki

    2015-04-10

    Polysulfides are a typical type of bound sulfur, which is physiologically stable form of sulfur species, derived from the hydrogen sulfide (H2S) that is generated endogenously in cells. We previously reported that bound sulfur protects neuronal cells from oxidative injury. In the present study, we demonstrated that polysulfides inhibited cell growth and promoted neurite outgrowth in mouse neuroblastoma Neuro2A (N2A) cells. However, Na2S showed no effect on neurite outgrowth in N2A cells. Furthermore, 2-APB and SKF96365, which are typical transient receptor potential (TRP) channel inhibitors, suppressed the neurite outgrowth induced by Na2S4. These new findings suggest that bound sulfur could induce neurite outgrowth and cell differentiation of N2A cells by accelerating calcium influx. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Chloroacetic acid induced neuronal cells death through oxidative stress-mediated p38-MAPK activation pathway regulated mitochondria-dependent apoptotic signals.

    Science.gov (United States)

    Chen, Chun-Hung; Chen, Sz-Jie; Su, Chin-Chuan; Yen, Cheng-Chieh; Tseng, To-Jung; Jinn, Tzyy-Rong; Tang, Feng-Cheng; Chen, Kuo-Liang; Su, Yi-Chang; Lee, kuan-I; Hung, Dong-Zong; Huang, Chun-Fa

    2013-01-07

    Chloroacetic acid (CA), a toxic chlorinated analog of acetic acid, is widely used in chemical industries as an herbicide, detergent, and disinfectant, and chemical intermediates that are formed during the synthesis of various products. In addition, CA has been found as a by-product of chlorination disinfection of drinking water. However, there is little known about neurotoxic injuries of CA on the mammalian, the toxic effects and molecular mechanisms of CA-induced neuronal cell injury are mostly unknown. In this study, we examined the cytotoxicity of CA on cultured Neuro-2a cells and investigated the possible mechanisms of CA-induced neurotoxicity. Treatment of Neuro-2a cells with CA significantly reduced the number of viable cells (in a dose-dependent manner with a range from 0.1 to 3mM), increased the generation of ROS, and reduced the intracellular levels of glutathione depletion. CA also increased the number of sub-G1 hypodiploid cells; increased mitochondrial dysfunction (loss of MMP, cytochrome c release, and accompanied by Bcl-2 and Mcl-1 down-regulation and Bax up-regulation), and activated the caspase cascades activations, which displayed features of mitochondria-dependent apoptosis pathway. These CA-induced apoptosis-related signals were markedly prevented by the antioxidant N-acetylcysteine (NAC). Moreover, CA activated the JNK and p38-MAPK pathways, but did not that ERK1/2 pathway, in treated Neuro-2a cells. Pretreatment with NAC and specific p38-MAPK inhibitor (SB203580), but not JNK inhibitor (SP600125) effectively abrogated the phosphorylation of p38-MAPK and attenuated the apoptotic signals (including: decrease in cytotoxicity, caspase-3/-7 activation, the cytosolic cytochrome c release, and the reversed alteration of Bcl-2 and Bax mRNA) in CA-treated Neuro-2a cells. Taken together, these data suggest that oxidative stress-induced p38-MAPK activated pathway-regulated mitochondria-dependent apoptosis plays an important role in CA-caused neuronal cell

  16. Apolipoprotein E4 (1–272 fragment is associated with mitochondrial proteins and affects mitochondrial function in neuronal cells

    Directory of Open Access Journals (Sweden)

    Michikawa Makoto

    2009-08-01

    Full Text Available Abstract Background Apolipoprotein E allele ε4 (apoE4 is a strong risk factor for developing Alzheimer's disease (AD. Secreted apoE has a critical function in redistributing lipids among central nervous system cells to maintain normal lipid homeostasis. In addition, previous reports have shown that apoE4 is cleaved by a protease in neurons to generate apoE4(1–272 fragment, which is associated with neurofibrillary tanglelike structures and mitochondria, causing mitochondrial dysfunction. However, it still remains unclear how the apoE fragment associates with mitochondria and induces mitochondrial dysfunction. Results To clarify the molecular mechanism, we carried out experiments to identify intracellular apoE-binding molecules and their functions in modulating mitochondria function. Here, we found that apoE4 binds to ubiquinol cytochrome c reductase core protein 2 (UQCRC2 and cytochrome C1, both of which are components of mitochondrial respiratory complex III, and cytochrome c oxidase subunit 4 isoform 1 (COX IV 1, which is a component of complex IV, in Neuro-2a cells. Interestingly, these proteins associated with apoE4(1–272 more strongly than intact apoE4(1–299. Further analysis showed that in Neuro-2a cells expressing apoE4(1–272, the enzymatic activities of mitochondrial respiratory complexes III and IV were significantly lower than those in Neuro-2a cells expressing apoE4(1–299. Conclusion ApoE4(1–272 fragment expressed in Neuro2a cells is associated with mitochondrial proteins, UQCRC2 and cytochrome C1, which are component of respiratory complex III, and with COX IV 1, which is a member of complex IV. Overexpression of apoE4(1–272 fragment impairs activities of complex III and IV. These results suggest that the C-terminal-truncated fragment of apoE4 binds to mitochondrial complexes and affects their activities, and thereby leading to neurodegeneration.

  17. Loss of the RNA helicase SKIV2L2 impairs mitotic progression and replication-dependent histone mRNA turnover in murine cell lines.

    Science.gov (United States)

    Onderak, Alexis M; Anderson, James T

    2017-06-01

    RNA surveillance via the nuclear exosome requires cofactors such as the helicase SKIV2L2 to process and degrade certain noncoding RNAs. This research aimed to characterize the phenotype associated with RNAi knockdown of Skiv2l2 in two murine cancer cell lines: Neuro2A and P19. SKIV2L2 depletion in Neuro2A and P19 cells induced changes in gene expression indicative of cell differentiation and reduced cellular proliferation by 30%. Propidium iodide-based cell-cycle analysis of Skiv2l2 knockdown cells revealed defective progression through the G2/M phase and an accumulation of mitotic cells, suggesting SKIV2L2 contributes to mitotic progression. Since SKIV2L2 targets RNAs to the nuclear exosome for processing and degradation, we identified RNA targets elevated in cells depleted of SKIV2L2 that could account for the observed twofold increase in mitotic cells. Skiv2l2 knockdown cells accumulated replication-dependent histone mRNAs, among other RNAs, that could impede mitotic progression and indirectly trigger differentiation. © 2017 Onderak and Anderson; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  18. Genome engineering of mammalian haploid embryonic stem cells using the Cas9/RNA system

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    Takuro Horii

    2013-12-01

    Full Text Available Haploid embryonic stem cells (ESCs are useful for studying mammalian genes because disruption of only one allele can cause loss-of-function phenotypes. Here, we report the use of haploid ESCs and the CRISPR RNA-guided Cas9 nuclease gene-targeting system to manipulate mammalian genes. Co-transfection of haploid ESCs with vectors expressing Cas9 nuclease and single-guide RNAs (sgRNAs targeting Tet1, Tet2, and Tet3 resulted in the complete disruption of all three genes and caused a loss-of-function phenotype with high efficiency (50%. Co-transfection of cells with vectors expressing Cas9 and sgRNAs targeting two loci on the same chromosome resulted in the creation of a large chromosomal deletion and a large inversion. Thus, the use of the CRISPR system in combination with haploid ESCs provides a powerful platform to manipulate the mammalian genome.

  19. A High-Throughput Microfluidic Platform for Mammalian Cell Transfection and Culturing

    Science.gov (United States)

    Woodruff, Kristina; Maerkl, Sebastian J.

    2016-01-01

    Mammalian synthetic biology could be augmented through the development of high-throughput microfluidic systems that integrate cellular transfection, culturing, and imaging. We created a microfluidic chip that cultures cells and implements 280 independent transfections at up to 99% efficiency. The chip can perform co-transfections, in which the number of cells expressing each protein and the average protein expression level can be precisely tuned as a function of input DNA concentration and synthetic gene circuits can be optimized on chip. We co-transfected four plasmids to test a histidine kinase signaling pathway and mapped the dose dependence of this network on the level of one of its constituents. The chip is readily integrated with high-content imaging, enabling the evaluation of cellular behavior and protein expression dynamics over time. These features make the transfection chip applicable to high-throughput mammalian protein and synthetic biology studies. PMID:27030663

  20. Combination of Id2 Knockdown Whole Tumor Cells and Checkpoint Blockade: A Potent Vaccine Strategy in a Mouse Neuroblastoma Model.

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    Lina Chakrabarti

    Full Text Available Tumor vaccines have held much promise, but to date have demonstrated little clinical success. This lack of success is conceivably due to poor tumor antigen presentation combined with immuno-suppressive mechanisms exploited by the tumor itself. Knock down of Inhibitor of differentiation protein 2 (Id2-kd in mouse neuroblastoma whole tumor cells rendered these cells immunogenic. Id2-kd neuroblastoma (Neuro2a cells (Id2-kd N2a failed to grow in most immune competent mice and these mice subsequently developed immunity against further wild-type Neuro2a tumor cell challenge. Id2-kd N2a cells grew aggressively in immune-compromised hosts, thereby establishing the immunogenicity of these cells. Therapeutic vaccination with Id2-kd N2a cells alone suppressed tumor growth even in established neuroblastoma tumors and when used in combination with immune checkpoint blockade eradicated large established tumors. Mechanistically, immune cell depletion studies demonstrated that while CD8+ T cells are critical for antitumor immunity, CD4+ T cells are also required to induce a sustained long-lasting helper effect. An increase in number of CD8+ T-cells and enhanced production of interferon gamma (IFNγ was observed in tumor antigen stimulated splenocytes of vaccinated mice. More importantly, a massive influx of cytotoxic CD8+ T-cells infiltrated the shrinking tumor following combined immunotherapy. These findings show that down regulation of Id2 induced tumor cell immunity and in combination with checkpoint blockade produced a novel, potent, T-cell mediated tumor vaccine strategy.

  1. Electroporation-mediated gene transfer of SOX trio to enhance chondrogenesis in adipose stem cells.

    Science.gov (United States)

    Im, G-I; Kim, H-J

    2011-04-01

    The aim of the present study was to determine if the electroporation-mediated gene transfer of SOX trio enhances the chondrogenic potential of adipose stem cells (ASCs). ASCs were transfected with SOX trio genes using an electroporation technique and cultured for 3 weeks. The pellets were analyzed for DNA and glycosaminoglycan (GAG) analysis, and the gene and protein expression of SOX-5, SOX-6, SOX-9, type 1 collagen (COL1Al), type 2 collagen (COL2Al) and type 10 collagen (COL10A1) using real-time PCR and Western blot analysis. Further in vivo studies were carried out by subcutaneous transplantation of pellets in severe combined immunodeficiency (SCID) mice for 3 weeks. The gene transfer efficiency was high (approximately 70%). Transfected ASCs showed high expression of corresponding genes after 21 days, and each SOX protein was detected in ASCs transfected with the corresponding gene. The chondrogenic differentiation of ASCs, as demonstrated by GAG levels and Safranin-O staining, showed significant enhancement when SOX trio were co-transfected, while subsets with single gene transfer of SOX-5, -6, or -9 did not show significant elevation. SOX trio co-transfection enhanced COL2A1 mRNA, but did not increase COL1A1 and COL10A1 mRNA. Type II collagen protein dramatically increased, and type X collagen decreased with co-transfection of the SOX trio. When pellets were implanted in the subcutaneous pouch of SCID mice for 3 weeks, ASCs co-transfected with SOX trio demonstrated abundant proteoglycan, significantly reduced mineralization. The electroporation-mediated transfection of SOX trio greatly enhances chondrogenesis from ASCs, while decreasing hypertrophy. Copyright © 2011 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  2. Imaging grafted cells with [18F]FHBG using an optimized HSV1-TK mammalian expression vector in a brain injury rodent model.

    Science.gov (United States)

    Salabert, Anne-Sophie; Vaysse, Laurence; Beaurain, Marie; Alonso, Mathieu; Arribarat, Germain; Lotterie, Jean-Albert; Loubinoux, Isabelle; Tafani, Mathieu; Payoux, Pierre

    2017-01-01

    Cell transplantation is an innovative therapeutic approach after brain injury to compensate for tissue damage. To have real-time longitudinal monitoring of intracerebrally grafted cells, we explored the feasibility of a molecular imaging approach using thymidine kinase HSV1-TK gene encoding and [18F]FHBG as a reporter probe to image enzyme expression. A stable neuronal cell line expressing HSV1-TK was developed with an optimised mammalian expression vector to ensure long-term transgene expression. After [18F]FHBG incubation under defined parameters, calibration ranges from 1 X 104 to 3 X 106 Neuro2A-TK cells were analysed by gamma counter or by PET-camera. In parallel, grafting with different quantities of [18F]FHBG prelabelled Neuro2A-TK cells was carried out in a rat brain injury model induced by stereotaxic injection of malonate toxin. Image acquisition of the rats was then performed with PET/CT camera to study the [18F]FHBG signal of transplanted cells in vivo. Under the optimised incubation conditions, [18F]FHBG cell uptake rate was around 2.52%. In-vitro calibration range analysis shows a clear linear correlation between the number of cells and the signal intensity. The PET signal emitted into rat brain correlated well with the number of cells injected and the number of surviving grafted cells was recorded via the in-vitro calibration range. PET/CT acquisitions also allowed validation of the stereotaxic injection procedure. Technique sensitivity was evaluated under 5 X 104 grafted cells in vivo. No [18F]FHBG or [18F]metabolite release was observed showing a stable cell uptake even 2 h post-graft. The development of this kind of approach will allow grafting to be controlled and ensure longitudinal follow-up of cell viability and biodistribution after intracerebral injection.

  3. Imaging grafted cells with [18F]FHBG using an optimized HSV1-TK mammalian expression vector in a brain injury rodent model.

    Directory of Open Access Journals (Sweden)

    Anne-Sophie Salabert

    Full Text Available Cell transplantation is an innovative therapeutic approach after brain injury to compensate for tissue damage. To have real-time longitudinal monitoring of intracerebrally grafted cells, we explored the feasibility of a molecular imaging approach using thymidine kinase HSV1-TK gene encoding and [18F]FHBG as a reporter probe to image enzyme expression.A stable neuronal cell line expressing HSV1-TK was developed with an optimised mammalian expression vector to ensure long-term transgene expression. After [18F]FHBG incubation under defined parameters, calibration ranges from 1 X 104 to 3 X 106 Neuro2A-TK cells were analysed by gamma counter or by PET-camera. In parallel, grafting with different quantities of [18F]FHBG prelabelled Neuro2A-TK cells was carried out in a rat brain injury model induced by stereotaxic injection of malonate toxin. Image acquisition of the rats was then performed with PET/CT camera to study the [18F]FHBG signal of transplanted cells in vivo.Under the optimised incubation conditions, [18F]FHBG cell uptake rate was around 2.52%. In-vitro calibration range analysis shows a clear linear correlation between the number of cells and the signal intensity. The PET signal emitted into rat brain correlated well with the number of cells injected and the number of surviving grafted cells was recorded via the in-vitro calibration range. PET/CT acquisitions also allowed validation of the stereotaxic injection procedure. Technique sensitivity was evaluated under 5 X 104 grafted cells in vivo. No [18F]FHBG or [18F]metabolite release was observed showing a stable cell uptake even 2 h post-graft.The development of this kind of approach will allow grafting to be controlled and ensure longitudinal follow-up of cell viability and biodistribution after intracerebral injection.

  4. Imaging grafted cells with [18F]FHBG using an optimized HSV1-TK mammalian expression vector in a brain injury rodent model

    Science.gov (United States)

    Beaurain, Marie; Alonso, Mathieu; Arribarat, Germain; Lotterie, Jean-Albert; Loubinoux, Isabelle; Tafani, Mathieu; Payoux, Pierre

    2017-01-01

    Introduction Cell transplantation is an innovative therapeutic approach after brain injury to compensate for tissue damage. To have real-time longitudinal monitoring of intracerebrally grafted cells, we explored the feasibility of a molecular imaging approach using thymidine kinase HSV1-TK gene encoding and [18F]FHBG as a reporter probe to image enzyme expression. Methods A stable neuronal cell line expressing HSV1-TK was developed with an optimised mammalian expression vector to ensure long-term transgene expression. After [18F]FHBG incubation under defined parameters, calibration ranges from 1 X 104 to 3 X 106 Neuro2A-TK cells were analysed by gamma counter or by PET-camera. In parallel, grafting with different quantities of [18F]FHBG prelabelled Neuro2A-TK cells was carried out in a rat brain injury model induced by stereotaxic injection of malonate toxin. Image acquisition of the rats was then performed with PET/CT camera to study the [18F]FHBG signal of transplanted cells in vivo. Results Under the optimised incubation conditions, [18F]FHBG cell uptake rate was around 2.52%. In-vitro calibration range analysis shows a clear linear correlation between the number of cells and the signal intensity. The PET signal emitted into rat brain correlated well with the number of cells injected and the number of surviving grafted cells was recorded via the in-vitro calibration range. PET/CT acquisitions also allowed validation of the stereotaxic injection procedure. Technique sensitivity was evaluated under 5 X 104 grafted cells in vivo. No [18F]FHBG or [18F]metabolite release was observed showing a stable cell uptake even 2 h post-graft. Conclusion The development of this kind of approach will allow grafting to be controlled and ensure longitudinal follow-up of cell viability and biodistribution after intracerebral injection. PMID:28926581

  5. Large-scale RNA interference screening in mammalian cells identifies novel regulators of mutant huntingtin aggregation.

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    Tomoyuki Yamanaka

    Full Text Available In polyglutamine (polyQ diseases including Huntington's disease (HD, mutant proteins containing expanded polyQ stretch form aggregates in neurons. Genetic or RNAi screenings in yeast, C. elegans or Drosophila have identified multiple genes modifying polyQ aggregation, a few of which are confirmed effective in mammals. However, the overall molecular mechanism underlying polyQ protein aggregation in mammalian cells still remains obscure. We here perform RNAi screening in mouse neuro2a cells to identify mammalian modifiers for aggregation of mutant huntingtin, a causative protein of HD. By systematic cell transfection and automated cell image analysis, we screen ∼ 12000 shRNA clones and identify 111 shRNAs that either suppress or enhance mutant huntingtin aggregation, without altering its gene expression. Classification of the shRNA-targets suggests that genes with various cellular functions such as gene transcription and protein phosphorylation are involved in modifying the aggregation. Subsequent analysis suggests that, in addition to the aggregation-modifiers sensitive to proteasome inhibition, some of them, such as a transcription factor Tcf20, and kinases Csnk1d and Pik3c2a, are insensitive to it. As for Tcf20, which contains polyQ stretches at N-terminus, its binding to mutant huntingtin aggregates is observed in neuro2a cells and in HD model mouse neurons. Notably, except Pik3c2a, the rest of the modifiers identified here are novel. Thus, our first large-scale RNAi screening in mammalian system identifies previously undescribed genetic players that regulate mutant huntingtin aggregation by several, possibly mammalian-specific mechanisms.

  6. HNF1 alpha activates the aminopeptidase N promoter in intestinal (Caco-2) cells

    DEFF Research Database (Denmark)

    Olsen, Jørgen; Laustsen, Lotte; Troelsen, J

    1994-01-01

    The importance of HNF1 binding proteins for intestinal aminopeptidase N expression was investigated using the Caco-2 cell-line. Aminopeptidase N promoter activity in Caco-2 cells depends on the HNF1 element (positions -85 to -58) and co-transfection with an HNF1 alpha expression vector demonstrates...... a direct activation of the promoter by HNF1 alpha through this element. Electrophoretic mobility shift assays using nuclear extracts from Caco-2 cells show the presence of high amounts of HNF1 binding proteins irrespective of their state of differentiation....

  7. Arsenic induces reactive oxygen species-caused neuronal cell apoptosis through JNK/ERK-mediated mitochondria-dependent and GRP 78/CHOP-regulated pathways.

    Science.gov (United States)

    Lu, Tien-Hui; Tseng, To-Jung; Su, Chin-Chuan; Tang, Feng-Cheng; Yen, Cheng-Chieh; Liu, Yu-Yun; Yang, Ching-Yao; Wu, Chin-Ching; Chen, Kuo-Liang; Hung, Dong-Zong; Chen, Ya-Wen

    2014-01-03

    Arsenic (As), a well-known high toxic metal, is an important environmental and industrial contaminant, and it induces oxidative stress, which causes many adverse health effects and diseases in humans, particularly in inorganic As (iAs) more harmful than organic As. Recently, epidemiological studies have suggested a possible relationship between iAs exposure and neurodegenerative disease development. However, the toxicological effects and underlying mechanisms of iAs-induced neuronal cell injuries are mostly unknown. The present study demonstrated that iAs significantly decreased cell viability and induced apoptosis in Neuro-2a cells. iAs also increased oxidative stress damage (production of malondialdehyde (MDA) and ROS, and reduction of Nrf2 and thioredoxin protein expression) and induced several features of mitochondria-dependent apoptotic signals, including: mitochondrial dysfunction, the activations of PARP and caspase cascades, and the increase in caspase-3 activity. Pretreatment with the antioxidant N-acetylcysteine (NAC) effectively reversed these iAs-induced responses. iAs also increased the phosphorylation of JNK and ERK1/2, but did not that p38-MAPK, in treated Neuro-2a cells. NAC and the specific JNK inhibitor (SP600125) and ERK1/2 inhibitor (PD98059) abrogated iAs-induced cell cytotoxicity, caspase-3/-7 activity, and JNK and ERK1/2 activation. Additionally, exposure of Neuro-2a cells to iAs triggered endoplasmic reticulum (ER) stress identified through several key molecules (GRP 78, CHOP, XBP-1, and caspase-12), which was prevented by NAC. Transfection with GRP 78- and CHOP-specific si-RNA dramatically suppressed GRP 78 and CHOP expression, respectively, and attenuated the activations of caspase-12, -7, and -3 in iAs-exposed cells. Therefore, these results indicate that iAs induces ROS causing neuronal cell death via both JNK/ERK-mediated mitochondria-dependent and GRP 78/CHOP-triggered apoptosis pathways. Copyright © 2013 Elsevier Ireland Ltd. All

  8. Chloroacetic acid triggers apoptosis in neuronal cells via a reactive oxygen species-induced endoplasmic reticulum stress signaling pathway.

    Science.gov (United States)

    Lu, Tien-Hui; Su, Chin-Chuan; Tang, Feng-Cheng; Chen, Chun-Hung; Yen, Cheng-Chieh; Fang, Kai-Min; Lee, kuan-I; Hung, Dong-Zong; Chen, Ya-Wen

    2015-01-05

    Chloroacetic acid (CA), a chlorinated analog of acetic acid and an environmental toxin that is more toxic than acetic, dichloroacetic, or trichloroacetic acids, is widely used in chemical industries. Furthermore, CA has been found to be the major disinfection by-products (DBPs) of drinking water. CA has been reported to be highly corrosive and to induce severe tissue injuries (including nervous system) that lead to death in mammals. However, the effects and underlying mechanisms of CA-induced neurotoxicity remain unknown. In the present study, we found that CA (0.5-2.0 mM) significantly increased LDH release, decreased the number of viable cells (cytotoxicity) and induced apoptotic events (including: increases in the numbers of apoptotic cells, the membrane externalization of phosphatidylserine (PS), and caspase-3/-7 activity) in Neuro-2a cells. CA (1.5 mM; the approximate to LD50) also triggered ER stress, which was identified by monitoring several key molecules that are involved in the unfolded protein responses (including the increase in the expressions of p-PERK, p-IRE-1, p-eIF2α, ATF-4, ATF-6, CHOP, XBP-1, GRP 78, GRP 94, and caspase-12) and calpain activity. Transfection of GRP 78- and GRP 94-specific si-RNA effectively abrogated CA-induced cytotoxicity, caspase-3/-7 and caspase-12 activity, and GRP 78 and GRP 94 expression in Neuro-2a cells. Additionally, pretreatment with 2.5 mM N-acetylcysteine (NAC; a glutathione (GSH) precursor) dramatically suppressed the increase in lipid peroxidation, cytotoxicity, apoptotic events, calpain and caspase-12 activity, and ER stress-related molecules in CA-exposed cells. Taken together, these results suggest that the higher concentration of CA exerts its cytotoxic effects in neuronal cells by triggering apoptosis via a ROS-induced ER stress signaling pathway. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  9. Botulinum Neurotoxin Serotype C Associates with Dual Ganglioside Receptors to Facilitate Cell Entry*

    Science.gov (United States)

    Karalewitz, Andrew P.-A.; Fu, Zhuji; Baldwin, Michael R.; Kim, Jung-Ja P.; Barbieri, Joseph T.

    2012-01-01

    Botulinum neurotoxins (BoNTs) cleave SNARE proteins in motor neurons that inhibits synaptic vesicle (SV) exocytosis, resulting in flaccid paralysis. There are seven BoNT serotypes (A–G). In current models, BoNTs initially bind gangliosides on resting neurons and upon SV exocytosis associate with the luminal domains of SV-associated proteins as a second receptor. The entry of BoNT/C is less clear. Characterizing the heavy chain receptor binding domain (HCR), BoNT/C was shown to utilize gangliosides as dual host receptors. Crystallographic and biochemical studies showed that the two ganglioside binding sites, termed GBP2 and Sia-1, were independent and utilized unique mechanisms to bind complex gangliosides. The GBP2 binding site recognized gangliosides that contained a sia5 sialic acid, whereas the Sia-1 binding site recognized gangliosides that contained a sia7 sialic acid and sugars within the backbone of the ganglioside. Utilizing gangliosides that uniquely recognized the GBP2 and Sia-1 binding sites, HCR/C entry into Neuro-2A cells required both functional ganglioside binding sites. HCR/C entered cells differently than the HCR of tetanus toxin, which also utilizes dual gangliosides as host receptors. A point-mutated HCR/C that lacked GBP2 binding potential retained the ability to bind and enter Neuro-2A cells. This showed that ganglioside binding at the Sia-1 site was accessible on the plasma membrane, suggesting that SV exocytosis may not be required to expose BoNT/C receptors. These studies highlight the utility of BoNT HCRs as probes to study the role of gangliosides in neurotransmission. PMID:23027864

  10. Folic Acid Inhibits Amyloid β-Peptide Production through Modulating DNA Methyltransferase Activity in N2a-APP Cells

    Directory of Open Access Journals (Sweden)

    Wen Li

    2015-10-01

    Full Text Available Alzheimer’s disease (AD is a common neurodegenerative disease resulting in progressive dementia, and is a principal cause of dementia among older adults. Folate acts through one-carbon metabolism to support the methylation of multiple substrates. We hypothesized that folic acid supplementation modulates DNA methyltransferase (DNMT activity and may alter amyloid β-peptide (Aβ production in AD. Mouse Neuro-2a cells expressing human APP695 were incubated with folic acid (2.8–40 μmol/L, and with or without zebularine (the DNMT inhibitor. DNMT activity, cell viability, Aβ and DNMTs expression were then examined. The results showed that folic acid stimulated DNMT gene and protein expression, and DNMT activity. Furthermore, folic acid decreased Aβ protein production, whereas inhibition of DNMT activity by zebularine increased Aβ production. The results indicate that folic acid induces methylation potential-dependent DNMT enzymes, thereby attenuating Aβ production.

  11. Metafectene is superior to lipofectamine in the transfection of G(s) alpha prostate cancer cells.

    Science.gov (United States)

    Iczkowski, Kenneth A; Omara-Opyene, A Levi; Klösel, Roland

    2004-10-01

    Transfection efficiency of the novel reagent metafectene has not been compared with that of lipofectamine in the published English literature. We used these agents to transfect two prostate cancer cell lines, PC3 and G(s) alpha, with a deoxyribonucleic acid (DNA) expression vector that generates double-stranded ribonucleic acid (RNA) for RNA interference (RNAi). Cotransfection of the green fluorescent protein (GFP) reporter gene revealed that the mean (+/- standard deviation) transfection efficiencies with lipofectamine were 5.8+/-0.4% for PC3 cells and 3.6+/-1.5% for G(s) alpha cells. Mean transfection efficiency with metafectene declined to 0.1+/-0% for PC3 cells but improved to 54.6+/-5.5% for G(s) alpha cells. With G(s) alpha cells, metafectene transfection of GFP plasmid alone yielded 46.9% positive cells, and cotransfection with CD44v9 expression vector yielded 45.9% positive cells. The visual impact of the transfected RNAi construct was detectable at the protein level 4 to 6 d posttransfection and was more dramatic after using metafectene than after using lipofectamine. Thus, in vitro, metafectene transfection efficiency was sufficient to allow us to assess the functional significance of our RNAi construct, suggesting metafectene as an excellent candidate for RNAi-mediated anticancer gene therapy.

  12. Electroporation-mediated transfer of SOX trio genes (SOX-5, SOX-6, and SOX-9) to enhance the chondrogenesis of mesenchymal stem cells.

    Science.gov (United States)

    Kim, Hye-Joung; Im, Gun-Il

    2011-12-01

    The purpose of this study was to test the hypothesis that the SOX trio genes (SOX-5, SOX-6, and SOX-9) have a lower level of expression during the chondrogenic differentiation of mesenchymal stem cells (MSCs) compared with chondrocytes and that the electroporation-mediated gene transfer of SOX trio promotes chondrogenesis from human MSCs. An in vitro pellet culture was carried out using MSCs or chondrocytes at passage 3 and analyzed after 7 and 21 days. Then, MSCs were transfected with SOX trio genes and analyzed for the expression of chondrogenic markers after 21 days of in vitro culture. Without transforming growth factor-β1, the untransfected MSCs had a lower level of SOX trio gene and protein expression than chondrocytes. However, the level of SOX-9 gene expression increased in MSCs when treated with transforming growth factor-β1. GAG level significantly increased 7-fold in MSCs co-transfected with SOX trio, which was corroborated by Safranin-O staining. SOX trio co-transfection significantly increased COL2A1 gene and protein and decreased COL10A1 protein in MSCs. It is concluded that the SOX trio have a significantly lower expression in human MSCs than in chondrocytes and that the electroporation-mediated co-transfection of SOX trio enhances chondrogenesis and suppresses hypertrophy of human MSCs.

  13. Reconstitution of glucotoxic HIT-T15 cells with somatostatin transcription factor-1 partially restores insulin promoter activity.

    Science.gov (United States)

    Harmon, J S; Tanaka, Y; Olson, L K; Robertson, R P

    1998-06-01

    We have reported that chronic culture of HIT-T15 cells in medium containing supraphysiologic glucose concentrations (11.1 mmol/l) causes a decrease in insulin mRNA levels, insulin content, and insulin release. Furthermore, decreases in insulin gene transcription and binding activity of two essential beta-cell transcription factors, somatostatin transcription factor-1 (STF-1; also known as GSTF, IDX-1, IPF-1, PDX-1, and GSF) and RIPE-3b1 activator, are associated with this glucotoxic effect. In this study, we observed that the loss of RIPE-3b1 occurs much earlier (79% decrease at passage [p]81) than the loss of STF-1 (65% decrease at p104), with abolishment of both factors by p122. Since the STF-1, but not the RIPE-3b1 activator, gene has been cloned, we examined its restorative effects on insulin gene promoter activity after reconstitution with STF-1 cDNA. Basal insulin promoter activities normalized to early (p71-74) passage cells (1.000 +/- 0.069) were 0.4066 +/- 0.093 and 0.142 +/- 0.034 for intermediate (p102-106) and late (p118-122) passage cells, respectively. Early, intermediate, and late passage cells, all chronically cultured in medium containing 11.1 mmol/l glucose, were transfected with STF-1 alone or cotransfected with E2-5, an E-box factor known to be synergistically associated with STF-1. Compared with basal levels, we observed a trend toward an increase in insulin promoter activity in intermediate passage cells with STF-1 transfection (1.43-fold) that became a significant increase when E2-5 was cotransfected (1.78-fold). In late passage cells, transfection of STF-1 alone significantly stimulated a 2.2-fold increase in the insulin promoter activity. Cotransfection of STF-1 and E2-5 in late passage cells stimulated insulin promoter activity 2.8-fold, which was 40% of the activity observed in early passage cells. Control studies in glucotoxic betaTC-6 cells deficient in RIPE-3b1 activator but not STF-1 did not demonstrate an increase in insulin promoter

  14. Positive autoregulation of the transcription factor Pax6 in response to increased levels of either of its major isoforms, Pax6 or Pax6(5a, in cultured cells

    Directory of Open Access Journals (Sweden)

    Mason John O

    2006-05-01

    Full Text Available Abstract Background Pax6 is a transcription factor essential for normal development of the eyes and nervous system. It has two major isoforms, Pax6 and Pax6(5a, and the ratios between their expression levels vary within narrow limits. We tested the effects of overexpressing either one or other isoform on endogenous Pax6 expression levels in Neuro2A and NIH3T3 cells. Results We found that both isoforms caused an up-regulation of endogenous Pax6 expression in cells with (Neuro2A or without (NIH3T3 constitutive Pax6 expression. Western blots showed that cells stably transfected with constructs expressing either Pax6 or Pax6(5a contained raised levels of both Pax6 and Pax6(5a. Quantitative RT-PCR confirmed an increase in levels of Pax6(5a mRNA in cells containing Pax6-expressing constructs and an increase in levels of Pax6 mRNA in cells containing Pax6(5a-expressing constructs. The fact that the introduction of constructs expressing only one isoform increased the cellular levels of not only that isoform but also the other indicates that activation of the endogenous Pax6 locus occurred. The ratio between the levels of the two isoforms was maintained close to physiological values. The overexpression of either isoform in neuroblastoma (Neuro2A cell lines also promoted morphological change and an increase in β-III-tubulin expression, indicating an increase in neurogenesis. Conclusion Our results demonstrate that Pax6 can up-regulate production of Pax6 protein from an entire intact endogenous Pax6 locus in its genomic environment. This adds to previous studies showing that Pax6 can up-regulate reporter expression driven by isolated Pax6 regulatory elements. Furthermore, our results suggest that an important function of positive feedback might be to stabilise the relative levels of Pax6 and Pax6(5a.

  15. Investigation of factors responsible for cell line cytoplasmic expression differences

    Directory of Open Access Journals (Sweden)

    Finn Jonathan D

    2005-05-01

    Full Text Available Abstract Background Previous work has described a novel cytoplasmic expression system that results in a 20-fold increase in the levels of gene expression over a standard CMV-based nuclear expression system, as compared with a 2–3 fold increase seen with previous similar systems. While this increase was seen with BHK and Neuro-2a cells, further studies revealed that some cell lines, such as COS-7, demonstrated relatively poor levels of cytoplasmic expression. The objective of this study was to determine what factors were responsible for the different expression levels between BHK (a high expressing cell line and COS-7 (a low expressing cell line. Results The main findings of this work are that the individual elements of the cytoplasmic expression system (such as the T7 RNAP gene and Internal Ribosome Entry Sequence are functioning similarly in both cell types. Both cell types were found to have the same amount of cytosolic nuclease activity, and that the cells appeared to have differences in the intra-cellular processing of DNA -cationic lipid complexes. Conclusion After exploring many factors, it was found that differences in the intra-cellular processing of the DNA-cationic lipid complex was the most probable factor responsible for the difference in cytoplasmic gene expression.

  16. Co-overexpression of Mgat1 and Mgat4 in CHO cells for production of highly sialylated albumin-erythropoietin.

    Science.gov (United States)

    Cha, Hyun-Myoung; Lim, Jin-Hyuk; Yeon, Jung-Heum; Hwang, Jeong-Min; Kim, Dong-Il

    2017-08-01

    Terminal sialic acids on N-glycan of recombinant human erythropoietin are very important for in vivo half-life, as this glycoprotein has three N-glycosylation sites. N-acetylglucosaminyltransferases I, II, IV, and V (i.e. Mgat1, Mgat2, Mgat4, and Mgat5) catalyze the formation of a glycan antennary structure. These enzymes display different reaction kinetics for a common substrate and generally show low expression in Chinese hamster ovary (CHO) cells. Therefore, genetic control of Mgat expression is an effective method to increase sialic acid contents by enhancing glycan antennarity. To produce highly sialylated albumin-erythropoietin (Alb-EPO), we co-overexpressed the Mgat1 and Mgat4 genes in CHO cells and determined the optimal ratio of Mgat1:Mgat4 gene expression. All transfected cell lines showed increased gene expression of Mgat4, including Mgat1 overexpressing cell line. Sialic acid content of Alb-EPO was highest in co-transfected cells with excess Mgat4 gene, and these cells showed a higher tri- and tetra-antennary structure than control cells. Based on these results, we suggest that co-transfection of the Mgat1 and Mgat4 genes at a ratio of 2:8 is optimal for extension of antennary structures. Also, regulation of Mgat gene expression in the glycan biosynthesis pathway can be a novel approach to increase the terminal sialic acids of N-glycans. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. U6 snRNA expression prevents toxicity in TDP-43-knockdown cells.

    Directory of Open Access Journals (Sweden)

    Masao Yahara

    Full Text Available Depletion of amyotrophic lateral sclerosis (ALS-associated transactivation response (TAR RNA/DNA-binding protein 43 kDa (TDP-43 alters splicing efficiency of multiple transcripts and results in neuronal cell death. TDP-43 depletion can also disturb expression levels of small nuclear RNAs (snRNAs as spliceosomal components. Despite this knowledge, the relationship between cell death and alteration of snRNA expression during TDP-43 depletion remains unclear. Here, we knocked down TDP-43 in murine neuroblastoma Neuro2A cells and found a time lag between efficient TDP-43 depletion and appearance of cell death, suggesting that several mechanisms mediate between these two events. The amount of U6 snRNA was significantly decreased during TDP-43 depletion prior to increase of cell death, whereas that of U1, U2, and U4 snRNAs was not. Downregulation of U6 snRNA led to cell death, whereas transient exogenous expression of U6 snRNA counteracted the effect of TDP-43 knockdown on cell death, and slightly decreased the mis-splicing rate of Dnajc5 and Sortilin 1 transcripts, which are assisted by TDP-43. These results suggest that regulation of the U6 snRNA expression level by TDP-43 is a key factor in the increase in cell death upon TDP-43 loss-of-function.

  18. Bax translocation into mitochondria during dihydroartemisinin(DHA)-induced apoptosis in human lung adenocarcinoma cells

    Science.gov (United States)

    Lu, Ying-ying; Chen, Tong-sheng; Qu, Jun-Le

    2009-02-01

    Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, isolated from the traditional Chinese herb Artemisia annua, has been shown to possess promising anticancer activities and induce cancer cell death through apoptotic pathways. However, the molecular mechanisms are not well understood. This study was investigated in human lung adenocarconoma ASTC-a-1 cell line and aimed to determine whether the apoptotic process was mediated by Bax activation and translocation during DHA-induced apoptosis. In this study, DHA induced a time-dependent apoptotic cell death, which was assayed by Cell Counting Kit (CCK-8) and Hoechst 33258 staining. Detection of Bax aggregation and translocation to mitochondria was observed in living cells which were co-transfected with GFP-Bax and Dsred-mito plasmid using confocal fluorescence microscope technique. Overall, these results demonstrated that Bax activation and translocation to mitochondria occurred during DHA-induced apoptosis.

  19. Cre Fused with RVG Peptide Mediates Targeted Genome Editing in Mouse Brain Cells In Vivo.

    Science.gov (United States)

    Zou, Zhiyuan; Sun, Zhaolin; Li, Pan; Feng, Tao; Wu, Sen

    2016-12-14

    Cell penetrating peptides (CPPs) are short peptides that can pass through cell membranes. CPPs can facilitate the cellular entry of proteins, macromolecules, nanoparticles and drugs. RVG peptide (RVG hereinafter) is a 29-amino-acid CPP derived from a rabies virus glycoprotein that can cross the blood-brain barrier (BBB) and enter brain cells. However, whether RVG can be used for genome editing in the brain has not been reported. In this work, we combined RVG with Cre recombinase for bacterial expression. The purified RVG-Cre protein cut plasmids in vitro and traversed cell membranes in cultured Neuro2a cells. By tail vein-injecting RVG-Cre into Cre reporter mouse lines mTmG and Rosa26lacZ, we demonstrated that RVG-Cre could target brain cells and achieve targeted somatic genome editing in adult mice. This direct delivery of the gene-editing enzyme protein into mouse brains with RVG is much safer than plasmid- or viral-based methods, holding promise for further applications in the treatment of various brain diseases.

  20. Generation of reactive oxygen species mediates butein-induced apoptosis in neuroblastoma cells.

    Science.gov (United States)

    Chen, Ya-Hui; Yeh, Chi-Wei; Lo, Hui-Chen; Su, Shih-Li; Hseu, You-Cheng; Hsu, Li-Sung

    2012-04-01

    Flavonoids exhibit chemopreventive and chemotherapeutic effects. Butein, a bioactive flavonoid isolated from numerous native plants, has been shown to induce apoptosis in human cancer cells. In the current study, the molecular mechanisms of butein action on cell proliferation and apoptosis of neuroblastoma cells were evaluated. Treatment with butein decreased the viability of Neuro-2A neuroblastoma cells in a dose- and time-dependent manner. The dose-dependent nature of butein-induced apoptosis was characterized by an increase in the sub-G1 phase population. Treatment with butein significantly increased intracellular reactive oxygen species (ROS)levels and reduced the Bcl-2/Bax ratio, triggering the cleavage of pro-caspase 3 and poly-(ADP-ribose) polymerase (PARP). Pre-treatment with the antioxidant agent, N-acetyl cysteine (NAC), blocks butein-induced ROS generation and cell death. NAC also recovers butein-induced apoptosis-related protein alteration. In conclusion, butein-triggered neuroblastoma cells undergo apoptosis via generation of ROS, alteration of the Bcl‑2/Bax ratio, and cleavage of pro-caspase 3 and PARP. Our results suggest that butein may serve as a potential therapeutic agent for the treatment of neuroblastoma.

  1. Improved antitumour immunity in murine neuroblastoma using a combination of IL-2 and IL-12.

    Science.gov (United States)

    Siapati, K E; Barker, S; Kinnon, C; Michalski, A; Anderson, R; Brickell, P; Thrasher, A J; Hart, S L

    2003-05-19

    Neuroblastoma immunotherapy using cytokine-modified tumour cells has been tested in clinical trials. However, because of the complex nature of antitumour immune responses, a number of therapies may be required for complete tumour eradication and generation of systemic immunity. We report here the improved antitumour effect of two cytokines, interleukin-2 (IL-2) and interleukin-12 (IL-12), when coexpressed by neuroblastoma cell lines. Initially, transfection of human and mouse neuroblastoma cell lines resulted in high expression levels of biologically active IL-2 and IL-12 in vitro. These cytokines when expressed by transfected Neuro-2A cells completely abolished their in vivo tumorigenicity in a syngeneic neuroblastoma model. Vaccination of established tumours with IL-12-producing cells exhibited a clear effect with reduced tumour growth in the presence of IL-2. In vivo depletion studies showed that CD4(+) and CD8(+) T cells mediate the response against cytokine-producing cells. These results suggest that IL-2 and IL-12, when cotransfected in tumour cells, are effective against established disease and provide a promising immunotherapeutic approach for the treatment of neuroblastoma.

  2. Novel anthranilamide-pyrazolo[1,5-a]pyrimidine conjugates modulate the expression of p53-MYCN associated micro RNAs in neuroblastoma cells and cause cell cycle arrest and apoptosis.

    Science.gov (United States)

    Ramaiah, M Janaki; Pushpavalli, Sreerangam N C V L; Lavanya, A; Bhadra, Kaustav; Haritha, V; Patel, Nibedita; Tamboli, Jaki R; Kamal, Ahmed; Bhadra, Utpal; Pal-Bhadra, Manika

    2013-10-15

    It has previously been shown that anthranilamide-pyrazolo[1,5-a]pyrimidine conjugates activate p53 and cause apoptosis in cervical cancer cells such as HeLa and SiHa. Here we establish the role of these conjugates in activating p53 pathway by phosphorylation at Ser15, 20 and 46 residues and downregulate key oncogenic proteins such as MYCN and Mdm2 in IMR-32 neuroblastoma cells. Compounds decreased the proliferation rate of neuroblastoma cells such as IMR-32, Neuro-2a, SK-N-SH. Compound treatment resulted in G2/M cell cycle arrest. The expression of p53 dependent genes such as p21, Bax, caspases was increased with concomitant decrease of the survival proteins as well as anti-apoptotic proteins such as Akt1, E2F1 and Bcl2. In addition the expression of important microRNAs such as miR-34a, c, miR-200b, miR-107, miR-542-5p and miR-605 were significantly increased that eventually lead to the activation of apoptotic pathway. Our data revealed that conjugates of this nature cause cell cycle arrest and apoptosis in IMR-32 cells [MYCN (+) with intact wild-type p53] by activating p53 signalling and provides a lead for the development of anti-cancer therapeutics. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. Antisense downregulation of mutant huntingtin in a cell model

    DEFF Research Database (Denmark)

    Hasholt, L.; Abell, K.; Norremolle, A.

    2003-01-01

    Background Huntington's disease (HD) is an inherited neurodegenerative disorder which is caused by an expansion of a CAG repeat sequence in the HD gene. The repeat encodes an expanded polyglutamine tract in the protein huntingtin. The still unknown pathological mechanisms leading to death...... transfected with plasmid constructs containing exon 1 of the HD gene with expanded CAG repeats in frame with the reporter protein EGFP. The transfected cell cultures were treated with a phosphorothioated antisense oligonucleotide (PS-ASHD/20+) or a control oligonucleotide either by cotransfection...... is a sensitive biological marker. The findings suggest that antisense knockdown of huntingtin could be a useful strategy for treatment of HD, and could also be suitable for studies of the normal and pathological function of huntingtin in different cellular model systems....

  4. Viola tricolor Induces Apoptosis in Cancer Cells and Exhibits Antiangiogenic Activity on Chicken Chorioallantoic Membrane

    Directory of Open Access Journals (Sweden)

    Hamid Reza Sadeghnia

    2014-01-01

    Full Text Available In the present study, the cytotoxic and apoptogenic properties of hydroalcoholic extract and ethyl acetate (EtOAc, n-butanol, and water fractions (0–800 μg/mL of Viola tricolor were investigated in Neuro2a mouse neuroblastoma and MCF-7 human breast cancer cells. In addition, antiangiogenic effect of EtOAc fraction was evaluated on chicken chorioallantoic membrane (CAM. The quality of EtOAc fraction was also characterized using high performance liquid chromatography (HPLC fingerprint. Cytotoxicity assay revealed that EtOAc fraction was the most potent among all fractions with maximal effect on MCF-7 and minimal toxicity against normal murine fibroblast L929 cells. Apoptosis induction by EtOAc fraction was confirmed by increased sub-G1 peak of propidium iodide (PI stained cells. This fraction triggered the apoptotic pathway by increased Bax/Bcl-2 ratio and cleaved caspase-3 level. Moreover, treatment with EtOAc fraction significantly decreased the diameter of vessels on CAM, while the number of newly formed blood vessels was not suppressed significantly. Analysis of quality of EtOAc fraction using HPLC fingerprint showed six major peaks with different retention times. The results of the present study suggest that V. tricolor has potential anticancer property by inducing apoptosis and inhibiting angiogenesis.

  5. Overexpression of protein tyrosine phosphatase-alpha (PTP-alpha) but not PTP-kappa inhibits translocation of GLUT4 in rat adipose cells

    DEFF Research Database (Denmark)

    Cong, L N; Chen, H; Li, Y

    1999-01-01

    Protein tyrosine phosphatases (PTPases) are likely to play important roles in insulin action. We recently demonstrated that the nontransmembrane PTPase PTP1B can act as a negative modulator of insulin-stimulated translocation of GLUT4. We now examine the role of PTP-alpha and PTP-kappa (two...... transmembrane PTPases) in this metabolic action of insulin. Rat adipose cells were transfected with either PTP-alpha or PTP-kappa and effects of these PTPases on the translocation of a cotransfected epitope-tagged GLUT4 were studied. Cells overexpressing wild-type PTP-alpha had significantly lower levels...... of cell surface GLUT4 in response to insulin and a threefold decrease in insulin sensitivity when compared with control cells expressing only tagged GLUT4. Co-overexpression of PTP-alpha and PTP1B did not have additive effects, suggesting that these PTPases share common substrates. Cells overexpressing...

  6. Neurite outgrowth stimulatory effects of culinary-medicinal mushrooms and their toxicity assessment using differentiating Neuro-2a and embryonic fibroblast BALB/3T3

    OpenAIRE

    Phan, Chia-Wei; David, Pamela; Naidu, Murali; Wong, Kah-Hui; Sabaratnam, Vikineswary

    2013-01-01

    Background Mushrooms are not only regarded as gourmet cuisine but also as therapeutic agent to promote cognition health. However, little toxicological information is available regarding their safety. Therefore, the aim of this study was to screen selected ethno-pharmacologically important mushrooms for stimulatory effects on neurite outgrowth and to test for any cytotoxicity. Methods The stimulatory effect of mushrooms on neurite outgrowth was assessed in differentiating mouse neuroblastoma (...

  7. Germline Gene Editing in Chickens by Efficient CRISPR-Mediated Homologous Recombination in Primordial Germ Cells.

    Directory of Open Access Journals (Sweden)

    Lazar Dimitrov

    Full Text Available The CRISPR/Cas9 system has been applied in a large number of animal and plant species for genome editing. In chickens, CRISPR has been used to knockout genes in somatic tissues, but no CRISPR-mediated germline modification has yet been reported. Here we use CRISPR to target the chicken immunoglobulin heavy chain locus in primordial germ cells (PGCs to produce transgenic progeny. Guide RNAs were co-transfected with a donor vector for homology-directed repair of the double-strand break, and clonal populations were selected. All of the resulting drug-resistant clones contained the correct targeting event. The targeted cells gave rise to healthy progeny containing the CRISPR-targeted locus. The results show that gene-edited chickens can be obtained by modifying PGCs in vitro with the CRISPR/Cas9 system, opening up many potential applications for efficient genetic modification in birds.

  8. Germline Gene Editing in Chickens by Efficient CRISPR-Mediated Homologous Recombination in Primordial Germ Cells.

    Science.gov (United States)

    Dimitrov, Lazar; Pedersen, Darlene; Ching, Kathryn H; Yi, Henry; Collarini, Ellen J; Izquierdo, Shelley; van de Lavoir, Marie-Cecile; Leighton, Philip A

    2016-01-01

    The CRISPR/Cas9 system has been applied in a large number of animal and plant species for genome editing. In chickens, CRISPR has been used to knockout genes in somatic tissues, but no CRISPR-mediated germline modification has yet been reported. Here we use CRISPR to target the chicken immunoglobulin heavy chain locus in primordial germ cells (PGCs) to produce transgenic progeny. Guide RNAs were co-transfected with a donor vector for homology-directed repair of the double-strand break, and clonal populations were selected. All of the resulting drug-resistant clones contained the correct targeting event. The targeted cells gave rise to healthy progeny containing the CRISPR-targeted locus. The results show that gene-edited chickens can be obtained by modifying PGCs in vitro with the CRISPR/Cas9 system, opening up many potential applications for efficient genetic modification in birds.

  9. Live-cell visualization of intracellular interaction between a nuclear migration protein (hNUDC and the thrombopoietin receptor (Mpl.

    Directory of Open Access Journals (Sweden)

    Yuan-Bin Zheng

    Full Text Available We previously demonstrated that endogenous hNUDC and Mpl co-localized in the perinuclear and cytoplasmic regions of megakaryocyte cells by indirect immunofluorescence. We further reported that hNUDC accumulated in the Golgi when NIH 3T3 cells were transfected with an hNUDC expression vector alone. However, co-transfection with hNUDC and Mpl expression vectors caused both proteins to co-localize predominantly in the cytosol. These observations led us to hypothesize that a complex containing hNUDC and Mpl may alter hNUDC subcellular location and induce its secretion. In the present study, we test this hypothesis by employing bimolecular fluorescence complementation (BiFC to detect and visualize the complex formation of hNUDC/Mpl in living cells. We further examined in detail the subcellular locations of the hNUDC/Mpl complex by co-transfection of BiFC chimeras with known subcellular markers. The distribution of hNUDC/Mpl in the endoplasmic reticulum (ER, Golgi and cell surface was determined. Furthermore, the N-terminal 159 amino acids of hNUDC, but not C-terminal half, bound to Mpl in vivo and exhibited a similar localization pattern to that of full-length hNUDC in Cos-1 cells. Adenovirus-mediated overexpression of hNUDC or its N-terminal 159 residues in a human megakaryocyte cell line (Dami resulted in increased levels of hNUDC or hNUDC(1-159 secretion. In contrast, depletion of Mpl by transfecting Dami cells with adenovirus bearing Mpl-targeting siRNA significantly blocked hNUDC secretion. Thus, we provide the first evidence that the N-terminal region of hNUDC contains all of the necessary information to complex with Mpl and traffic through the secretory pathway.

  10. Counterselection and co-delivery of transposon and transposase functions for Sleeping Beauty-mediated transposition in cultured mammalian cells.

    Science.gov (United States)

    Converse, Andrea D; Belur, Lalitha R; Gori, Jennifer L; Liu, Geyi; Amaya, Felipe; Aguilar-Cordova, Estuardo; Hackett, Perry B; McIvor, R Scott

    2004-12-01

    Sleeping Beauty (SB) is a gene-insertion system reconstructed from transposon sequences found in teleost fish and is capable of mediating the transposition of DNA sequences from transfected plasmids into the chromosomes of vertebrate cell populations. The SB system consists of a transposon, made up of a gene of interest flanked by transposon inverted repeats, and a source of transposase. Here we carried out a series of studies to further characterize SB-mediated transposition as a tool for gene transfer to chromosomes and ultimately for human gene therapy. Transfection of mouse 3T3 cells, HeLa cells, and human A549 lung carcinoma cells with a transposon containing the neomycin phosphotransferase (NEO) gene resulted in a several-fold increase in drug-resistant colony formation when co-transfected with a plasmid expressing the SB transposase. A transposon containing a methotrexate-resistant dihydrofolate reductase gene was also found to confer an increased frequency of methotrexate-resistant colony formation when co-transfected with SB transposase-encoding plasmid. A plasmid containing a herpes simplex virus thymidine kinase gene as well as a transposon containing a NEO gene was used for counterselection against random recombinants (NEO+TK+) in medium containing G418 plus ganciclovir. Effective counterselection required a recovery period of 5 days after transfection before shifting into medium containing ganciclovir to allow time for transiently expressed thymidine kinase activity to subside in cells not stably transfected. Southern analysis of clonal isolates indicated a shift from random recombination events toward transposition events when clones were isolated in medium containing ganciclovir as well as G418. We found that including both transposon and transposase functions on the same plasmid substantially increased the stable gene transfer frequency in Huh7 human hepatoma cells. The results from these experiments contribute technical and conceptual insight into

  11. Comparative Study of Various Delivery Methods for the Supply of Alpha-Ketoglutarate to the Neural Cells for Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Tanushree Vishnoi

    2013-01-01

    Full Text Available Delivery of growth factors or bioactive molecules plays an important role in tissue engineering, as the duration to which these are supplied can modulate the cell fate. Thus, the delivery method plays an important role, and the same is presented in this work wherein the exogenous supply of alpha-ketoglutarate (α-KG gave better results for fast proliferating cells as compared to delivery by microspheres or microspheres incorporated scaffolds which can be used while culturing slow growing cells. All these studies were performed in two dimensional (2D and three dimensional (3D setups in which chitosan-gelatin-polypyrrole has been used as 3-D scaffolds. Chitosan and gelatin microspheres alone as well as incorporated in the cryogels were characterized. MTT assay done using neuro-2a cell line showed approximately 42% and 70% increment in cellular proliferation when gelatin and chitosan microspheres were added in a 3-D setup, respectively, as compared to the control. Biochemical analysis of ammonia showed 6-fold reductions in ammonia level in a 3-D setup compared to the control. We also studied the synthesis of a neurotransmitter-like glutamate and found that its concentration increased up to 0.25 mg/ml when the microspheres were added exogenously in a 3-D system.

  12. Withanone, an Active Constituent from Withania somnifera, Affords Protection Against NMDA-Induced Excitotoxicity in Neuron-Like Cells.

    Science.gov (United States)

    Dar, Nawab John; Bhat, Javeed Ahmad; Satti, Naresh Kumar; Sharma, Parduman Raj; Hamid, Abid; Ahmad, Muzamil

    2017-09-01

    Withania somnifera has immense pharmacologic and clinical uses. Owing to its similar pharmacologic activity as that of Korean Ginseng tea, it is popularly called as Indian ginseng. In most cases, extracts of this plant have been evaluated against various diseases or models of disease. However, little efforts have been made to evaluate individual constituents of this plant for neurodegenerative disorders. Present study was carried out to evaluate Withanone, one of the active constituents of Withania somnifera against NMDA-induced excitotoxicity in retinoic acid, differentiated Neuro2a cells. Cells were pre-treated with 5, 10 and 20 μM doses of Withanone and then exposed to 3-mM NMDA for 1 h. MK801, a specific NMDA receptor antagonist, was used as positive control. The results indicated that NMDA induces significant death of cells by accumulation of intracellular Ca2+, generation of reactive oxygen species (ROS), loss of mitochondrial membrane potential, crashing of Bax/Bcl-2 ratio, release of cytochrome c, increased caspase expression, induction of lipid peroxidation as measured by malondialdehyde levels and cleavage of poly(ADP-ribose) polymerase-1 (Parp-1), which is indicative of DNA damage. All these parameters were attenuated with various doses of Withanone pre-treatment. These results suggest that Withanone may serve as potential neuroprotective agent.

  13. Myosin IIB isoform plays an essential role in the formation of two distinct types of macropinosomes

    OpenAIRE

    Jiang, Jun; Kolpak, Adrianne L.; Bao, Zheng-Zheng

    2010-01-01

    The function and mechanism of macropinocytosis in cells outside of the immune system remain poorly understood. We used a neuroblastoma cell line, Neuro-2a, to study macropinocytosis in neuronal cells. We found phorbol 12-myristate 13-acetate (PMA) and insulin-like growth factor 1 (IGF-1) induced two dinstinct types of macropinocytosis in the Neuro-2a cells. IGF-1-induced macropinocytosis occurs mostly around the cell bodies and requires phosphoinositide 3-kinase (PI3K), while PMA-induced macr...

  14. New cell line development for antibody-producing Chinese hamster ovary cells using split green fluorescent protein

    Directory of Open Access Journals (Sweden)

    Kim Yeon-Gu

    2012-05-01

    Full Text Available Abstract Background The establishment of high producer is an important issue in Chinese hamster ovary (CHO cell culture considering increased heterogeneity by the random integration of a transfected foreign gene and the altered position of the integrated gene. Fluorescence-activated cell sorting (FACS-based cell line development is an efficient strategy for the selection of CHO cells in high therapeutic protein production. Results An internal ribosome entry site (IRES was introduced for using two green fluorescence protein (GFP fragments as a reporter to both antibody chains, the heavy chain and the light chain. The cells co-transfected with two GFP fragments showed the emission of green fluorescence by the reconstitution of split GFP. The FACS-sorted pool with GFP expression had a higher specific antibody productivity (qAb than that of the unsorted pool. The qAb was highly correlated with the fluorescence intensity with a high correlation coefficient, evidenced from the analysis of median GFP and qAb in individual selected clones. Conclusions This study proved that the fragment complementation for split GFP could be an efficient indication for antibody production on the basis of high correlation of qAb with reconstitution of GFP. Taken together, we developed an efficient FACS-based screening method for high antibody-producing CHO cells with the benefits of the split GFP system.

  15. PEGylated and targeted extracellular vesicles display enhanced cell specificity and circulation time.

    Science.gov (United States)

    Kooijmans, S A A; Fliervoet, L A L; van der Meel, R; Fens, M H A M; Heijnen, H F G; van Bergen En Henegouwen, P M P; Vader, P; Schiffelers, R M

    2016-02-28

    Extracellular vesicles (EVs) are increasingly being recognized as candidate drug delivery systems due to their ability to functionally transfer biological cargo between cells. However, the therapeutic applicability of EVs may be limited due to a lack of cell-targeting specificity and rapid clearance of exogenous EVs from the circulation. In order to improve EV characteristics for drug delivery to tumor cells, we have developed a novel method for decorating EVs with targeting ligands conjugated to polyethylene glycol (PEG). Nanobodies specific for the epidermal growth factor receptor (EGFR) were conjugated to phospholipid (DMPE)-PEG derivatives to prepare nanobody-PEG-micelles. When micelles were mixed with EVs derived from Neuro2A cells or platelets, a temperature-dependent transfer of nanobody-PEG-lipids to the EV membranes was observed, indicative of a 'post-insertion' mechanism. This process did not affect EV morphology, size distribution, or protein composition. After introduction of PEG-conjugated control nanobodies to EVs, cellular binding was compromised due to the shielding properties of PEG. However, specific binding to EGFR-overexpressing tumor cells was dramatically increased when EGFR-specific nanobodies were employed. Moreover, whereas unmodified EVs were rapidly cleared from the circulation within 10min after intravenous injection in mice, EVs modified with nanobody-PEG-lipids were still detectable in plasma for longer than 60min post-injection. In conclusion, we propose post-insertion as a novel technique to confer targeting capacity to isolated EVs, circumventing the requirement to modify EV-secreting cells. Importantly, insertion of ligand-conjugated PEG-derivatized phospholipids in EV membranes equips EVs with improved cell specificity and prolonged circulation times, potentially increasing EV accumulation in targeted tissues and improving cargo delivery. Copyright © 2015. Published by Elsevier B.V.

  16. Influence of RNA interference on the mitochondrial subcellular localization of alpha-synuclein and on the formation of Lewy body-like inclusions in the cytoplasm of human embryonic kidney 293 cells induced by the overexpression of alpha-synuclein☆

    Science.gov (United States)

    Chen, Tao; Liao, Xiaoping; Wen, Guoqiang; Deng, Yidong; Guo, Min; Long, Zhigang; Ouyang, Feng

    2012-01-01

    The specific and effective α-synuclein RNA interference (RNAi) plasmids, and the α-synuclein-pEGFP recombinant plasmids were co-transfected into human embryonic kidney 293 (HEK293) cells using the lipofectamine method. Using an inverted fluorescence microscope, α-synuclein proteins were observed to aggregate in the cytoplasm and nucleus. Wild-type α-synuclein proteins co-localized with mitochondria. Hematoxylin-eosin staining revealed round eosinophilic bodies (Lewy body-like inclusions) in the cytoplasm of some cells transfected with α-synuclein-pEGFP plasmid. However, the formation of Lewy body-like inclusions was not observed following transfection with the RNAi pSYN-1 plasmid. RNAi blocked Lewy body-like inclusions in the cytoplasm of HEK293 cells induced by wild-type α-synuclein overexpression, but RNAi did not affect the subcellular localization of wild-type α-synuclein in mitochondria. PMID:25767480

  17. Endocrine disrupting potentials of Bisphenol A, Bisphenol A dimethacrylate, 4-n-Nonyl-phenol and 4-Octylphenol assessed in cell model systems for effects on the estrogen-, androgen-, aryl hydrocarbon-receptor and aromatase activity

    DEFF Research Database (Denmark)

    Bonefeld-Jørgensen, Eva Cecilie; Long, Manhai; Hofmeister, Marlene V

      An array of plastic components is known to possess endocrine disruption (ED) potentials. Bisphenol A (BPA) and BPA-Dimethacrylate (BPA-DM) are monomers used to high extent in the plastic industry and as dental sealants. Alkylphenols such as 4-n-nonylphenol (NP) and 4-octylphenol (OP) are widely...... used as surfactants. We have investigated the effect in vitro of these four plasticizers in four cell culture model systems.The estrogenic potencies were analyzed using the stable ERE-luciferase transfected cell line MVLN measuring the relative estrogen receptor (ER) transactivated luciferase units...... (RLU). Effects on the Androgen receptor (AR) trans-activation were investigated by co-transfection of the CHO-K1 hamster cells using the reporter vector MMTV-LUC and the AR expression vector pSVAR0. Effects on the CYP19 aromatase, an important enzyme in the steroid synthesis pathway involved...

  18. [Establishment and identification of mouse lymphoma cell line EL4 expressing red fluorescent protein].

    Science.gov (United States)

    Li, Yan-Jie; Cao, Jiang; Chen, Chong; Wang, Dong-Yang; Zeng, Ling-Yu; Pan, Xiu-Ying; Xu, Kai-Lin

    2010-02-01

    This study was purposed to construct a lentiviral vector encoding red fluorescent protein (DsRed) and transfect DsRed into EL4 cells for establishing mouse leukemia/lymphoma model expressing DsRed. The bicistronic SIN lentiviral transfer plasmid containing the genes encoding neo and internal ribosomal entry site-red fluorescent protein (IRES-DsRed) was constructed. Human embryonic kidney 293FT cells were co-transfected with the three plasmids by liposome method. The viral particles were collected and used to transfect EL4 cells, then the cells were selected by G418. The results showed that the plasmid pXZ208-neo-IRES-DsRed was constructed successfully, and the viral titer reached to 10(6) U/ml. EL4 cells were transfected by the viral solution efficiently. The transfected EL4 cells expressing DsRed survived in the final concentration 600 microg/ml of G418. The expression of DsRed in the transfected EL4 cells was demonstrated by fluorescence microscopy and flow cytometry. In conclusion, the EL4/DsRed cell line was established successfully.

  19. Polo-like kinase (Plk)1 depletion induces apoptosis in cancer cells.

    Science.gov (United States)

    Liu, Xiaoqi; Erikson, Raymond L

    2003-05-13

    Elevated expression of mammalian polo-like kinase (Plk)1 occurs in many different types of cancers, and Plk1 has been proposed as a novel diagnostic marker for several tumors. We used the recently developed vector-based small interfering RNA technique to specifically deplete Plk1 in cancer cells. We found that Plk1 depletion dramatically inhibited cell proliferation, decreased viability, and resulted in cell-cycle arrest with 4 N DNA content. The formation of dumbbell-like chromatin structure suggests the inability of these cells to completely separate the sister chromatids at the onset of anaphase. Plk1 depletion induced apoptosis, as indicated by the appearance of subgenomic DNA in fluorescence-activated cell-sorter (FACS) profiles, the activation of caspase 3, and the formation of fragmented nuclei. Plk1-depletion-induced apoptosis was partially reversed by cotransfection of nondegradable mouse Plk1 constructs. In addition, the p53 pathway was shown to be involved in Plk1-depletion-induced apoptosis. DNA damage occurred in Plk1-depleted cells and inhibition of ATM strongly potentiated the lethality of Plk1 depletion. Although p53 is stabilized in Plk1-depleted cells, DNA damage also occurs in p53(-/-) cells. These data support the notion that disruption of Plk1 function could be an important application in cancer therapy.

  20. Decreased long non-coding RNA MTM contributes to gastric cancer cell migration and invasion via modulating MT1F.

    Science.gov (United States)

    Lin, Zhenghua; Lai, Sanchuan; He, Xingkang; Zhuo, Wei; Wang, Lan; Si, Jianmin; Chen, Shujie

    2017-11-14

    The role of long non-coding RNAs (lncRNA) on gastric cancer (GC) are an emerging field. Here, we focused on a cancer-related lncRNA MTM and tried to explore its correlation with the development of GC. The expression of MTM was detected by qRT-PCR in GC cell lines and tissues. The relationship between MTM level and clinicopathological factors was then analyzed. Cell biological assays with overexpression or co-transfection approaches were examined to probe the functional relevance of this lncRNA and its potential targets. The results showed that MTM expression was significantly lower in GC cell lines and tissues, and closely correlated with lymphatic metastasis, invasive depth, tumor staging and overall survival. Overexpression of MTM significantly inhibited GC cell migration and invasion, suppressed cell proliferation and induced cell apoptosis. In addition, we found a positive correlation between the expression level of MTM and MT1F both in cell and tissue samples. MT1F overexpression decreased GC cell migration and invasion, while knockdown of MT1F restored cell migration and invasion in MTM-overexpressing GC cells, suggesting MT1F as a key target of MTM. Conclusively, abnormal decreased expression of MTM was observed in human GC, which might contribute to gastric carcinogenesis by modulating MT1F expression.

  1. Quercetin improves the activity of the ubiquitin-proteasomal system in 150Q mutated huntingtin-expressing cells but exerts detrimental effects on neuronal survivability.

    Science.gov (United States)

    Chakraborty, J; Rajamma, U; Jana, N; Mohanakumar, K P

    2015-10-01

    Quercetin, a strong free radical scavenger, is investigated for neuroprotective effects in a Neuro 2a cell line conditionally transfected with 16Q huntingtin (Htt) and 150Q Htt, which express the protein upon stimulation. Cells were protected from death by a 20-µM dose of quercetin on the second day of Htt induction, but 30-100-µM doses of the drug caused further toxicity in both 16Q and 150Q cells, as indicated by MTT assay and by significant reductions in the number of cells bearing neurites on the second day. A significant decrease in the number of cells containing aggregate was seen in induced 150Q cells treated with 20 µM but not for those treated with 40 or 50 µM quercetin up to 4 days of induction. Mutated Htt (mHtt)-induced reduction in proteasomal activity of the ubiquitin-proteasomal system (UPS) was significantly attenuated by 20 µM quercetin. However, neither mitochondrial membrane potential loss nor colocalization of 20S proteasome with mHtt aggregate was corrected by quercetin treatment. Our results imply that the neuroprotective effect of quercetin arises out of the upregulation of UPS activity, which causes a decrease in the number of mHtt aggregate-harboring cells. The increased neurotoxicity could result from the continued association of mHtt with 20S proteasome and the failure of quercetin to correct mitochondrial membrane potential loss. These results suggest that, although quercetin at a low dose protects against mHtt-mediated cell death, higher doses are toxic to the cells, clearly demarcating a narrow therapeutic window for this dietary flavonoid. © 2015 Wiley Periodicals, Inc.

  2. Real-time single cell analysis of Bid cleavage and translocation in cisplatin-induced apoptosis

    Science.gov (United States)

    Liu, Lei; Xing, Da; Pei, Yihui; Chen, Wei R.

    2007-02-01

    Cancer cell apoptosis can be induced by cisplatin, an efficient anticancer agent. However, its mechanism is not fully understood. Bcl-2 homology domain (BH) 3-only proteins couple stress signals to mitochondrial apoptotic pathways. Calpain-mediated cleavage of the BH3-only protein Bid into a 14 kD truncated protein (tBid) has been implicated in cisplatin-induced apoptotic pathway. We utilized a recombinant fluorescence resonance energy transfer (FRET) Bid probe to determine the kinetics of Bid cleavage during cisplatin-induced apoptosis in ASTC-a-1 cells. The cells were also co-transfected with Bid-CFP and DsRed-Mit to dynamically detect tBid translocation. Cells showed a cleavage of the Bid-FRET probe occurring at about 4-5 h after treated with 20 µM cisplatin. Cleavage of the Bid-FRET probe coincided with a translocation of tBid from the cytosolic to the mitochondria, and the translocation lasted about 1.5 h. Using real-time single-cell analysis, we first observed the kinetics of Bid cleavage and translocation to mitochondria in living cells during cisplatin-induced apoptosis.

  3. Dynamic imaging of interaction between protein 14-3-3 and Bid in living cells

    Science.gov (United States)

    Chen, Tongsheng; Xing, Da; Wang, Jinjun

    2006-02-01

    The 14-3-3 proteins are known to sequester certain pro-apoptotic members of this family. BH3- interacting domain death agonist (Bid) may contribute to tumor necrosis factor α(TNF-α)-induced neuronal death, although regulation by 14-3-3 has not been reported. In this study we examined whether 14-3-3 proteins interact with Bid/tBid during TNF-α-induced cell death. The TNF-αtriggered Bid cleavage and tBid translocated to mitochondria. Human lung adenocarcinoma cells were co-transfected with both CFP-Bid and 14-3-3-YFP plasmids, and the dynamical interaction between the Bid/tBid and 14-3-3 were performed on laser confocal fluorescence microscope in single living cell during TNF-α-induced cell apoptosis. The Bid distribute equally only in the cytoplasm of healthy cells, and the 14-3-3 protein distribute not only in the cytoplasm but also in the nucleus of healthy cells. Our data showed that the tBid aggregate, but the 14-3-3 protein does not aggregate as the tBid, and the 14-3-3 protein separate from the aggregated tBid, implying that the 14-3-3 proteins do not interact with the aggregated tBid after TNF-αtreatment.

  4. PI3-kinase in concert with Src promotes the S-phase entry of oestradiol-stimulated MCF-7 cells

    Science.gov (United States)

    Castoria, Gabriella; Migliaccio, Antimo; Bilancio, Antonio; Di Domenico, Marina; de Falco, Antonietta; Lombardi, Maria; Fiorentino, Roberto; Varricchio, Lilian; Barone, Maria Vittoria; Auricchio, Ferdinando

    2001-01-01

    The p85-associated phosphatidylinositol (PI) 3-kinase/Akt pathway mediates the oestradiol-induced S-phase entry and cyclin D1 promoter activity in MCF-7 cells. Experiments with Src, p85α and Akt dominant-negative forms indicate that in oestradiol-treated cells these signalling effectors target the cyclin D1 promoter. Oestradiol acutely increases PI3-kinase and Akt activities in MCF-7 cells. In NIH 3T3 cells expressing ERα, a dominant-negative p85 suppresses hormone stimulation of Akt. The Src inhibitor, PP1, prevents hormone stimulation of Akt and PI3-kinase activities in MCF-7 cells. In turn, stimulation of Src activity is abolished in ERα-expressing NIH 3T3 fibroblasts by co-transfection of the dominant-negative p85α and in MCF-7 cells by the PI3-kinase inhibitor, LY294002. These findings indicate a novel reciprocal cross-talk between PI3-kinase and Src. Hormone stimulation of MCF-7 cells rapidly triggers association of ERα with Src and p85. In vitro these proteins are assembled in a ternary complex with a stronger association than that of the binary complexes composed by the same partners. The ternary complex probably favours hormone activation of Src- and PI3-kinase-dependent pathways, which converge on cell cycle progression. PMID:11689445

  5. Co-transfection of decorin and interleukin-10 modulates pro-fibrotic extracellular matrix gene expression in human tenocyte culture

    Science.gov (United States)

    Abbah, Sunny A.; Thomas, Dilip; Browne, Shane; O'Brien, Timothy; Pandit, Abhay; Zeugolis, Dimitrios I.

    2016-02-01

    Extracellular matrix synthesis and remodelling are driven by increased activity of transforming growth factor beta 1 (TGF-β1). In tendon tissue repair, increased activity of TGF-β1 leads to progressive fibrosis. Decorin (DCN) and interleukin 10 (IL-10) antagonise pathological collagen synthesis by exerting a neutralising effect via downregulation of TGF-β1. Herein, we report that the delivery of DCN and IL-10 transgenes from a collagen hydrogel system supresses the constitutive expression of TGF-β1 and a range of pro-fibrotic extracellular matrix genes.

  6. MicroRNA-1185 Induces Endothelial Cell Apoptosis by Targeting UVRAG and KRIT1

    Directory of Open Access Journals (Sweden)

    Haoyuan Deng

    2017-04-01

    Full Text Available Background/Aims: Atherosclerosis is a multifactorial chronic disease and is the main cause of death and impairment in the world. Endothelial injury and apoptosis play a crucial role in the onset and development of atherosclerosis. MicroRNAs (miRNAs have been proven to be involved in the pathogenesis of atherosclerosis. However, studies of the functional role of apoptosis-related miRNAs in the endothelium during atherogenesis are limited. Methods: Cell injury and apoptosis were measured in five types of cells transfected with miR-1185 or co-transfected with miR-1185 and its inhibitor. Bioinformatics analysis and a luciferase reporter assay were used to confirm the targets of miR-1185. The effects of the targets of miR-1185 on endothelial apoptosis were determined using small-interfering RNA. Results: In this study, we first report that miR-1185 significantly promoted apoptosis in endothelial cells but not in vascular smooth muscle cells and macrophages. A mechanistic analysis showed that ultraviolet irradiation resistance-associated gene (UVRAG and krev1 interaction trapped gene 1 (KRIT1, targets of miR-1185, mediated miR-1185-induced endothelial cell apoptosis. Conclusion: The results revealed the impact of miR-1185 on endothelial apoptosis, suggesting that miR-1185 may be a potential target for the prevention and treatment of atherosclerosis.

  7. Drosophila S2 Schneider cells: a useful tool for rebuilding and redesigning approaches in synthetic biology.

    Science.gov (United States)

    Yang, Jianying; Reth, Michael

    2012-01-01

    Synthetic biology is an engineering approach to biology. A synthetic biologist wants to describe biological molecules and their subdomains as well-defined parts of a molecular machine. To achieve this goal, synthetic biologists rebuild minimal functional biological systems from well-defined parts or they design new molecules that do not exist in nature but have new and useful functions. In short, these engineering approaches can be summarized as "rebuild, alter, and understand." The Drosophila S2 Schneider cell is a useful tool for both rebuilding and redesigning approaches. S2 cells are phagocytic cells that easily take up large amounts of DNA from the cell culture. They, thus, have a high cotransfection rate, allowing the coexpression of up to 12 different proteins. We have developed a transient transfection protocol allowing the rapid and parallel analysis of wild-type and altered forms of a biological system. This chapter describes our methods to rebuild and better understand mammalian signaling systems in the evolutionary distant environment of Drosophila S2 cells.

  8. Mesenchymal stem cells alleviate Japanese encephalitis virus-induced neuroinflammation and mortality.

    Science.gov (United States)

    Bian, Peiyu; Ye, Chuantao; Zheng, Xuyang; Yang, Jing; Ye, Wei; Wang, Yuan; Zhou, Yun; Ma, Hongwei; Han, Peijun; Zhang, Hai; Zhang, Ying; Zhang, Fanglin; Lei, Yingfeng; Jia, Zhansheng

    2017-02-16

    Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis in Asia. Japanese encephalitis (JE) caused by JEV is characterized by extensive inflammatory cytokine secretion, microglia activation, blood-brain barrier (BBB) breakdown, and neuronal death, all of which contribute to the vicious cycle of inflammatory damage. There are currently no effective treatments for JE. Mesenchymal stem cells (MSCs) have been demonstrated to have a therapeutic effect on many central nervous system (CNS) diseases by regulating inflammation and other mechanisms. In vivo, 8- to 10-week-old mice were infected intraperitoneally with JEV and syngeneic bone marrow MSCs were administered through the caudal vein at 1 and 3 days post-infection. The mortality, body weight, and behavior were monitored daily. Brains from each group were harvested at the indicated times for hematoxylin and eosin staining, immunohistochemical observation, flow cytometric analysis, TUNEL staining, Western blot, quantitative real-time polymerase chain reaction, and BBB permeability assays. In vitro, co-culture and mixed culture experiments of MSCs with either microglia or neurons were performed, and then the activation state of microglia and survival rate of neurons were tested 48 h post-infection. MSC treatment reduced JEV-induced mortality and improved the recovery from JE in our mouse model. The inflammatory response, microglia activation, neuronal damage, BBB destruction, and viral load (VL) were significantly decreased in the MSC-treated group. In co-culture experiments, MSCs reprogrammed M1-to-M2 switching in microglia and improved neuron survival. Additionally, the VL was decreased in Neuro2a cells in the presence of MSCs accompanied by increased expression of interferon-α/β. MSC treatment alleviated JEV-induced inflammation and mortality in mice.

  9. Using intron splicing trick for preferential gene expression in transduced cells: an approach for suicide gene therapy.

    Science.gov (United States)

    Pourzadegan, F; Shariati, L; Taghizadeh, R; Khanahmad, H; Mohammadi, Z; Tabatabaiefar, M A

    2016-01-01

    Suicide gene therapy is one of the most innovative approaches in which a potential toxic gene is delivered to the targeted cancer cell by different target delivery methods. We constructed a transfer vector to express green fluorescent protein (GFP) in transduced cells but not in packaging cells. We placed gfp under the control of the cytomegalovirus (CMV) promoter, which is positioned between the two long-terminal repeats in reverse direction. The intron-2 sequence of the human beta globin gene with two poly-A signals and several stop codons on the antisense strand was placed on the leading strand between the CMV promoter and gfp. For lentiviral production, the HEK293T and line were co-transfected with the PMD2G, psPAX2 and pLentiGFP-Ins2 plasmids. The HEK293T and line were transduced with this virus. PCR was performed for evaluation of intron splicing in transduced cells. The GFP expression was seen in 65% of the cells transduced. The PCR amplification of the genomic DNA of transduced cells confirmed the splicing of intron 2. The strategy is significant to accomplish our goal for preserving the packaging cells from the toxic gene expression during viral assembly and the resultant reduction in viral titration. Also it serves to address several other issues in the gene therapy.

  10. The regulation of CD5 expression in murine T cells

    Directory of Open Access Journals (Sweden)

    Herzenberg Leonard A

    2001-05-01

    Full Text Available Abstract Background CD5 is a pan-T cell surface marker that is also present on a subset of B cells, B-1a cells.Functional and developmental subsets of T cells express characteristic CD5 levels that vary over roughly a 30-fold range. Previous investigators have cloned a 1.7 Kb fragment containing the CD5 promoter and showed that it can confer similar lymphocyte-specific expression pattern as observed for endogenous CD5 expression. Results We further characterize the CD5 promoter and identify minimal and regulatory regions on the CD5 promoter. Using a luciferase reporter system, we show that a 43 bp region on the CD5 promoter regulates CD5 expression in resting mouse thymoma EL4 T cells and that an Ets binding site within the 43 bp region mediates the CD5 expression. In addition, we show that Ets-1, a member of the Ets family of transcription factors, recognizes the Ets binding site in the electrophoretic mobility shift assay (EMSA. This Ets binding site is directly responsible for the increase in reporter activity when co-transfected with increasing amounts of Ets-1 expression plasmid. We also identify two additional evolutionarily-conserved regions in the CD5 promoter (CD5X and CD5Y and demonstrate the respective roles of the each region in the regulation of CD5 transcription. Conclusion Our studies define a minimal and regulatory promoter for CD5 and show that the CD5 expression level in T cells is at least partially dependent on the level of Ets-1 protein. Based on the findings in this report, we propose a model of CD5 transcriptional regulation in T cells.

  11. StearoylCoA desaturase-5: a novel regulator of neuronal cell proliferation and differentiation.

    Directory of Open Access Journals (Sweden)

    Debora I Sinner

    Full Text Available Recent studies have demonstrated that human stearoylCoA desaturase-1 (SCD1, a Δ9-desaturase that converts saturated fatty acids (SFA into monounsaturated fatty acids, controls the rate of lipogenesis, cell proliferation and tumorigenic capacity in cancer cells. However, the biological function of stearoylCoA desaturase-5 (SCD5, a second isoform of human SCD that is highly expressed in brain, as well as its potential role in human disease, remains unknown. In this study we report that the constitutive overexpression of human SCD5 in mouse Neuro2a cells, a widely used cell model of neuronal growth and differentiation, displayed a greater n-7 MUFA-to-SFA ratio in cell lipids compared to empty-vector transfected cells (controls. De novo synthesis of phosphatidylcholine and cholesterolesters was increased whereas phosphatidylethanolamine and triacylglycerol formation was reduced in SCD5-expressing cells with respect to their controls, suggesting a differential use of SCD5 products for lipogenic reactions. We also observed that SCD5 expression markedly accelerated the rate of cell proliferation and suppressed the induction of neurite outgrowth, a typical marker of neuronal differentiation, by retinoic acid indicating that the desaturase plays a key role in the mechanisms of cell division and differentiation. Critical signal transduction pathways that are known to modulate these processes, such epidermal growth factor receptor (EGFRAkt/ERK and Wnt, were affected by SCD5 expression. Epidermal growth factor-induced phosphorylation of EGFR, Akt and ERK was markedly blunted in SCD5-expressing cells. Furthermore, the activity of canonical Wnt was reduced whereas the non-canonical Wnt was increased by the presence of SCD5 activity. Finally, SCD5 expression increased the secretion of recombinant Wnt5a, a non-canonical Wnt, whereas it reduced the cellular and secreted levels of canonical Wnt7b. Our data suggest that, by a coordinated modulation of key

  12. Generation of murine tumor cell lines deficient in MHC molecule surface expression using the CRISPR/Cas9 system.

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    Krishna Das

    Full Text Available In this study, the CRISPR/Cas9 technology was used to establish murine tumor cell lines, devoid of MHC I or MHC II surface expression, respectively. The melanoma cell line B16F10 and the murine breast cancer cell line EO-771, the latter stably expressing the tumor antigen NY-BR-1 (EO-NY, were transfected with an expression plasmid encoding a β2m-specific single guide (sgRNA and Cas9. The resulting MHC I negative cells were sorted by flow cytometry to obtain single cell clones, and loss of susceptibility of peptide pulsed MHC I negative clones to peptide-specific CTL recognition was determined by IFNγ ELISpot assay. The β2m knockout (KO clones did not give rise to tumors in syngeneic mice (C57BL/6N, unless NK cells were depleted, suggesting that outgrowth of the β2m KO cell lines was controlled by NK cells. Using sgRNAs targeting the β-chain encoding locus of the IAb molecule we also generated several B16F10 MHC II KO clones. Peptide loaded B16F10 MHC II KO cells were insusceptible to recognition by OT-II cells and tumor growth was unaltered compared to parental B16F10 cells. Thus, in our hands the CRISPR/Cas9 system has proven to be an efficient straight forward strategy for the generation of MHC knockout cell lines. Such cell lines could serve as parental cells for co-transfection of compatible HLA alleles together with human tumor antigens of interest, thereby facilitating the generation of HLA matched transplantable tumor models, e.g. in HLAtg mouse strains of the newer generation, lacking cell surface expression of endogenous H2 molecules. In addition, our tumor cell lines established might offer a useful tool to investigate tumor reactive T cell responses that function independently from MHC molecule surface expression by the tumor.

  13. Transcriptional regulation by Barhl1 and Brn-3c in organ-of-Corti-derived cell lines.

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    Sud, Richa; Jones, Chris M; Banfi, Sandro; Dawson, Sally J

    2005-11-30

    Barhl1 and Brn-3c have been identified as transcription factors that are essential for survival and maintenance of hair cells of the inner ear. Little is known about the mechanism of how Brn-3c or Barhl1 may regulate transcription in the inner ear. In this study, the transcriptional function of both Brn-3c and Barhl1 was investigated in the organ-of-Corti-derived cell lines, OC-1 and OC-2. We examined regulatory domains in these transcription factors by linking regions of Barhl1 and Brn-3c to the DNA binding domain of the heterologous transcription factor GAL4 and assayed their effect on a heterologous promoter containing GAL4 DNA binding sites by co-transfection into OC-1 and OC-2 cell lines. Brn-3c was found to contain an independent N-terminal activation domain that is sufficient to activate gene transcription in the organ of corti derived cell lines. Barhl1 on the other hand was found to act as a transcriptional repressor with repressive activity not restricted to a particular domain of Barhl1. In addition, we analyzed the effect of Barhl1 on the promoters of the neurotrophin genes NT-3 and BDNF in OC-1 and OC-2 cell lines. However, Barhl1 was not found to directly regulate neurotrophin promoter constructs in these cells.

  14. [Cloning and expression of 3C protease gene from foot-and-mouth disease virus in insect cell].

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    Cao, Yimei; Lu, Zengjun; Sun, Pu; Sun, Jiachuan; Liu, Zaixin

    2008-03-01

    The 3C protease from foot-and-mouth disease virus (FMDV 3Cpro) is critical for viral pathogenesis, has vital roles in both processing of the polyprotein precursor and RNA replication, and is a potential anti-viral drug target. In the study, 3C gene of FMDV from serotype Asia I was obtained through Polymerase Chain Reaction (PCR), and subcloned into baculovirus transfer vector pMelBac-B. The recombinant transfer plasmid and linearized baculovirus DNA were co-transfected into sf9 insect cell, and the recombinant baculovirus were screened by plaque cloning and PCR identification. After amplification of recombinant baculovirus on cell passages, the recombinant virus were seeded on sf9 cell with 10 multiplicity of infection (MOI), and cells were harvested 72 hours after infection. The expressing product of 3C gene in insect cells was detected by Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis (SDS-PAGE) and Western blot. The result demonstrated that the 3C gene was successfully expressed in insect cells. The product was a 23 kDa protein and could be recognized by anti-FMDV serum in western blot. The results provide a basis for research of the assembly of FMDV empty capsids in vitro and the design of antivirus drug.

  15. TMEPAI genome editing in triple negative breast cancer cells

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    Bantari W.K. Wardhani

    2017-05-01

    Full Text Available Background: Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9 is a powerful genome editing technique. It consists of RNA-guided DNA endonuclease Cas9 and single guide RNA (gRNA. By combining their expressions, high efficiency cleavage of the target gene can be achieved, leading to the formation of DNA double-strand break (DSB at the genomic locus of interest which will be repaired via NHEJ (non-homologous end joining or HDR (homology-directed repair and mediate DNA alteration. We aimed to apply the CRISPR/Cas9 technique to knock-out the transmembrane prostate androgen-induced protein (TMEPAI gene in the triple negative breast cancer cell line.Methods: Designed gRNA which targets the TMEPAI gene was synthesized, annealed, and cloned into gRNA expression vector. It was co-transfected into the TNBC cell line using polyethylenimine (PEI together with Cas9-GFP and puromycin resistant gene vector. At 24-hours post-transfection, cells were selected by puromycin for 3 days before they were cloned. Selected knock-out clones were subsequently checked on their protein levels by western blotting.Results: CRISPR/Cas9, a genome engineering technique successfully knocked-out TMEPAI in the Hs578T TNBC cell line. Sequencing shows a frameshift mutation in TMEPAI. Western blot shows the absence of TMEPAI band on Hs578T KO cells.Conclusion: TMEPAI gene was deleted in the TNBC cell line using the genomic editing technique CRISPR/Cas9. The deletion was confirmed by genome and protein analysis.

  16. Down-regulation of alpha-synuclein expression can rescue dopaminergic cells from cell death in the substantia nigra of Parkinson's disease rat model.

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    Hayashita-Kinoh, Hiromi; Yamada, Masanori; Yokota, Takanori; Mizuno, Yoshikuni; Mochizuki, Hideki

    2006-03-24

    Fibrillization and aggregation of alpha-synuclein may play a critical role in neurodegenerative diseases like Parkinson's diseases. Adeno-associated virus (AAV) vector delivery of an alpha-synuclein ribozyme was tested for its silencing effect on degenerating nigrostriatal neurons in the MPP(+) model of Parkinson's disease. We designed alpha-synuclein ribozyme against human alpha-synuclein gene expression and constructed alpha-synuclein ribozymes-carrying rAAV vector (designated rAAV-SynRz). Co-transfection of rAAV-SynRz and rAAV-alpha-synuclein into HEK293 cells resulted in down-regulation of alpha-synuclein protein expression in vitro. Then, rAAV-SynRz was injected into the substantia nigra (SN) of MPP(+)-treated rats. Cell counts of TH-positive neurons in the SN revealed that rAAV-SynRz significantly protected TH-positive cells against apoptotic death, compared with those of rAAV-EGFP or no rAAV injected rats. Our results indicate that the use of rAAV-SynRz allowed the survival of higher number of TH-positive neurons in SN in the MPP(+) model. Down-regulation of alpha-synuclein expression could be potentially a suitable target for gene therapy of Parkinson's disease.

  17. Myostatin gene knockout mediated by Cas9-D10A nickase in chicken DF1 cells without off-target effect

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    Jeong Hyo Lee

    2017-05-01

    Full Text Available Objective Based on rapid advancement of genetic modification techniques, genomic editing is expected to become the most efficient tool for improvement of economic traits in livestock as well as poultry. In this study, we examined and verified the nickase of mutated CRISPR-associated protein 9 (Cas9 to modulate the specific target gene in chicken DF1 cells. Methods Chicken myostatin which inhibits muscle cell growth and differentiation during myogenesis was targeted to be deleted and mutated by the Cas9-D10A nickase. After co-transfection of the nickase expression vector with green fluorescent gene (GFP gene and targeted multiplex guide RNAs (gRNAs, the GFP-positive cells were sorted out by fluorescence-activated cell sorting procedure. Results Through the genotyping analysis of the knockout cells, the mutant induction efficiency was 100% in the targeted site. Number of the deleted nucleotides ranged from 2 to 39 nucleotide deletion. There was no phenotypic difference between regular cells and knockout cells. However, myostatin protein was not apparently detected in the knockout cells by Western blotting. Additionally, six off-target sites were predicted and analyzed but any non-specific mutation in the off-target sites was not observed. Conclusion The knockout technical platform with the nickase and multiplex gRNAs can be efficiently and stablely applied to functional genomics study in poultry and finally adapted to generate the knockout poultry for agribio industry.

  18. Interaction of NF-κB and IκBα, IκBαM, IκBα243N or IκBα244C studied with fluorescent fusion proteins by FRET in living cells

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    Li, Xian; Chen, Xiaojia; Tang, Yonghong

    2006-09-01

    In this paper, the location and interaction of NF-κB and IκBα (IκBαM, IκBα243N, or IκBα244C) in vivo is investigated by fluorescence resonance energy transfer (FRET). Co-transfection of a YFP-p65 construct with CFP- IκBα, C.FP-lid3aM (S32,36A), or CFP-IκBα243N(i-243) resulted in cytosolic localization of both proteins in almost all of the transfected cells. Co-transfection of YFP-p65 construct with CFP-Iw.Ba244C showed a predominant nuclear fluorescence of the proteins. The interaction between YFP-p65 and CFP-IκBα, CFP-IκBαM, CFP-IκBα243N or CFP-IκBα244C were further studied by acceptor bleaching experiments. When YFP-p65 were bleached, the fluorescence of CFP-IκBα, CFP-IκBm, CFP-IκBα243N increased. However, YFP-p65 and CFP-IκBα244C didn't have FRET and the fluorescence of CFP-IκBα244C were not influenced when YFP-p65 were bleached. This observation suggests that NF-κB interacted with the ankyrin repeat domain of IκBα, and our study domonstrates that the application of fluorescent fusion protein, FRET and acceptor bleaching technique to investigate protein-protein interactions in living cells might expand our understanding of these interactions considerably.

  19. Both nitric oxide and nitrite prevent homocysteine-induced endoplasmic reticulum stress and subsequent apoptosis via cGMP-dependent pathway in neuronal cells.

    Science.gov (United States)

    Jeong, Sun-Oh; Son, Yong; Lee, Ju Hwan; Choi, Seung Won; Kim, Sung Hun; Cheong, Yong-Kwan; Chung, Hun-Taeg; Pae, Hyun-Ock

    2017-11-04

    Growing evidence indicates that endoplasmic reticulum (ER) stress and/or ER stress-mediated apoptosis may play a role in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease. The present study investigated the effects of non-cytotoxic concentrations of nitric oxide (NO) and nitrite, a metabolite of NO, on ER stress and ER stress-mediated apoptosis in Neuro-2a cells exposed to homocysteine (Hcy), an endogenous ER stress inducer. Hcy induced ER stress, as confirmed by inositol-requiring enzyme 1α (IRE1α) phosphorylation and X-box-binding protein-1 (Xbp1) mRNA splicing as well as C/EBP homologous protein (CHOP) expression, and apoptosis, as verified by Annexin V-positive cells. Surprisingly, non-cytotoxic NO (S-nitrosoglutathione) and nitrite markedly reduced Hcy-induced IRE1α phosphorylation, Xbp1 mRNA splicing, CHOP expression, and Annexin V-positive cells, indicating the cytoprotection of NO and nitrite against Hcy-induced ER stress and apoptosis. Moreover, inhibition of sGC/cGMP pathway abolished the cytoprotective effects of NO and nitrite, whereas cellular elevation of cGMP levels mimicked the cytoprotective actions of NO and nitrite. These findings provide the first evidence showing that both NO and nitrite can reduce ER stress and subsequent apoptosis via NO-sGC-cGMP pathway in neuronal cells and suggesting that NO and/or nitrite may have therapeutic value in the treatment of ER stress-associated neurodegenerative diseases. Copyright © 2017. Published by Elsevier Inc.

  20. Efficient production of an avian adeno-associated virus vector using insect cell/baculovirus expression system.

    Science.gov (United States)

    Wang, Anping; Wang, Yongjuan; Wu, Shuang; Zuo, Weiyong; Guo, Changming; Hong, Weiming; Zhu, Shanyuan

    2017-02-01

    Recombinant avian adeno-associated virus (rAAAV) is a promising gene transfer vector for avian cells. Although rAAAV can be produced by co-transfection of HEK293 cells with three plasmids, both scalability and productivity of the transient transfection method can not meet the demand for large-scale in vivo experiments. In this study, a scalable rAAAV production method was established by using insect cell/baculovirus expression system. Three recombinant baculoviruses, namely BacARep, BacAVP and BacAGFP, were generated by transfection of Sf9 cells with the three plasmids expressing AAAV Rep genes, modified VP gene or the inverted terminal repeats-flanked green fluorescent protein (GFP) gene. After demonstration of the correct expression of AAAV genes, rAAAV-GFP was produced by triple infection of insect cells or triple transfection of HEK293 cells for comparison purpose. Electron microscopy revealed the formation of typical AAAV particles in the insect cells. Western blotting showed the correct assembly of rAAAV particles with a VP protein ratio similar to that of AAAV. Quantitative PCR showed that the insect cell-produced rAAAV yield was almost 25-fold higher than that produced by HEK293 cells. Fluorescent microscopy showed that the insect cell-produced rAAAV could transfer GFP reporter gene into two avian cell types with similar transfer efficiency to that of HEK293 cell-produced rAAAV. These data suggest that insect cell/baculovirus expression system could be used for scalable production of rAAAV, and the viral vector produced could be used as the gene transfer vehicle for avian cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. NF-κB is involved in the regulation of CD154 (CD40 ligand) expression in primary human T cells

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    Srahna, M; Remacle, J E; Annamalai, K; Pype, S; Huylebroeck, D; Boogaerts, M A; Vandenberghe, P

    2001-01-01

    Cognate interactions between CD154 (CD40 ligand, CD40L) on activated T cells and its receptor CD40 on various antigen-presenting cells are involved in thymus-dependent humoral immune responses and multiple other cell-mediated immune responses. We have studied the regulation of CD154 expression in human T cells after activation with anti-CD3 and anti-CD28 antibodies or after pharmacological activation of protein kinase C with phorbol 12-myristate 13-acetate, and the calcium ionophore ionomycin. Under these conditions, transcription of the CD154 gene was rapidly induced without requiring de novo protein synthesis. Pharmacological inhibitors of NF-κB activation down-regulated CD154 mRNA and protein levels. Cyclosporin A, an inhibitor of NF-AT activation, acted similarly, and the effects of both inhibitors were additive. A potential NF-κB binding site is present within the CD154 promoter at positions −1190 to −1181. In electrophoretic mobility shift assays, this sequence was specifically bound by NF-κB present in nuclear extracts from activated T cells. Furthermore, in transient co-transfection of Jurkat T cells, p65 activated the transcription of a reporter construct containing a multimer of this NF-κB binding site. These observations demonstrate a role of NF-κB transcription factors in the regulation of CD40L expression in activated primary human T cells. PMID:11529914

  2. {beta}-Catenin/LEF1 activated enamelin expression in ameloblast-like cells

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    Tian, Hua; Lv, Ping [Department of Cariology and Endodontology, Peking University School and Hospital of Stomatology, Beijing 100081 (China); Ma, Kangtao [Department of Biochemistry and Molecular Biology, Peking University School of Basic Medical Sciences, Beijing 100191 (China); Zhou, Chunyan, E-mail: chunyanzhou@bjmu.edu.cn [Department of Biochemistry and Molecular Biology, Peking University School of Basic Medical Sciences, Beijing 100191 (China); Gao, Xuejun, E-mail: kqxjgao@bjmu.edu.cn [Department of Cariology and Endodontology, Peking University School and Hospital of Stomatology, Beijing 100081 (China)

    2010-07-30

    Research highlights: {yields} {beta}-Catenin/LEF1 complex could activate enamelin gene transcription. {yields} {beta}-Catenin/LEF1 can directly bind to enamelin 5' regulatory region. {yields} Wnt/{beta}-catenin signaling can upregulate enamelin expression in ameloblast-like cells. -- Abstract: Enamelin is an ameloblast-specific matrix protein believed to play essential roles in enamel formation. However, mechanisms of enamelin transcription regulation are not clear. {beta}-Catenin/LEF1 is a key transcriptional complex involved in tooth development. In this study, the role of {beta}-catenin/LEF1 in enamelin expression was investigated. The 5'-flanking region of the mouse enamelin gene was analyzed and cloned. Co-transfection analysis and mutation assays revealed that two conserved LEF1 responsive elements located at -1002 and -597 bp upstream of the enamelin translation initiation site could augment transcriptional activity of the enamelin. The interaction between the enamelin elements and {beta}-catenin/LEF1 was further confirmed by electrophoresis mobility shift assays and chromatin immunoprecipitation assays. In addition, LiCl treatment induced nuclear translocation of {beta}-catenin and elevated endogenous enamelin expression in mouse ameloblast-like cells. The results suggested that Wnt/{beta}-catenin signaling could function in enamelin gene expression by direct interaction through two conserved LEF1 responsive elements on the enamelin gene in ameloblast-like cells.

  3. A Lentiviral Vector Expressing Desired Gene Only in Transduced Cells: An Approach for Suicide Gene Therapy.

    Science.gov (United States)

    Mohammadi, Zahra; Shariati, Laleh; Khanahmad, Hossein; Kolahdouz, Mahsa; Kianpoor, Fariborz; Ghanbari, Jahan Afrooz; Hejazi, Zahra; Salehi, Mansoor; Nikpour, Parvaneh; Tabatabaiefar, Mohammad Amin

    2015-09-01

    Suicide gene therapy is a therapeutic strategy, in which cell suicide inducing transgenes are introduced into target cells. Inserting a toxin-encoding gene into a lentiviral vector leads to decreased efficiency of virus production due to lethal effect of toxin on packaging cells. In this study, we designed and constructed a transfer vector to express the toxin in transduced cells but not in packaging cells. Plasmid pLenti-F/GFP was constructed by cutting out R 5'LTR-R 3'LTR fragment with the AflII restriction endonuclease from a plasmid pLenti4-GW/H1/TO-laminshRNA, followed by ligating R 5'LTR-R 3'LTR fragment, constructed by three PCR stages. The promoter and GFP CDS were inserted in opposite strand. For lentiviral production, the HEK293T cell line was co-transfected with the PMD2G, psPAX2, and pLenti-F/GFP plasmids (envelope, packaging, and transfer plasmids).Viral vector titers were assayed. The HEK293T cell line was transduced with this virus. PCR was performed to confirm the presence of the promoter fragment between the R and U5 in 3'LTR. The lentivirus titers were approximately 2 × 10(5). The GFP expression was seen in 51 % of the HEK293T cells transduced with lentivirus. The PCR product size was 1440 bp confirming the promoter fragment position between the R and U5 in 3'LTR. The strategy enables us to use a broad spectrum of toxin genes in gene therapy and helps avoid the death of the packaging cells with lentiviral vectors carrying a toxin-encoding gene, thereby increasing the efficiency of viral production in packaging cells.

  4. Ets-2 regulates cell apoptosis via the Akt pathway, through the regulation of urothelial cancer associated 1, a long non-coding RNA, in bladder cancer cells.

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    Wenjing Wu

    Full Text Available The majority of the human genome is transcribed and generates non-coding RNAs (ncRNAs that fail to encode protein information. Long non-coding RNAs (lncRNAs are emerging as a novel class of ncRNAs, but our knowledge about these ncRNAs is limited. Previously, our laboratory has identified that a lncRNA, Urothelial cancer associated 1 (UCA1, played an important role in bladder cancer. Despite the recent interest in UCA1 as a diagnostic marker for bladder cancer, little is known about its transcriptional regulation. To elucidate the regulation of UCA1 gene expression, we have characterized the human UCA1 gene promoter. A 2.0-kb fragment of its 5' flanking region was cloned into a luciferase reporter vector. Deletion and mutation analysis suggested that an Ets-2 binding site was critical for UCA1 gene promoter activity. Further analysis of this site by gel shifting, chromatin immune precipitation (ChIP, and co-transfection experiments showed that transcription factor Ets-2 directly bound to the UCA1 promoter region and stimulated UCA1 promoter activity in bladder cancer cells. Taking into account the anti-apoptosis function of Ets-2, our data suggested that Ets-2 regulates apoptosis process by regulating the expression of UCA1, moreover UCA1 may be involved in the activation of Akt signaling pathway by Ets-2 in bladder cancer cells.

  5. MicroRNA-218 inhibits cell invasion and migration of pancreatic cancer via regulating ROBO1.

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    He, Hang; Hao, Si-Jie; Yao, Lie; Yang, Feng; Di, Yang; Li, Ji; Jiang, Yong-Jian; Jin, Chen; Fu, De-Liang

    2014-10-01

    miRNA-218 is a highlighted tumor suppressor and its underlying role in tumor progression is still unknown. Here, we restored the expression of miRNA-218 in pancreatic cancer to clarify the function and potent downstream pathway of miRNA-218. The expressions of both miRNA-218 and its potent target gene ROBO1 were revealed by RT-PCR and western blotting analysis. Transfection of miRNA-218 precursor mimics and luciferase assay were performed to elucidate the regulation mechanism between miRNA-218 and ROBO1. Cells, stably expressing miRNA-218 followed by forced expression of mutant ROBO1, were established through co-transfections of both lentivirus vector and plasmid vector. The cell migration and invasion abilities were evaluated by migration assay and invasion assay respectively. An increased expression of ROBO1 was revealed in cell BxPC-3-LN compared with cell BxPC-3. Elevated expression of miRNA-218 would suppress the expression of ROBO1 via complementary binding to a specific region within 3'UTR of ROBO1 mRNA (sites 971-978) in pancreatic cancer cells. Stably restoring the expression of miRNA-218 in pancreatic cancer significantly downregulated the expression of ROBO1 and effectively inhibited cell migration and invasion. Forced expression of mutant ROBO1 could reverse the repression effects of miRNA-218 on cell migration and invasion. Consequently, miRNA-218 acted as a tumor suppressor in pancreatic cancer by inhibiting cell invasion and migration. ROBO1 was a functional target of miRNA-218's downstream pathway involving in cell invasion and migration of pancreatic cancer.

  6. Plasmids encoding PKI(1-31), a specific inhibitor of cAMP-stimulated gene expression, inhibit the basal transcriptional activity of some but not all cAMP-regulated DNA response elements in JEG-3 cells.

    Science.gov (United States)

    Grove, J R; Deutsch, P J; Price, D J; Habener, J F; Avruch, J

    1989-11-25

    Plasmids that encode a bioactive amino-terminal fragment of the heat-stable inhibitor of the cAMP-dependent protein kinase, PKI(1-31), were employed to characterize the role of this protein kinase in the control of transcriptional activity mediated by three DNA regulatory elements in the JEG-3 human placental cell line. The 5'-flanking sequence of the human collagenase gene contains the heptameric sequence, 5'-TGAGTCA-3', previously identified as a "phorbol ester" response element. Reporter genes containing either the intact 1.2-kilobase 5'-flanking sequence from the human collagenase gene or just the 7-base pair (bp) response element, when coupled to an enhancerless promoter, each exhibit both cAMP and phorbol ester-stimulated expression in JEG-3 cells. Cotransfection of either construct with plasmids encoding PKI(1-31) inhibits cAMP-stimulated but not basal- or phorbol ester-stimulated expression. Pretreatment of cells with phorbol ester for 1 or 2 days abrogates completely the response to rechallenge with phorbol ester but does not alter the basal expression of either construct; cAMP-stimulated expression, while modestly inhibited, remains vigorous. The 5'-flanking sequence of the human chorionic gonadotropin-alpha subunit (HCG alpha) gene has two copies of the sequence, 5'-TGACGTCA-3', contained in directly adjacent identical 18-bp segments, previously identified as a cAMP-response element. Reporter genes containing either the intact 1.5 kilobase of 5'-flanking sequence from the HCG alpha gene, or just the 36-bp tandem repeat cAMP response element, when coupled to an enhancerless promoter, both exhibit a vigorous cAMP stimulation of expression but no response to phorbol ester in JEG-3 cells. Cotransfection with plasmids encoding PKI(1-31) inhibits both basal and cAMP-stimulated expression in a parallel fashion. The 5'-flanking sequence of the human enkephalin gene mediates cAMP-stimulated expression of reporter genes in both JEG-3 and CV-1 cells. Plasmids

  7. The use of fluorescent intrabodies to detect endogenous gankyrin in living cancer cells

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    Rinaldi, Anne-Sophie; Freund, Guillaume; Desplancq, Dominique; Sibler, Annie-Paule; Baltzinger, Mireille [Ecole Supérieure de Biotechnologie de Strasbourg, UMR 7242, CNRS/Université de Strasbourg, boulevard Sébastien Brant, 67412 Illkirch (France); Rochel, Natacha [Institut de Génétique et de Biologie Moléculaire et Cellulaire, UMR 7104, CNRS/INSERM/Université de Strasbourg, rue Laurent Fries, 67404 Illkirch (France); Mély, Yves; Didier, Pascal [Faculté de Pharmacie, UMR 7213, CNRS/Université de Strasbourg, route du Rhin, 67401 Illkirch (France); Weiss, Etienne, E-mail: eweiss@unistra.fr [Ecole Supérieure de Biotechnologie de Strasbourg, UMR 7242, CNRS/Université de Strasbourg, boulevard Sébastien Brant, 67412 Illkirch (France)

    2013-04-01

    Expression of antibody fragments in mammalian cells (intrabodies) is used to probe the target protein or interfere with its biological function. We previously described the in vitro characterisation of a single-chain Fv (scFv) antibody fragment (F5) isolated from an intrabody library that binds to the oncoprotein gankyrin (GK) in solution. Here, we have isolated several other scFvs that interact with GK in the presence of F5 and tested whether they allow, when fused to fluorescent proteins, to detect by FRET endogenous GK in living cells. The binding of pairs of scFvs to GK was analysed by gel filtration and the ability of each scFv to mediate nuclear import/export of GK was determined. Binding between scFv-EGFP and RFP-labelled GK in living cells was detected by fluorescence lifetime imaging microscopy (FLIM). After co-transfection of two scFvs fused to EGFP and RFP, respectively, which form a tri-molecular complex with GK in vitro, FRET signal was measured. This system allowed us to observe that GK is monomeric and distributed throughout the cytoplasm and nucleus of several cancer cell lines. Our results show that pairs of fluorescently labelled intrabodies can be monitored by FLIM–FRET microscopy and that this technique allows the detection of lowly expressed endogenous proteins in single living cells. Highlights: ► Endogenous GK in living cells was targeted with pairs of fluorescently-tagged scFvs. ► Tri-molecular complexes containing two scFvs and one molecule GK were formed. ► GK was detected using fluorescence lifetime-based FRET imaging. ► GK is monomeric and homogeneously distributed in several cancer cell lines. ► This technique may have many applications in live-cell imaging of endogenous proteins.

  8. TNF-α contributes to caspase-3 independent apoptosis in neuroblastoma cells: role of NFAT.

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    Susana Alvarez

    Full Text Available There is increasing evidence that soluble factors in inflammatory central nervous system diseases not only regulate the inflammatory process but also directly influence electrophysiological membrane properties of neurons and astrocytes. In this context, the cytokine TNF-α (tumor necrosis factor-α has complex injury promoting, as well as protective, effects on neuronal viability. Up-regulated TNF-α expression has also been found in various neurodegenerative diseases such as cerebral malaria, AIDS dementia, Alzheimer's disease, multiple sclerosis, and stroke, suggesting a potential pathogenic role of TNF-α in these diseases as well. We used the neuroblastoma cells SK-N-MC. Transcriptional activity was measured using luciferase reporter gene assays by using lipofectin. We performed cotransfection experiments of NFAT (nuclear factor of activated T cells promoter constructed with a dominant negative version of NFAT (dn-NFAT. Cell death was performed by MTT (3-(4,5-dimethylthiazol-2-yl5,5-diphenyltetrazolium bromide and TUNEL assays. NFAT translocation was confirmed by Western blot. Involvement of NFAT in cell death was assessed by using VIVIT. P53, Fas-L, caspase-3, and caspase-9 expressions were carried out by Western blot. The mechanisms involved in TNF-α-induced cell death were assessed by using microarray analysis. TNF-α causes neuronal cell death in the absence of glia. TNF-α treatment results in nuclear translocation of NFAT through activation of calcineurin in a Ca(2+ independent manner. We demonstrated the involvement of FasL/Fas, cytochrome c, and caspase-9 but the lack of caspase-3 activation. NB cell death was absolutely reverted in the presence of VIVIT, and partially diminished by anti-Fas treatment. These data demonstrate that TNF-α promotes FasL expression through NFAT activation in neuroblastoma cells and this event leads to increased apoptosis through independent caspase-3 activation.

  9. Beta-catenin/LEF1 activated enamelin expression in ameloblast-like cells.

    Science.gov (United States)

    Tian, Hua; Lv, Ping; Ma, Kangtao; Zhou, Chunyan; Gao, Xuejun

    2010-07-30

    Enamelin is an ameloblast-specific matrix protein believed to play essential roles in enamel formation. However, mechanisms of enamelin transcription regulation are not clear. beta-Catenin/LEF1 is a key transcriptional complex involved in tooth development. In this study, the role of beta-catenin/LEF1 in enamelin expression was investigated. The 5'-flanking region of the mouse enamelin gene was analyzed and cloned. Co-transfection analysis and mutation assays revealed that two conserved LEF1 responsive elements located at -1002 and -597bp upstream of the enamelin translation initiation site could augment transcriptional activity of the enamelin. The interaction between the enamelin elements and beta-catenin/LEF1 was further confirmed by electrophoresis mobility shift assays and chromatin immunoprecipitation assays. In addition, LiCl treatment induced nuclear translocation of beta-catenin and elevated endogenous enamelin expression in mouse ameloblast-like cells. The results suggested that Wnt/beta-catenin signaling could function in enamelin gene expression by direct interaction through two conserved LEF1 responsive elements on the enamelin gene in ameloblast-like cells. Copyright 2010 Elsevier Inc. All rights reserved.

  10. Suppression of Glypican 3 Inhibits Growth of Hepatocellular Carcinoma Cells through Up-Regulation of TGF-β2

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    Chris K. Sun

    2011-08-01

    Full Text Available Glypican 3 (GPC3 is a valuable diagnostic marker and a potential therapeutic target in hepatocellular carcinoma (HCC. To evaluate the efficacy of targeting GPC3 at the translational level, we used RNA interference to examine the biologic and molecular effects of GPC3 suppression in HCC cells in vitro and in vivo. Transfection of Huh7 and HepG2 cells with GPC3-specific small interfering RNA (siRNA inhibited cell proliferation (P < .001 together with cell cycle arrest at the G1 phase, down-regulation of antiapoptotic protein (Bcl-2, Bcl-xL, and Mcl-1, and replicative senescence. Gene expression analysis revealed that GPC3 suppression significantly correlated with transforming growth factor beta receptor (TGFBR pathway (P = 4.57e-5 and upregulated TGF-β2 at both RNA and protein levels. The effects of GPC3 suppression by siRNA can be recapitulated by addition of human recombinant TGF-β2 to HCC cells in culture, suggesting the possible involvement of TGF-β2 in growth inhibition of HCC cells. Cotransfection of siRNA-GPC3 with siRNA-TGF-β2 partially attenuated the effects of GPC3 suppression on cell proliferation, cell cycle progression, apoptosis, and replicative senescence, confirming the involvement of TGF-β2 in siRNA-GPC3-mediated growth suppression. In vivo, GPC3 suppression significantly inhibited the growth of orthotopic xenografts of Huh7 and HepG2 cells (P < .05, accompanied by increased TGF-β2 expression, reduced cell proliferation (observed by proliferating cell nuclear antigen staining, and enhanced apoptosis (by TUNEL staining. In conclusion, molecular targeting of GPC3 at the translational level offers an effective option for the clinical management of GPC3-positive HCC patients.

  11. High-throughput cell-based screening reveals a role for ZNF131 as a repressor of ERalpha signaling

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    Du Peige

    2008-10-01

    Full Text Available Abstract Background Estrogen receptor α (ERα is a transcription factor whose activity is affected by multiple regulatory cofactors. In an effort to identify the human genes involved in the regulation of ERα, we constructed a high-throughput, cell-based, functional screening platform by linking a response element (ERE with a reporter gene. This allowed the cellular activity of ERα, in cells cotransfected with the candidate gene, to be quantified in the presence or absence of its cognate ligand E2. Results From a library of 570 human cDNA clones, we identified zinc finger protein 131 (ZNF131 as a repressor of ERα mediated transactivation. ZNF131 is a typical member of the BTB/POZ family of transcription factors, and shows both ubiquitous expression and a high degree of sequence conservation. The luciferase reporter gene assay revealed that ZNF131 inhibits ligand-dependent transactivation by ERα in a dose-dependent manner. Electrophoretic mobility shift assay clearly demonstrated that the interaction between ZNF131 and ERα interrupts or prevents ERα binding to the estrogen response element (ERE. In addition, ZNF131 was able to suppress the expression of pS2, an ERα target gene. Conclusion We suggest that the functional screening platform we constructed can be applied for high-throughput genomic screening candidate ERα-related genes. This in turn may provide new insights into the underlying molecular mechanisms of ERα regulation in mammalian cells.

  12. Transfection of CTGF siRNA inhibits transdifferentiation in human lens epithelium cell line B3 in vitro

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    Hua Zhuang

    2017-08-01

    Full Text Available AIM: To investigate the expression of connective tissue growth factor(CTGFand α-SMA in human lens epithelium cell(HLECline B3 after transfection by liposome-coated siRNA targeting CTGF. METHODS: HLECs were transfected with small interfering RNA(siRNAtargeting CTGF, labeled with 5'- fluorescein isothiocyanate(5'-FITCand coated with lipofectamine. The transfection ratio was evaluated via fluorescence intensity. Cell counting kit-8(CCK-8assay was performed to assess cytoviability of both non-transfected and transfected HLECs. Quantitative RT- PCR, cell immunochemistry and Western blot analysis were conducted to detect the expression changes of CTGF and α-SMA after transfection. RESULTS: A highly effective transfection ratio was observed in siRNA co-transfected with lipofectamine. The transfection ratio reached 95% at 24h. The proliferation of HLECs was inhibited by siRNA after 72h transfection. The expression of CTGF and α-SMA significantly decreased in HLECs after transfected by CTGF siRNA for 24h. This effect was not found in negative control siRNA. CONCLUSIONS: SiRNA targeting CTGF decreased CTGF and α-SMA expression in HLECs, which is a potential therapeutic strategy for posterior capsular opacification.

  13. Rapid establishment of a HEK 293 cell line expressing FVIII-BDD using AAV site-specific integration plasmids.

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    Liu, Xiaomei; Ping, Han; Zhang, Chun

    2014-09-10

    Stable human cell lines have gradually become the preferred system for large scale production of recombinant proteins for clinical applications because of their capacity of proper protein post-translational modification and low immunogenicity. However, human cell line development technologies are commonly based on random genome integration of protein expressing genes. It is required to screen large numbers of cell clones to identify stable high producer cell clones and the cell line development process usually takes 6 to 12 months. Adeno-associated virus type 2 (AAV2) Rep protein is known to induce rAAV DNA integration into a specific site (AAVS1) of the human chromosome 19 and integrated transgenes can stably express proteins. We take advantage of this AAV unique feature to develop a rapid protocol to clone a stable recombinant protein expression human cell line. We have constructed two plasmids. One plasmid, pSVAV2, contains the AAV rep gene for the synthesis of integrase; the second plasmid, pTRP5GFPFVIII-BDD, contains B-domain-deleted factor VIII (FVIII-BDD) and GFP gene flanked by AAV ITRs. Human embryonic kidney (HEK) 293 cells were co-transfected with the two plasmids and the cells were screened by green fluorescence to establish the recombinant FVIII-BDD cell line. PCR analysis showed that the FVIII-BDD gene has been integrated into the AAVS1 site of human chromosome 19. The FVIII-BDD protein secreted into the extracellular media exhibited coagulant activity. We developed a method of rapid establishment of human HEK 293 cell line expressing recombinant FVIII-BDD protein with AAV site-specific integration plasmids.

  14. The antagonistic regulation of human MUC4 and ErbB-2 genes by the Ets protein PEA3 in pancreatic cancer cells: implications for the proliferation/differentiation balance in the cells.

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    Fauquette, Valérie; Perrais, Michael; Cerulis, Sylvain; Jonckheere, Nicolas; Ducourouble, Marie-Paule; Aubert, Jean-Pierre; Pigny, Pascal; Van Seuningen, Isabelle

    2005-02-15

    The human transmembrane mucin MUC4 is aberrantly expressed in 75% of pancreatic ductal adenocarcinomas, whereas no expression is found in normal pancreas. Therefore MUC4 appears as a useful biological marker for the diagnosis of ductal adenocarcinomas. Since rat Muc4 was shown to interact with ErbB-2 tyrosine kinase receptor and to either promote cell survival and differentiation or cell proliferation, it is postulated that MUC4 may also participate in pancreatic carcinogenesis. Our aim was to investigate in parallel the role of the Ets factor PEA3 in MUC4 and ErbB-2 transcriptional regulation in pancreatic cancer cells. Two MUC4-expressing WD (well-differentiated) (CAPAN-1 and -2) and one MUC4-non-expressing poorly differentiated (PANC-1) cell lines were used. The three cell lines express ErbB-2 at different levels. By co-transfection and site-directed mutagenesis, we show that PEA3 is a transactivator of the MUC4 promoter and that the -216 and -2368 PEA3 binding sites of the MUC4 promoter are essential. We also demonstrate that PEA3 acts in synergy with c-Jun and specificity protein 1 to transactivate the proximal region of the MUC4 promoter and increase MUC4 mRNA levels in WD cells. These results suggest that MUC4 is a new target gene of the Ets factor PEA3 in pancreatic cancer cells. In contrast, PEA3 represses the transcriptional activity of two fragments of the ErbB-2 promoter in a dose-dependent manner and decreases the endogenous ErbB-2 mRNA levels in WD cell lines. Thus, PEA3, by its capacity to up-regulate the epithelial marker MUC4 and to down-regulate the ErbB-2 oncogene, appears as a key regulator of the differentiation/proliferation balance in pancreatic cancer cells.

  15. The human mucin MUC4 is transcriptionally regulated by caudal-related homeobox, hepatocyte nuclear factors, forkhead box A, and GATA endodermal transcription factors in epithelial cancer cells.

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    Jonckheere, Nicolas; Vincent, Audrey; Perrais, Michaël; Ducourouble, Marie-Paule; Male, Anita Korteland-van; Aubert, Jean-Pierre; Pigny, Pascal; Carraway, Kermit L; Freund, Jean-Noël; Renes, Ingrid B; Van Seuningen, Isabelle

    2007-08-03

    The human gene MUC4 encodes a large transmembrane mucin that is developmentally regulated and expressed along the undifferentiated pseudostratified epithelium, as early as 6.5 weeks during fetal development. Immunohistochemical analysis of Muc4 expression in developing mouse lung and gastrointestinal tract showed a different spatio-temporal pattern of expression before and after cytodifferentiation. The molecular mechanisms governing MUC4 expression during development are, however, unknown. Hepatocyte nuclear factors (HNF), forkhead box A (FOXA), GATA, and caudal-related homeobox transcription factors (TFs) are known to control cell differentiation of gut endoderm derived-tissues during embryonic development. They also control the expression of cell- and tissue-specific genes and may thus control MUC4 expression. To test this hypothesis, we studied and deciphered the molecular mechanisms responsible for MUC4 transcriptional regulation by these TFs. Experiments using small interfering RNA, cell co-transfection, and site-directed mutagenesis indicated that MUC4 is regulated at the transcriptional level by CDX-1 and -2, HNF-1 alpha and -1 beta, FOXA1/A2, HNF-4 alpha and -4 gamma, and GATA-4, -5, and -6 factors in a cell-specific manner. Binding of TFs was assessed by chromatin immunoprecipitation, and gel-shift assays. Altogether, these results demonstrate that MUC4 is a target gene of endodermal TFs and thus point out an important role for these TFs in regulating MUC4 expression during epithelial differentiation during development, cancer, and repair.

  16. Resistin promotes tumor metastasis by down-regulation of miR-519d through the AMPK/p38 signaling pathway in human chondrosarcoma cells

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    Huang, Ho-Ning; Hung, Chih-Hung; Hsu, Chin-Jung; Fong, Yi-Chin; Hsu, Horng-Chaung; Huang, Yuan-Li; Tang, Chih-Hsin

    2015-01-01

    Resistin is a recently discovered adipocyte-secreting adipokine, which may play a critical role in modulating cancer pathogenesis. Chondrosarcoma is a highly malignant tumor known to frequently metastasize; however, the role of resistin in the metastasis of human chondrosarcoma is largely unknown. Here, we found that the expression of resistin was higher in chondrosarcoma biopsy tissues than in normal cartilage. Moreover, treatment with resistin increased matrix metalloproteinase (MMP)-2 expression and promoted cell migration in human chondrosarcoma cells. Co-transfection with microRNA (miR)-519d mimic resulted in reversed resistin-mediated cell migration and MMP-2 expression. Additionally, AMP-activated protein kinase (AMPK) and p38 inhibitors or siRNAs reduced the resistin-increased cell migration and miR-519d suppression, and inhibition of resistin expression resulted in suppression of MMP-2 expression and lung metastasis in vivo. Taken together, our results indicate that resistin promotes chondrosarcoma metastasis and MMP-2 expression through activation of the AMPK/p38 signaling pathway and down-regulation of miR-519d expression. Therefore, resistin may represent a potential novel molecular therapeutic target in chondrosarcoma metastasis. PMID:25404641

  17. Interleukin 8 haplotypes drive divergent responses in uterine endometrial cells and are associated with somatic cell score in Holstein-Friesian cattle.

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    Stojkovic, Bojan; Mullen, Michael P; Donofrio, Gaetano; McLoughlin, Rachel M; Meade, Kieran G

    2017-02-01

    Interleukin 8 is a proinflammatory chemokine involved in neutrophil recruitment and activation in response to infection and also in the resolution of inflammation. Our previous studies identified a number of genetic polymorphisms in the bovine IL8 promoter region which segregate into two haplotypes, with balanced frequencies in the Holstein-Friesian (HF). We subsequently showed that these haplotypes confer divergent IL8 activity both in vitro in mammary epithelial cells and in vivo in response to LPS. In this study, we hypothesised that the balanced frequency of IL8 haplotype in HF could be explained by divergent selection pressures acting on this locus. To address this hypothesis, an association study was carried out aiming to identify a putative link between the IL8 haplotype and somatic cell score (SCS) in 5746 Holstein-Friesian dairy cows. In addition, the basal and inducible promoter activity of the two IL8 haplotypes was characterised in bovine endometrial epithelial (BEND) cells and in monocyte-derived macrophages. Results showed a significant association between IL8 haplotype 2 (IL8-h2) with increased SCS (P<0.05). Functional analysis showed that the same haplotype was a more potent inducer of IL8 expression in BEND cells in response to LPS and TNFα stimulation. In contrast, co-transfection of the BEND cells with a DNA construct encoding a bovine herpesvirus 4 antigen, induced significantly higher IL8 expression from IL8-h1. The present study sheds light on the molecular mechanisms underlying selection for SCS and provides evidence that the balanced frequencies of the two IL8 haplotypes in HF cattle may occur as a result of opposing directional selection pressures of both bacterial and viral infection. Copyright © 2016. Published by Elsevier B.V.

  18. Intensified Beclin-1 Mediated by Low Expression of Mir-30a-5p Promotes Chemoresistance in Human Small Cell Lung Cancer

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    Xiang Yang

    2017-10-01

    Full Text Available Background/Aims: Although small cell lung cancer (SCLC is sensitive to initial chemotherapy, patients experience tumor recurrence and metastasis, leading to treatment failure. Autophagy as a protective pattern for cell survival in the harsh environment plays an important role in chemoresistance. However, the role of Beclin-1, a key regulator of autophagy in the drug-resistance of SCLC cells is still poorly understood. In the current study, we focused on the effect and regulation of Beclin-1 in chemoresistance of SCLC cells. Methods: We analyzed the levels of Beclin-1 in etoposide/cisplatin (EP -resistant and -sensitive cell lines, as well as the relationship between Beclin-1 and patients’ chemosensitivity. The function of Beclin-1 in chemoresistant SCLC cells in vitro was measured by MTT, WB, colony formation and flow cytometric analysis. Further rescue experiment was performed after co-transfected with siBeclin-1 and miR-30a mimics or inhibitor. Results: Beclin-1 was upregulated in drug-resistant cells and patients with lower sensitivity to etoposide/cisplatin therapy. Downregulated Beclin-1 attenuated drug sensitivity and colony formation ability of chemoresistant cells. Moreover, inhibition of Beclin-1 resulted in a dramatic decline of autophagy and increase of apoptosis in drug-resistant cells, accompanied by a remarkable reduction in S phase and a raise in G2/M phase of cell cycle. The transfection with miR-30a-5p mimics exhibited an opposite effect. In addition, inhibition of Beclin-1 could partly reverse the effect induced by miR-30a-5p suppression in drug-sensitive cells. Conclusion: Beclin-1 regulated by miR-30a-5p plays a notable role in the drug-resistance of SCLC. Inhibition of Beclin-1 by induction of miR-30a-5p may improve the therapeutic outcome via resensitizing the drug-resistant cells to chemotherapy in SCLC.

  19. Artificial Induction of Native Aquaporin-1 Expression in Human Salivary Cells.

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    Wang, Z; Pradhan-Bhatt, S; Farach-Carson, M C; Passineau, M J

    2017-04-01

    Gene therapy for dry mouth disorders has transitioned in recent years from theoretical to clinical proof of principle with the publication of a first-in-man phase I/II dose escalation clinical trial in patients with radiation-induced xerostomia. This trial used a prototype adenoviral vector to express aquaporin-1 (AQP1), presumably in the ductal cell layer and/or in surviving acinar cells, to drive transcellular flux of interstitial fluid into the labyrinth of the salivary duct. As the development of this promising gene therapy continues, safety considerations are a high priority, particularly those that remove nonhuman agents (i.e., viral vectors and genetic sequences of bacterial origin). In this study, we applied 2 emerging technologies, artificial transcriptional complexes and epigenetic editing, to explore whether AQP1 expression could be achieved by activating the native gene locus in a human salivary ductal cell line and primary salivary human stem/progenitor cells (hS/PCs), as opposed to the conventional approach of cytomegalovirus promoter-driven expression from an episomal vector. In our first study, we used a cotransfection strategy to express the components of the dCas9-SAM system to create an artificial transcriptional complex at the AQP1 locus in A253 and hS/PCs. We found that AQP1 expression was induced at a magnitude comparable to adenoviral infection, suggesting that AQP1 is primarily silenced through pretranscriptional mechanisms. Because earlier literature suggested that pretranscriptional silencing of AQP1 in salivary glands is mediated by methylation of the promoter, in our second study, we performed global, chemical demethylation of A253 cells and found that demethylation alone induced robust AQP1 expression. These results suggest the potential for success by inducing AQP1 expression in human salivary ductal cells through epigenetic editing of the native promoter.

  20. Cell surface serine protease matriptase-2 suppresses fetuin-A/AHSG-mediated induction of hepcidin.

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    Stirnberg, Marit; Maurer, Eva; Arenz, Katharina; Babler, Anne; Jahnen-Dechent, Willi; Gütschow, Michael

    2015-01-01

    Matriptase-2 is a type II transmembrane serine protease controlling the expression of hepcidin, the key regulator of iron homeostasis. By cleaving hemojuvelin, matriptase-2 suppresses bone morphogenetic protein/sons of mothers against decapentaplegic signaling. So far, the only known putative substrates of matriptase-2 are hemojuvelin and matriptase-2 itself. In this study, fetuin-A (α2-Heremans-Schmid glycoprotein) was identified in vitro as a substrate of matriptase-2. The protease-substrate interaction was validated by isolating matriptase-2 via the affinity to fetuin-A. Fetuin-A is a liver-derived plasma protein with multiple functions, which is proteolytically processed to yield a disulfide-linked two-chain form. In co-transfected cells, a matriptase-2-dependent conversion of unprocessed fetuin-A into a two-chain form was detected. Conversely, downregulation of endogenously expressed matriptase-2 stabilized fetuin-A. Arg and Lys residues located within the 40 residue spanning connecting peptide of fetuin-A were identified as cleavage sites for matriptase-2. Analysis of hepcidin expression revealed an inductive effect of fetuin-A, which was abolished by matriptase-2. Fetuin-A deficiency in mice resulted in decreased hepcidin mRNA levels. These findings implicate a role of fetuin-A in iron homeostasis and provide new insights into the mechanism of how matriptase-2 might modulate hepcidin expression.

  1. Resveratrol-induced transcriptional up-regulation of ASMase (SMPD1) of human leukemia and cancer cells

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    Mizutani, Naoki [Department of Pathophysiological Laboratory Science, Nagoya University Graduate School of Medicine, Nagoya (Japan); College of Life and Health Sciences, Chubu University, Kasugai (Japan); Omori, Yukari [Department of Pathophysiological Laboratory Science, Nagoya University Graduate School of Medicine, Nagoya (Japan); Kawamoto, Yoshiyuki; Sobue, Sayaka; Ichihara, Masatoshi [College of Life and Health Sciences, Chubu University, Kasugai (Japan); Suzuki, Motoshi [Division of Molecular Carcinogenesis, Nagoya University Graduate School of Medicine, Nagoya (Japan); Kyogashima, Mamoru [Department of Microbiology and Molecular Biology, Nihon Pharmaceutical University, Saitama (Japan); Nakamura, Mitsuhiro [Department of Drug Information, Gifu Pharmaceutical University, Gifu (Japan); Tamiya-Koizumi, Keiko [College of Life and Health Sciences, Chubu University, Kasugai (Japan); Nozawa, Yoshinori [Tokai Gakuin University, Kakamigahara (Japan); Murate, Takashi, E-mail: murate@isc.chubu.ac.jp [College of Life and Health Sciences, Chubu University, Kasugai (Japan)

    2016-02-19

    Resveratrol (RSV) is a plant-derived phytoalexin present in plants, whose pleiotropic effects for health benefits have been previously reported. Its anti-cancer activity is among the current topics for novel cancer treatment. Here, effects of RSV on cell proliferation and the sphingolipid metabolism of K562, a human leukemia cell line, were analyzed. Some experiments were also performed in HCT116, a human colon cancer cell line. RSV inhibited cell proliferation of both cell lines. Increased cellular ceramide and decreased sphingomyelin and S1P by RSV were observed in RSV-treated K562 cells. Further analysis revealed that acid sphingomyelinase mRNA and enzyme activity levels were increased by RSV. Desipramine, a functional ASMase inhibitor, prevented RSV-induced ceramide increase. RSV increased ATF3, EGR1, EGR3 proteins and phosphorylated c-Jun and FOXO3. However, co-transfection using these transcription factor expression vectors and ASMase promoter reporter vector revealed positive effects of EGR1 and EGR3 but not others. Electrophoresis mobility shift assay (EMSA) and Chromatin immunoprecipitation (ChIP) assay demonstrated the direct binding of EGR1/3 transcription factors with ASMase 5′-promoter. These results indicate that increased EGR1/3 and ASMase expression play an important role in cellular ceramide increase by RSV treatment. - Highlights: • Resveratrol inhibited cell proliferation of K562 and HCT116 cells. • Resveratrol increased cellular ceramide and decreased sphingomyelin and S1P. • ASMase mRNA and activity were increased with resveratrol. • ASMase inhibition suppressed RSV-induced ceramide accumulation. • Increased ASMase transcription was at least partially due to EGR family proteins.

  2. Attempts on producing lymphoid cell line from Penaeus monodon by induction with SV40-T and 12S EIA oncogenes.

    Science.gov (United States)

    Puthumana, Jayesh; Prabhakaran, Priyaja; Philip, Rosamma; Singh, I S Bright

    2015-12-01

    In an attempt of in vitro transformation, transfection mediated expression of Simian virus-40 (T) antigen (SV40-T) and transduction mediated expression of Adenovirus type 12 early region 1A (12S E1A) oncogene were performed in Penaeus monodon lymphoid cells. pSV3-neo vector encoding SV40-T oncogene and a recombinant baculovirus BacP2-12S E1A-GFP encoding 12S E1A oncogene under the control of hybrid promoters were used. Electroporation and lipofection mediated transformation of SV40-T in lymphoid cells confirmed the transgene expression by phenotypic variation and the expression of GFP in co-transfection experiment. The cells transfected by lipofection (≥ 5%) survived for 14 days with lower toxicity (30%), whilst on electroporation, most of the cells succumbed to death (60%) and survived cells lived up to 7 days. Transduction efficiency in primary lymphoid cells was more than 80% within 14 days of post-transduction, however, an incubation period of 7 days post-transduction was observed without detectable expression of 12S E1A. High level of oncogenic 12S E1A expression were observed after 14 day post-transduction and the proliferating cells survived for more than 90 days with GFP expression, however, without in vitro transformation and immortalization. The study put forth the requirement of transduction mediated 'specific' oncogene expression along with telomerase activation and epigenetic induction for the immortalization and establishment of shrimp cell line. Copyright © 2015. Published by Elsevier Ltd.

  3. Statins Activate Human PPAR Promoter and Increase PPAR mRNA Expression and Activation in HepG2 Cells

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    Makoto Seo

    2008-01-01

    Full Text Available Statins increase peroxisome proliferator-activated receptor (PPAR mRNA expression, but the mechanism of this increased PPAR production remains elusive. To examine the regulation of PPAR production, we examined the effect of 7 statins (atorvastatin, cerivastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin on human PPAR promoter activity, mRNA expression, nuclear protein levels, and transcriptional activity. The main results are as follows. (1 Majority of statins enhanced PPAR promoter activity in a dose-dependent manner in HepG2 cells transfected with the human PPAR promoter. This enhancement may be mediated by statin-induced HNF-4. (2 PPAR mRNA expression was increased by statin treatment. (3 The PPAR levels in nuclear fractions were increased by statin treatment. (4 Simvastatin, pravastatin, and cerivastatin markedly enhanced transcriptional activity in 293T cells cotransfected with acyl-coenzyme A oxidase promoter and PPAR/RXR expression vectors. In summary, these data demonstrate that PPAR production and activation are upregulated through the PPAR promoter activity by statin treatment.

  4. Fusion and differentiation of murine C2C12 skeletal muscle cells that express Trichinella spiralis p43 protein.

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    Jasmer, Douglas P; Kwak, Dongmi

    2006-02-01

    The ability of a 43 kDa stichocyte protein from Trichinella spiralis (Tsp43) to interfere with mammalian skeletal muscle gene expression was investigated. A MYC-tagged Tsp43 construct was expressed as a recombinant protein in C2C12 myoblasts. Transfection with low amounts of expression plasmid was required for successful expression of the protein. This construct had apparent toxic effects on transfected myoblasts and ectopic green fluorescent protein expression was suppressed in myoblasts co-transfected with the Tsp43 construct. These effects may result from similarities of Tsp43 to DNase II. Use of the general DNase inhibitor aurintricarboxylic acid (ATA) enhanced expression of MYC-Tsp43 in transfected muscle cells. Myoblasts transfected with Tsp43 did not fuse well when cultured under differentiation conditions without ATA. In contrast, transfected myoblasts transiently cultured with ATA underwent fusion and differentiation. Under short-term differentiation conditions without ATA, unfused myoblasts nevertheless expressed both MYC-Tsp43 and myosin heavy chain. Collectively, the results support that Tsp43 has a role in the T. spiralis life cycle that is distinct from repressing muscle gene expression during the muscle phase of infection. While the function of Tsp43 as a DNase is under debate, the effects of ATA on transfected muscle cells were consistent with this possibility.

  5. Magel2, a Prader-Willi syndrome candidate gene, modulates the activities of circadian rhythm proteins in cultured cells

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    Devos Julia

    2011-12-01

    Full Text Available Abstract Background The Magel2 gene is most highly expressed in the suprachiasmatic nucleus of the hypothalamus, where its expression cycles in a circadian pattern comparable to that of clock-controlled genes. Mice lacking the Magel2 gene have hypothalamic dysfunction, including circadian defects that include reduced and fragmented total activity, excessive activity during the subjective day, but they have a normal circadian period. Magel2 is a member of the MAGE family of proteins that have various roles in cellular function, but the specific function of Magel2 is unknown. Methods We used a variety of cell-based assays to determine whether Magel2 modifies the properties of core circadian rhythm proteins. Results Magel2 represses the activity of the Clock:Bmal1 heterodimer in a Per2-luciferase assay. Magel2 interacts with Bmal1 and with Per2 as measured by co-immunoprecipitation in co-transfected cells, and exhibits a subcellular distribution consistent with these interactions when visualized by immunofluorescence. As well, Magel2 induces the redistribution of the subcellular localization of Clock towards the cytoplasm, in contrast to the nucleus-directed effect of Bmal1 on Clock subcellular localization. Conclusion Consistent with the blunted circadian rhythm observed in Magel2-null mice, these data suggest that Magel2 normally promotes negative feedback regulation of the cellular circadian cycle, through interactions with key core circadian rhythm proteins.

  6. Extracellular Na+ levels regulate formation and activity of the NaX/alpha1-Na+/K+-ATPase complex in neuronal cells.

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    Emmanuelle eBerret

    2014-12-01

    Full Text Available MnPO neurons play a critical role in hydromineral homeostasis regulation by acting as sensors of extracellular sodium concentration ([Na+]out. The mechanism underlying Na+-sensing involves Na+-flow through the NaX channel, directly regulated by the Na+/K+-ATPase α1-isoform which controls Na+-influx by modulating channel permeability. Together, these two partners form a complex involved in the regulation of intracellular sodium ([Na+]in. Here we aim to determine whether environmental changes in Na+ could actively modulate the NaX/Na+/K+-ATPase complex activity.We investigated the complex activity using patch-clamp recordings from rat MnPO neurons and Neuro2a cells. When the rats were fed with a high-salt-diet, or the [Na+] in the culture medium was increased, the activity of the complex was up-regulated. In contrast, drop in environmental [Na+] decreased the activity of the complex. Interestingly under hypernatremic condition, the colocalization rate and protein level of both partners were up-regulated. Under hyponatremic condition, only NaX protein expression was increased and the level of NaX/Na+/K+-ATPase remained unaltered. This unbalance between NaX and Na+/K+-ATPase pump proportion would induce a bigger portion of Na+/K+-ATPase-control-free NaX channel. Thus we suggest that hypernatremic environment increases NaX/Na+/K+-ATPase α1-isoform activity by increasing the number of both partners and their colocalization rate, whereas hyponatremic environment down-regulates complex activity via a decrease in the relative number of NaX channels controlled by the pump.

  7. [miR-143 inhibits cell proliferation through targeted regulating the expression of K-ras gene in HeLa cells].

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    Qin, H X; Cui, H K; Pan, Y; Hu, R L; Zhu, L H; Wang, S J

    2016-12-23

    Objective: To explore the effect of microRNA miR-143 on the proliferation of cervical cancer HeLa cells through targeted regulating the expression of K-ras gene. Methods: The luciferase report carrier containing wild type 3'-UTR of K-ras gene (K-ras-wt) or mutated 3'-UTR of the K-ras (K-ras-mut) were co-transfected with iR-143 mimic into the HeLa cells respectively, and the targeting effect of miR-143 in the transfectants was verified by the dual luciferase report system. HeLa cells were also transfected with miR-143 mimic (miR-143 mimic group), mimic control (negative control group), and miR-143 mimic plus K-ras gene (miR-143 mimic+ K-ras group), respectively. The expression of miR-143 in the transfected HeLa cells was detected by real-time PCR (RT-PCR), and the expression of K-ras protein was detected by Western blot. The cell proliferation activity of each group was examined by MTT assay. In addition, human cervical cancer tissue samples (n=5) and cervical intraepithelial neoplasia tissue samples (n=5) were also examined for the expression of miR-143 and K-ras protein by RT-PCR and Western blot, respectively. Results: The luciferase report assay showed that co-transfection with miR-143 mimic decreased the luciferase activity of the K-ras-wt significantly, but did not inhibit the luciferase activity of the K-ras-mut. The expression of miR-143 in the HeLa cells transfected with miR-143 mimic was significantly higher than that in the HeLa cells transfected with the mimic control (3.31±0.45 vs 0.97±0.22, Pras group was also significantly lower than the control group (Pras protein in the miR-143 mimic group, the negative control group and the miR-143 mimic+ K-ras group were lowest, moderate, and highest, respectively (115.27±34.08, 521.36±41.89, and 706.52±89.44, all Pras protein expression in the cervical cancer group was significantly higher than that in the cervical intraepithelial neoplasia group (584.39±72.34 vs. 114.23±25.82, Pras gene. In human cervical

  8. Direct interaction with contactin targets voltage-gated sodium channel Na(v)1.9/NaN to the cell membrane.

    Science.gov (United States)

    Liu, C J; Dib-Hajj, S D; Black, J A; Greenwood, J; Lian, Z; Waxman, S G

    2001-12-07

    The mechanisms that target various sodium channels within different regions of the neuronal membrane, which they endow with different physiological properties, are not yet understood. To examine this issue we studied the voltage-gated sodium channel Na(v)1.9/NaN, which is preferentially expressed in small sensory neurons of dorsal root ganglia and trigeminal ganglia and the nonmyelinated axons that arise from them. Our results show that the cell adhesion molecule contactin binds directly to Na(v)1.9/NaN and recruits tenascin to the protein complex in vitro. Na(v)1.9/NaN and contactin co-immunoprecipitate from dorsal root ganglia and transfected Chinese hamster ovary cell line, and co-localize in the C-type neuron soma and along nonmyelinated C-fibers and at nerve endings in the skin. Co-transfection of Chinese hamster ovary cells with Na(v)1.9/NaN and contactin enhances the surface expression of the sodium channel over that of Na(v)1.9/NaN alone. Thus contactin binds directly to Na(v)1.9/NaN and participates in the surface localization of this channel along nonmyelinated axons.

  9. CCL5 promotes vascular endothelial growth factor expression and induces angiogenesis by down-regulating miR-199a in human chondrosarcoma cells.

    Science.gov (United States)

    Liu, Guan-Ting; Huang, Yuan-Li; Tzeng, Huey-En; Tsai, Chun-Hao; Wang, Shih-Wei; Tang, Chih-Hsin

    2015-02-28

    Chondrosarcoma is a primary malignant bone cancer, with a potent capacity to invade locally and cause distant metastasis. Angiogenesis is a critical step in tumor growth and metastasis. Chemokine CCL5 (previously called RANTES) has been shown to facilitate tumor progression and metastasis. However, the relationship of CCL5 with vascular endothelial growth factor (VEGF) expression and angiogenesis in human chondrosarcoma is mostly unknown. In this study, CCL5 increased VEGF expression and also promoted chondrosarcoma medium-mediated angiogenesis in vitro as well as angiogenesis effects in the chick chorioallantoic membrane and Matrigel plug nude mice model in vivo. MicroRNA analysis was performed in CCL5-treated chondrosarcoma cells versus control cells to investigate the mechanism of CCL5-mediated promotion of chondrosarcoma angiogenesis. Among the miRNAs regulated by CCL5, miR-199a was the most downregulated miRNA after CCL5 treatment. In addition, co-transfection with miR-199a mimic reversed the CCL5-mediated VEGF expression and angiogenesis in vitro and in vivo. Moreover, overexpression of CCL5 increased tumor-associated angiogenesis and tumor growth by downregulating miR-199a in the xenograft tumor angiogenesis model. Taken together, these results demonstrated that CCL5 promotes VEGF-dependent angiogenesis in human chondrosarcoma cells by downregulating miR-199a. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  10. Dietary Estrogens Act through Estrogen Receptor-Mediated Processes and Show No Antiestrogenicity in Cultured Breast Cancer Cells.

    Science.gov (United States)

    Makela; Davis; Tally; Korkman; Salo; Vihko; Santti; Korach

    1994-06-01

    Dietary estrogens are believed to exert their estrogenic or antiestrogenic (chemopreventive) action in estrogen responsive cells by interacting with the estrogen receptor (ER). The present study was undertaken to evaluate a direct role of ER in estrogenic or antiestrogenic activities of three dietary estrogens (coumestrol, genistein and zearalenone). HeLa cells were transiently co-transfected with an expression vector for ER and an estrogen-responsive reporter gene construct. Coumestrol, genistein, and zearalenone all increased the activity of the reporter gene, only in the presence of the ER, and the activation was blocked with the ER antagonist ICI 164,384, demonstrating an ER-specific, agonist response. In addition, in MCF-7 cells, coumestrol and zearalenone increased the expression of the estrogen-responsive pS2 gene. Coumestrol and genistein inhibited the purified estrogen-specific 17ß-hydroxysteroid oxidoreductase enzyme and the conversion of estrone to 17ß-estradiol in T-47D cells, which contain this enzyme. However, they did not inhibit the estrone-induced proliferation of T-47D cells. In conclusion, coumestrol, genistein, and zearalenone are all potent estrogens in vitro, and they act through ER mediated mechanism. Our findings give no evidence to support the idea that these compounds act as antiestrogens through competition for the binding sites of ER or by inhibition of the conversion of estrone to 17ß-estradiol in breast cancer cells, since this effect was nullified by their agonist action on cell proliferation. Therefore, their suggested chemopreventive action in estrogen-related cancers must be mediated through other mechanisms.

  11. Interleukin-17 upregulates vascular endothelial growth factor by activating the JAK/STAT pathway in nucleus pulposus cells.

    Science.gov (United States)

    Hu, Bo; Wang, Jianxi; Wu, Xiaodong; Chen, Yu; Yuan, Wen; Chen, Huajiang

    2017-05-01

    Intervertebral disc (IVD) related diseases and age-related IVD degeneration are responsible for significant morbidity. Inflammatory mediators and pro-inflammatory cytokines, including interleukin (IL)-17, show elevated expression in degenerated disc tissue. IL-17 is reported to transduce signals across the cell membrane predominantly via the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signal transduction pathway, leading to transcriptional activation of target genes. In this study, we investigated whether the JAK/STAT pathway plays a role in IL-17-mediated signaling in the nucleus pulposus (NP) cells of IVDs. Vascular endothelial growth factor (VEGF) and IL-17 were found to be highly expressed in human degenerated NP tissue. In isolated rat NP cells, IL-17-induced VEGF expression in a time- and dose-dependent manner. Rat NP cells were co-transfected with VEGF promoter plasmid along with constitutively active STAT1, STAT3 or JAK2 plasmid. VEGF promoter activity was found to be increased by STAT1, STAT3 and JAK2 in IL-17-treated cells. Transfection of cultured rat NP cells with STAT1 or STAT3 lentiviral short hairpin RNAs or treatment with the JAK2 inhibitor AG490 significantly reduced IL-17-stimulated VEGF expression. IL-17 upregulated VEGF expression in rat NP cells mediated by the JAK/STAT pathway, and elevated levels of IL-17 and VEGF are present in human degenerated NP tissue. These findings provide new insight into the pathology of IVD degeneration. Copyright © 2016 Société française de rhumatologie. Published by Elsevier SAS. All rights reserved.

  12. Contactin associates with sodium channel Nav1.3 in native tissues and increases channel density at the cell surface.

    Science.gov (United States)

    Shah, Bhaval S; Rush, Anthony M; Liu, Shujun; Tyrrell, Lynda; Black, Joel A; Dib-Hajj, Sulayman D; Waxman, Stephen G

    2004-08-18

    The upregulation of voltage-gated sodium channel Na(v)1.3 has been linked to hyperexcitability of axotomized dorsal root ganglion (DRG) neurons, which underlies neuropathic pain. However, factors that regulate delivery of Na(v)1.3 to the cell surface are not known. Contactin/F3, a cell adhesion molecule, has been shown to interact with and enhance surface expression of sodium channels Na(v)1.2 and Na(v)1.9. In this study we show that contactin coimmunoprecipitates with Na(v)1.3 from postnatal day 0 rat brain where this channel is abundant, and from human embryonic kidney (HEK) 293 cells stably transfected with Na(v)1.3 (HEK-Na(v)1.3). Purified GST fusion proteins of the N and C termini of Na(v)1.3 pull down contactin from lysates of transfected HEK 293 cells. Transfection of HEK-Na(v)1.3 cells with contactin increases the amplitude of the current threefold without changing the biophysical properties of the channel. Enzymatic removal of contactin from the cell surface of cotransfected cells does not reduce the elevated levels of the Na(v)1.3 current. Finally, we show that, similar to Na(v)1.3, contactin is upregulated in axotomized DRG neurons and accumulates within the neuroma of transected sciatic nerve. We propose that the upregulation of contactin and its colocalization with Na(v)1.3 in axotomized DRG neurons may contribute to the hyper-excitablity of the injured neurons.

  13. Antibody-directed double suicide gene therapy targeting of MUC1- positive leukemia cells in vitro and in vivo.

    Science.gov (United States)

    Dong, Xiao-Ya; Wang, Wen-Qian; Zhao, Yu; Li, Xu-Dong; Fang, Zhi-Gang; Lin, Dong-Jun; Xiao, Ruo-Zhi; Huang, Ren-Wei; Pan, Guang-Jin; Liu, Jia-Jun

    2013-10-01

    Our aim was to specifically transfer the cytosine deaminase (CD) and thymidine kinase (TK) genes into mucin 1 (MUC1)-positive leukemia cells by anti-MUC1 antibody directed infection of replication-defective lentivirus and to evaluate the targeted cytotoxicity of double suicide genes to leukemia. The target gene vector (containing CD and TK) and envelope (containing GFP and anti-MUC1) and packaging plasmids were cotransfected into 293T cells to produce the recombinant lentivirus. Suicide genes in virus-infected leukemia cells (U937, Jurkat, and K562) were detected by western blot. The cytotoxicity and bystander effect in vitro and the therapeutic effect in vivo were detected after treatment with the prodrugs. The results revealed that combined treatment with prodrug 5-fluorocytosine (5-FC) and ganciclovir (GCV) inhibited leukemia cell growth and caused significant bystander effect than treatment with either prodrug alone. TK/GCV treatment alone induced degeneration and cell death while the effect of CD/5-FC alone mainly caused vacuolar degeneration and necrosis. The addictive effects of combinatorial use of GCV and 5-FC mainly induced swelling of the mitochondria followed by necrosis of the leukemia cells. In vivo experiments revealed that both single and combinatorial prodrug treatments could prolong the survival time of leukemic mice. In summary, anti-MUC1 antibody directed lentiviral vector successfully transduced dual suicide genes and exerted targeted cytotoxicity against MUC1 positive leukemia cells. This targeted lentiviral dual suicide gene delivering system provides a promising approach for clinical treatment of leukemia in future.

  14. Nuclear marginalization of host cell chromatin associated with expansion of two discrete virus-induced subnuclear compartments during baculovirus infection.

    Science.gov (United States)

    Nagamine, Toshihiro; Kawasaki, Yu; Abe, Atsushi; Matsumoto, Shogo

    2008-07-01

    Chromatin structure is strictly regulated during the cell cycle. DNA viruses occasionally disturb the spatial organization of the host cell chromatin due to formation of the viral DNA replication compartment. To examine chromatin behavior in baculovirus-infected cells, we constructed recombinant plasmids expressing fluorescent protein-tagged histone H4 molecules and visualized the intracellular localization of chromatin by their transient expression in live infected cells. Similar to other DNA viruses, the baculovirus Bombyx mori nucleopolyhedrovirus induced marginal relocation of chromatin within the nuclei of BmN cells, simultaneously with expansion of the viral DNA replication compartment, the virogenic stroma (VS). In the late stage of infection, however, the peristromal region (PR), another virus-induced subnuclear compartment, was also excluded from the chromatin-localizing area. Provided that late-gene products such as PR proteins (e.g., envelope proteins of the occlusion-derived virus) were expressed, blockage of viral DNA synthesis failed to inhibit chromatin relocation, despite abrogation of VS expansion. Instead, chromatin became marginalized concomitantly with PR expansion, suggesting that the PR contributes directly to chromatin replacement. In addition, chromatin was excluded from relatively large subnuclear structures that were induced in uninfected cells by cotransfection with four baculovirus genes, ie1, lef3, p143, and hr. Omission of any of the four genes, however, failed to result in formation of the large structures or chromatin exclusion. This correlation between compartmentalization and chromatin exclusion suggests the possibility that a chromatin-exclusive property of viral molecules, at least in part, supports nuclear compartmentalization of virus-infected cells.

  15. Nuclear Marginalization of Host Cell Chromatin Associated with Expansion of Two Discrete Virus-Induced Subnuclear Compartments during Baculovirus Infection▿

    Science.gov (United States)

    Nagamine, Toshihiro; Kawasaki, Yu; Abe, Atsushi; Matsumoto, Shogo

    2008-01-01

    Chromatin structure is strictly regulated during the cell cycle. DNA viruses occasionally disturb the spatial organization of the host cell chromatin due to formation of the viral DNA replication compartment. To examine chromatin behavior in baculovirus-infected cells, we constructed recombinant plasmids expressing fluorescent protein-tagged histone H4 molecules and visualized the intracellular localization of chromatin by their transient expression in live infected cells. Similar to other DNA viruses, the baculovirus Bombyx mori nucleopolyhedrovirus induced marginal relocation of chromatin within the nuclei of BmN cells, simultaneously with expansion of the viral DNA replication compartment, the virogenic stroma (VS). In the late stage of infection, however, the peristromal region (PR), another virus-induced subnuclear compartment, was also excluded from the chromatin-localizing area. Provided that late-gene products such as PR proteins (e.g., envelope proteins of the occlusion-derived virus) were expressed, blockage of viral DNA synthesis failed to inhibit chromatin relocation, despite abrogation of VS expansion. Instead, chromatin became marginalized concomitantly with PR expansion, suggesting that the PR contributes directly to chromatin replacement. In addition, chromatin was excluded from relatively large subnuclear structures that were induced in uninfected cells by cotransfection with four baculovirus genes, ie1, lef3, p143, and hr. Omission of any of the four genes, however, failed to result in formation of the large structures or chromatin exclusion. This correlation between compartmentalization and chromatin exclusion suggests the possibility that a chromatin-exclusive property of viral molecules, at least in part, supports nuclear compartmentalization of virus-infected cells. PMID:18434402

  16. Stat3 activates the receptor tyrosine kinase like orphan receptor-1 gene in chronic lymphocytic leukemia cells.

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    Ping Li

    Full Text Available BACKGROUND: The receptor tyrosine kinase like orphan receptor (ROR-1 gene is overexpressed in chronic lymphocytic leukemia (CLL. Because Stat3 is constitutively activated in CLL and sequence analysis revealed that the ROR1 promoter harbors gamma-interferon activation sequence-like elements typically activated by Stat3, we hypothesized that Stat3 activates ROR1. METHODOLOGY/PRINCIPAL FINDINGS: Because IL-6 induced Stat3 phosphorylation and upregulated Ror1 protein levels in MM1 cells, we used these cells as a model. We transfected MM1 cells with truncated ROR1 promoter luciferase reporter constructs and found that IL-6 induced luciferase activity of ROR1-195 and upstream constructs. Co-transfection with Stat3 siRNA reduced the IL-6-induced luciferase activity, suggesting that IL-6 induced luciferase activity by activating Stat3. EMSA and the ChIP assay confirmed that Stat3 binds ROR1, and EMSA studies identified two Stat3 binding sites. In CLL cells, EMSA and ChIP studies determined that phosphorylated Stat3 bound to the ROR1 promoter at those two ROR1 promoter sites, and ChIP analysis showed that Stat3 co-immunoprecipitated DNA of STAT3, ROR1, and several Stat3-regulated genes. Finally, like STAT3-siRNA in MM1 cells, STAT3-shRNA downregulated STAT3, ROR1, and STAT3-regulated genes and Stat3 and Ror1 protein levels in CLL cells. CONCLUSION/SIGNIFICANCE: Our data suggest that constitutively activated Stat3 binds to the ROR1 promoter and activates ROR1 in CLL cells.

  17. Establishment of cell strains stably-transfected with luciferase gene mediated by retrovirus and their detection with bioluminescence imaging system

    Directory of Open Access Journals (Sweden)

    Hai-juan WANG

    2012-05-01

    Full Text Available Objective  To establish cell strains stably transfected with Luciferase gene (Luc2, which was mediated by retrovirus, and explore the relationship between the number of Luc2-positive cells and light flux from bioluminescence imaging system by experiments in vitro and in vivo. Methods  We co-transfected pMX-Luc2 plasmid and pMD.G plasmid into 293T gag-pol cells to get retrovirus expressing Luc2 gene. Stable Luc2 positive cell lines were generated and screened by transduction of Retro-Luc2 in mouse colon cancer cell line CT26, human non-small cell lung cancer cell line NCI-H446, human colon cancer cell line HT-29, human ovarian carcinoma cell line SKOV3 and human hepatocellular carcinoma cell line SMMC-7721, all of them were identified by bioluminescence imaging system. Different numbers of SKOV3-Luc2 cells ranging from 10 to 10000 were plated onto culture dishes. Two xenograft models of ovarian cancer were reproduced by subcutaneous injection of 200μl SKOV3-Luc2 cell suspension with different concentrations (1×107, 5×106, 2.5×106, 1×106, 5×105, 2.5×105, 1×105 and 5×104/ml into 16 sites on the back of 4 nude mice, or intravenous injection of 1×106 or 3 ×106 SKOV3-Luc2 cells into the tail vein. Light flux value of SKOV3-Luc2 cells in dishes and in mice was measured by bioluminescence imaging system. Results  Retro-Luc2 was constructed successfully and expressed Luc2 stably in transduced CT26, NCI-H446, HT-29, SKOV3 and SMMC-7721 cell lines. Light flux was correlated in a linear manner with the number of Luc2-positive cells in dishes and in mice (R2=0.944, β=0.972; R2=0.991, β=0.996; R2=0.351, β=0.628; P < 0.01. Conclusion  Luc2-positive cell lines could be established rapidly and accurately by infecting tumor cells with retrovirus expressing Luc2. The number of Luc2 positive cells is significantly related in a linear manner to light flux from bioluminescence imaging system in vitro and in vivo.

  18. Expression patterns and miRNA regulation of DNA methyltransferases in chicken primordial germ cells.

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    Deivendran Rengaraj

    Full Text Available DNA methylation is widespread in most species, from bacteria to mammals, and is crucial for genomic imprinting, gene expression, and embryogenesis. DNA methylation occurs via two major classes of enzymatic reactions: maintenance-type methylation catalyzed by DNA (cytosine-5--methyltransferase (DNMT 1, and de novo methylation catalyzed by DNMT 3 alpha (DNMT3A and -beta (DNMT3B. The expression pattern and regulation of DNMT genes in primordial germ cells (PGCs and germ line cells has not been sufficiently established in birds. Therefore, we employed bioinformatics, RT-PCR, real-time PCR, and in situ hybridization analyses to examine the structural conservation and conserved expression patterns of chicken DNMT family genes. We further examined the regulation of a candidate de novo DNA methyltransferase gene, cDNMT3B by cotransfection of cDNMT3B 3'UTR- and cDNMT3B 3'UTR-specific miRNAs through a dual fluorescence reporter assay. All cDNMT family members were differentially detected during early embryonic development. Of interest, cDNMT3B expression was highly detected in early embryos and in PGCs. During germ line development and sexual maturation, cDNMT3B expression was reestablished in a female germ cell-specific manner. In the dual fluorescence reporter assay, cDNMT3B expression was significantly downregulated by four miRNAs: gga-miR-15c (25.82%, gga-miR-29b (30.01%, gga-miR-383 (30.0%, and gga-miR-222 (31.28%. Our data highlight the structural conservation and conserved expression patterns of chicken DNMTs. The miRNAs investigated in this study may induce downregulation of gene expression in chicken PGCs and germ cells.

  19. The Notch-2 gene is regulated by Wnt signaling in cultured colorectal cancer cells.

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    Jonas Ungerbäck

    Full Text Available BACKGROUND: Notch and Wnt pathways are key regulators of intestinal homeostasis and alterations in these pathways may lead to the development of colorectal cancer (CRC. In CRC the Apc/β-catenin genes in the Wnt signaling pathway are frequently mutated and active Notch signaling contributes to tumorigenesis by keeping the epithelial cells in a proliferative state. These pathways are simultaneously active in proliferative adenoma cells and a crosstalk between them has previously been suggested in normal development as well as in cancer. PRINCIPAL FINDINGS: In this study, in silico analysis of putative promoters involved in transcriptional regulation of genes coding for proteins in the Notch signaling pathway revealed several putative LEF-1/TCF sites as potential targets for β-catenin and canonical Wnt signaling. Further results from competitive electrophoretic mobility-shift assay (EMSA studies suggest binding of several putative sites in Notch pathway gene promoters to in vitro translated β-catenin/Lef-1. Wild type (wt-Apc negatively regulates β-catenin. By induction of wt-Apc or β-catenin silencing in HT29 cells, we observed that several genes in the Notch pathway, including Notch-2, were downregulated. Finally, active Notch signaling was verified in the Apc(Min/+ mouse model where Hes-1 mRNA levels were found significantly upregulated in intestinal tumors compared to normal intestinal mucosa. Luciferase assays showed an increased activity for the core and proximal Notch-2 promoter upon co-transfection of HCT116 cells with high expression recombinant Tcf-4, Lef-1 or β-catenin. CONCLUSIONS: In this paper, we identified Notch-2 as a novel target for β-catenin-dependent Wnt signaling. Furthermore our data supports the notion that additional genes in the Notch pathway might be transcriptionally regulated by Wnt signaling in colorectal cancer.

  20. Migration of turkey muscle satellite cells is enhanced by the syndecan-4 cytoplasmic domain through the activation of RhoA.

    Science.gov (United States)

    Shin, Jonghyun; McFarland, Douglas C; Velleman, Sandra G

    2013-03-01

    Syndecan-4 (S4) is a cell membrane-associated heparan sulfate proteoglycan that forms oligomers in muscle satellite cells. The S4 oligomers activate protein kinase Cα (PKCα) through the S4 cytoplasmic domain and may regulate the activation of ras homolog gene family member A (RhoA), a signal transduction molecule down-stream of PKCα which is thought to influence cell migration. However, little is known about the function of the S4 cytoplasmic domain in satellite cell migration and RhoA activation. The objective of the current study was to determine the function of S4 and its cytoplasmic domain in cell migration and RhoA activation. To study the objective, clones of S4 and S4 without the cytoplasmic domain (S4C) were used in overexpression studies, and small interference RNAs targeting S4 or RhoA were used in knockdown studies. Satellite cell migration was increased by S4 overexpression, but decreased by the knockdown or deletion of the S4 cytoplasmic domain. The RhoA protein was activated by the overexpression of S4, but not with the deletion of the S4 cytoplasmic domain. The treatment of Rho activator II or the knockdown of RhoA also modulated satellite cell migration. Finally, co-transfection (S4 overexpression and RhoA knockdown) and rescue (the knockdown of S4 and the treatment with Rho activator II) studies demonstrated that S4-mediated satellite cell migration was regulated through the activation of RhoA. The cytoplasmic domain of S4 is required for cell migration and RhoA activation which will affect muscle fiber formation.

  1. Co-operation of the transcription factor hepatocyte nuclear factor-4 with Sp1 or Sp3 leads to transcriptional activation of the human haem oxygenase-1 gene promoter in a hepatoma cell line.

    Science.gov (United States)

    Takahashi, Shigeru; Matsuura, Naomi; Kurokawa, Takako; Takahashi, Yuji; Miura, Takashi

    2002-11-01

    We reported previously that the 5'-flanking region (nucleotides -1976 to -1655) of the human haem oxygenase-1 ( hHO-1 ) gene enhances hHO-1 promoter activity in human hepatoma HepG2 cells, but not in HeLa cells [Takahashi, Takahashi, Ito, Nagano, Shibahara and Miura (1999) Biochim. Biophys. Acta 1447, 231-235]. To define more precisely the regulatory elements involved, in the present study we have functionally dissected this region and localized the enhancer to a 50 bp fragment (-1793 to -1744). Site-direct mutagenesis analysis revealed that two regions were responsible for this enhancer activity, i.e. a hepatocyte nuclear factor-4 (HNF-4) homologous region and a GC box motif homologous region. Mutation in either region alone moderately decreased enhancer activity. However, mutations in both regions reduced promoter activity to the basal level. Electrophoretic mobility-shift assays demonstrated that the P5-2 fragment (-1793 to -1744) interacted with at least two nuclear factors, i.e. HNF-4 and Sp1/Sp3. Co-transfection experiments using Drosophila SL2 cells revealed that HNF-4 and Sp1/Sp3 synergistically stimulated the enhancer activity of the P5-2 fragment. These results indicate that co-operation of HNF-4 with Sp1 or Sp3 leads to the activation of hHO-1 gene expression in hepatoma cells.

  2. EFEMP1 Suppresses Growth and Invasion of Lung Cancer Cells 
by Downregulating Matrix Metalloproteinase-7 Expression

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    Yuanyuan LANG

    2015-02-01

    Full Text Available Background and objective EFEMP1, a member of fibulin family proteins, is a very important extracellular matrix protein which is involved in cell metabolism and its role in tumor occurrence and progression is still poorly understood. The aim of this study is to investigate the functional effect and mechanism of EFEMP1 in lung cancer cell growth and invasion. Methods EFEMP1 expression in lung cancer cells was determined by Western blot. The promoter methylation status of EFEMP1 was detected by methylation-specific PCR (MSP. After transfection of control or EFEMP1 vector in lung cancer cells, the ability of colony formation and invasion was detected by colony formation experiment and matrigel invasion method. Western blot and real-time PCR were used to detect matrix metalloproteinase-7 (MMP-7 expression. Luciferase assay was used to detect expression of MMP-7 reporter construct transfected with or without EFEMP1 in lung cancer cells. Results Western blot result showed EFEMP1 expression was downregulated in lung cancer cells. The promoter region of EFEMP1 was methylated in A549 and H1299 and after treatment with 5-aza-2’-deoxycytidine, the EFEMP1 expression was upregulated. The growth and invasion of A549 and H1299 were all significantly suppressed by transfecting with EFEMP1 and the MMP-7 expression was dowanregulated by EFEMP1 as well. Expression activity of MMP-7 reporter construct was decreased by cotransfecting with EFEMP1. Conclusion Collectively, these results suggest that EFEMP1 functions as a suppressor of lung cancer growth and invasion. Epigenetic silencing of EFEMP1 promotes lung cancer invasion and metastasis by activating MMP-7 expression.

  3. Modulation of RANTES expression by HCV core protein in liver derived cell lines

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    Rapicetta Maria

    2007-06-01

    Full Text Available Abstract Background Hepatitis C virus (HCV infection is associated with high percentage of chronicity which implies the ability of the virus to evade or modulate host cell immune system. Modulation of chemokines, such as RANTES may be part of the virus induced pathogenicity. We examined the effect of core and structural proteins of HCV on RANTES expression in two liver derived cell lines, HepG2 and Chang Liver (CHL. Methods HepG2 and Chang Liver (CHL cell lines were established and selected for constitutive expression of HCV core and structural genes. Flow cytometry and quantitative RT-PCR analysis were performed to examine the effect of HCV core protein on RANTES expression. Luciferase analysis after RANTES-Luc-promoter transfection of established cell lines was assayed by luminometer measurements (RLU of RANTES promoter activity. IRF-1 and IRF-7 expression was then examined by immunoblotting analysis. Results Results of flow cytometry and RT-PCR analysis indicated that RANTES is differentially regulated by HCV core protein in the two cell lines examined as its expression was inhibited in HepG2 cells, by a reduction of RANTES promoter activity. Conversely, RANTES protein and mRNA were induced by the core protein in CHL cells, through the induction of the promoter. Since HCV genome modulates IRF-1 and IRF-7 in replicon system and IRF-1, IRF-3 and IRF-7 have been reported to regulate RANTES promoter in various cell systems, analysis of the mechanism underlying RANTES modulation by the core protein revealed that IRF-1 expression was induced in HepG2 cells by the core protein, whereas in CHL cells it was expressed at a very low level that was not influenced by transfection with the core protein construct. This suggested that IRF-1 level may mediate the expression of RANTES in cell lines of liver origin. The effect of the core protein on RANTES promoter was countered by co-transfection with NF90, a double-stranded-RNA binding protein that activates

  4. Comparison of abnormal isoform of prion protein in prion-infected cell lines and primary-cultured neurons by PrPSc-specific immunostaining.

    Science.gov (United States)

    Tanaka, Misaki; Fujiwara, Ai; Suzuki, Akio; Yamasaki, Takeshi; Hasebe, Rie; Masujin, Kentaro; Horiuchi, Motohiro

    2016-08-01

    We established abnormal isoform of prion protein (PrPSc)-specific double immunostaining using mAb 132, which recognizes aa 119-127 of the PrP molecule, and novel PrPSc-specific mAb 8D5, which recognizes the N-terminal region of the PrP molecule. Using the PrPSc-specific double immunostaining, we analysed PrPSc in immortalized neuronal cell lines and primary cerebral-neuronal cultures infected with prions. The PrPSc-specific double immunostaining showed the existence of PrPSc positive for both mAbs 132 and 8D5, as well as those positive only for either mAb 132 or mAb 8D5. This indicated that double immunostaining detects a greater number of PrPSc species than single immunostaining. Double immunostaining revealed cell-type-dependent differences in PrPSc staining patterns. In the 22 L prion strain-infected Neuro2a (N2a)-3 cells, a subclone of N2a neuroblastoma cell line, or GT1-7, a subclone of the GT1 hypothalamic neuronal cell line, granular PrPSc stains were observed at the perinuclear regions and cytoplasm, whereas unique string-like PrPSc stains were predominantly observed on the surface of the 22 L strain-infected primary cerebral neurons. Only 14 % of PrPSc in the 22 L strain-infected N2a-3 cells were positive for mAb 8D5, indicating that most of the PrPSc in N2a-3 lack the N-terminal portion. In contrast, nearly half PrPSc detected in the 22 L strain-infected primary cerebral neurons were positive for mAb 8D5, suggesting the abundance of full-length PrPSc that possesses the N-terminal portion of PrP. Further analysis of prion-infected primary neurons using PrPSc-specific immunostaining will reveal the neuron-specific mechanism for prion propagation.

  5. A common multiple cloning site in a set of vectors for expression of eukaryotic genes in mammalian, insect and bacterial cells

    DEFF Research Database (Denmark)

    Pallisgaard, N; Pedersen, FS; Birkelund, Svend

    1994-01-01

    Here, we describe the construction of plasmid vectors facilitating expression of cloned genes in bacteria and in cells of mammalian and insect origin. Two types of multiple cloning site (MCS) were designed based on the MCS in the expression vector lambda gt11Sfi-Not. In the first set of vectors...... a start Met codon was included in the same reading frame as in lambda gt11Sfi-Not to support expression of partial cDNA clones. Thus a cDNA insert of lambda gt11Sfi-Not could be shuttled among the new vectors for expression. The other set of vectors without a start codon were suitable for expression of cDNA...... carrying their own start Met codon. By Western blot analysis and by transactivation of a reporter plasmid in co-transfections we show that cDNA is very efficiently expressed in NIH 3T3 cells under control of the elongation factor 1 alpha promoter....

  6. Transplantation of rat embryonic stem cell-derived retinal progenitor cells preserves the retinal structure and function in rat retinal degeneration.

    Science.gov (United States)

    Qu, Zepeng; Guan, Yuan; Cui, Lu; Song, Jian; Gu, Junjie; Zhao, Hanzhi; Xu, Lei; Lu, Lixia; Jin, Ying; Xu, Guo-Tong

    2015-11-09

    Degenerative retinal diseases like age-related macular degeneration (AMD) are the leading cause of blindness. Cell transplantation showed promising therapeutic effect for such diseases, and embryonic stem cell (ESC) is one of the sources of such donor cells. Here, we aimed to generate retinal progenitor cells (RPCs) from rat ESCs (rESCs) and to test their therapeutic effects in rat model. The rESCs (DA8-16) were cultured in N2B27 medium with 2i, and differentiated to two types of RPCs following the SFEBq method with modifications. For rESC-RPC1, the cells were switched to adherent culture at D10, while for rESC-RPC2, the suspension culture was maintained to D14. Both RPCs were harvested at D16. Primary RPCs were obtained from P1 SD rats, and some of them were labeled with EGFP by infection with lentivirus. To generate Rax::EGFP knock-in rESC lines, TALENs were engineered to facilitate homologous recombination in rESCs, which were cotransfected with the targeting vector and TALEN vectors. The differentiated cells were analyzed with live image, immunofluorescence staining, flow cytometric analysis, gene expression microarray, etc. RCS rats were used to mimic the degeneration of retina and test the therapeutic effects of subretinally transplanted donor cells. The structure and function of retina were examined. We established two protocols through which two types of rESC-derived RPCs were obtained and both contained committed retina lineage cells and some neural progenitor cells (NPCs). These rESC-derived RPCs survived in the host retinas of RCS rats and protected the retinal structure and function in early stage following the transplantation. However, the glia enriched rESC-RPC1 obtained through early and longer adherent culture only increased the b-wave amplitude at 4 weeks, while the longer suspension culture gave rise to evidently neuronal differentiation in rESC-RPC2 which significantly improved the visual function of RCS rats. We have successfully differentiated

  7. Human gastrin mRNA expression up-regulated by Helicobacter pylori CagA through MEK/ERK and JAK2-signaling pathways in gastric cancer cells.

    Science.gov (United States)

    Zhou, Jianjiang; Xie, Yuan; Zhao, Yan; Wang, Shu; Li, Yu

    2011-10-01

    Helicobacter pylori-cytotoxin-associated protein A (CagA) and gastrin are believed to play an important role in gastric carcinogenesis, but their interaction has been incompletely clear. We constructed a eukaryotic expression vector pcDNA3.1/cagA and a luciferase reporter vector pGL/gastrin promoter, and then co-transfected them into gastric cancer AGS and SGC-7901 cells. The two kinds of gastric cancer cells were, respectively, infected with cagA-positive H. pylori NCTC11637, and then the gastrin promoter activity and gastrin mRNA level were detected with a Dual-Luciferase reporter assay system and quantitative reverse transcription polymerase chain reaction (RT-PCR), respectively. Next, after the MEK/ERK and JAK2-signaling pathway inhibitors, U0126 and AG490, were used to treat the two cell lines, or the ERK1 and JAK2 genes were knocked down by siRNAs (small interference RNAs) in the two cell lines, the gastrin promoter activity and gastrin mRNA level were observed again. The results indicated that CagA could activate the gastrin promoter and up-regulate gastrin mRNA expression in AGS and SGC-7901 cells, but these effects could be inhibited by the inhibitors U0126 and AG490, and the CagA-induced gastrin mRNA expression was down-regulated in the cells whose ERK1 or JAK2 gene was knocked down. Gastrin promoter may be the transcriptional target of CagA, and CagA activates the gastrin promoter to up-regulate gastrin mRNA expression through the MEK/ERK and JAK1-signaling pathway in gastric cancer cells.

  8. Drosophila S2 cells are non-permissive for vaccinia virus DNA replication following entry via low pH-dependent endocytosis and early transcription.

    Directory of Open Access Journals (Sweden)

    Zain Bengali

    Full Text Available Vaccinia virus (VACV, a member of the chordopox subfamily of the Poxviridae, abortively infects insect cells. We have investigated VACV infection of Drosophila S2 cells, which are useful for protein expression and genome-wide RNAi screening. Biochemical and electron microscopic analyses indicated that VACV entry into Drosophila S2 cells depended on the VACV multiprotein entry-fusion complex but appeared to occur exclusively by a low pH-dependent endocytic mechanism, in contrast to both neutral and low pH entry pathways used in mammalian cells. Deep RNA sequencing revealed that the entire VACV early transcriptome, comprising 118 open reading frames, was robustly expressed but neither intermediate nor late mRNAs were made. Nor was viral late protein synthesis or inhibition of host protein synthesis detected by pulse-labeling with radioactive amino acids. Some reduction in viral early proteins was noted by Western blotting. Nevertheless, synthesis of the multitude of early proteins needed for intermediate gene expression was demonstrated by transfection of a plasmid containing a reporter gene regulated by an intermediate promoter. In addition, expression of a reporter gene with a late promoter was achieved by cotransfection of intermediate genes encoding the late transcription factors. The requirement for transfection of DNA templates for intermediate and late gene expression indicated a defect in viral genome replication in VACV-infected S2 cells, which was confirmed by direct analysis. Furthermore, VACV-infected S2 cells did not support the replication of a transfected plasmid, which occurs in mammalian cells and is dependent on all known viral replication proteins, indicating a primary restriction of DNA synthesis.

  9. Expression of particulate-form of Japanese encephalitis virus envelope protein in a stably transfected Drosophila cell line

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    Zhang Li

    2007-02-01

    Full Text Available Abstract Background Japanese encephalitis virus (JEV, a member of the family Flaviviridae, is an important mosquito-borne human pathogen. Its envelope glycoprotein (E is the major determinant of the pathogenicity and host immune responses. In the present study, we explored the feasibility of producing recombinant JEV E protein in the virus-free Drosophila expression system. Results The coding sequence for the signal sequence of premembrane and E protein was cloned into the Drosophila expression vector pAc5.1/V5-His. A Drosophila cell line S2 was cotransfected with this construct as well as a plasmid providing hygromycin B resistance. A cell line expressing the JEV E protein was selected by immunofluoresence, confocal microscopy, and western blot analysis using three different monoclonal antibodies directed against JEV E protein. This cell line was stable in the yield of JEV E protein during two months in vitro maintenance in the presence of hygromycin B. The results showed that the recombinant E protein had an expected molecular weight of about 50 kilodalton, was immunoreactive with all three monoclonal antibodies, and found in both the cytoplasm and culture supernatant. Sucrose gradient ultracentrifugation analysis revealed that the secreted E protein product was in a particulate form. It migrated to the sucrose fraction with a density of 1.13 g/ml. Balb/c mice immunised with the sucrose fraction containing the E protein particles developed specific antibodies. These data show that functioning JEV E protein was expressed in the stable S2 cell line. Conclusion The Drosophila expression system is a more convenient, cheaper and safer approach to the production of vaccine candidates and diagnostic reagents for JEV.

  10. Enhancing the Breadth of Efficacy of Therapeutic Vaccines for Breast Cancer

    Science.gov (United States)

    2015-10-01

    provides information about the identity of individual T cells by analyzing the TCR chains as a pair, eliminating the need for computer -based...language and environment for statistical computing . Vienna, Austria: R Foundation for Statistical Computing . Available: http://www.R-project.org/. 13...co-transfect SF9 insect cells. Co-transfection of SF9 cells is initially assesed by survival of the cells compared to un-infected controls. The

  11. The B-Cell Specific Transcription Factor, Oct-2, Promotes Epstein-Barr Virus Latency by Inhibiting the Viral Immediate-Early Protein, BZLF1

    Science.gov (United States)

    Robinson, Amanda R.; Kwek, Swee Sen; Kenney, Shannon C.

    2012-01-01

    The Epstein-Barr virus (EBV) latent-lytic switch is mediated by the BZLF1 immediate-early protein. EBV is normally latent in memory B cells, but cellular factors which promote viral latency specifically in B cells have not been identified. In this report, we demonstrate that the B-cell specific transcription factor, Oct-2, inhibits the function of the viral immediate-early protein, BZLF1, and prevents lytic viral reactivation. Co-transfected Oct-2 reduces the ability of BZLF1 to activate lytic gene expression in two different latently infected nasopharyngeal carcinoma cell lines. Furthermore, Oct-2 inhibits BZLF1 activation of lytic EBV promoters in reporter gene assays, and attenuates BZLF1 binding to lytic viral promoters in vivo. Oct-2 interacts directly with BZLF1, and this interaction requires the DNA-binding/dimerization domain of BZLF1 and the POU domain of Oct-2. An Oct-2 mutant (Δ262–302) deficient for interaction with BZLF1 is unable to inhibit BZLF1-mediated lytic reactivation. However, an Oct-2 mutant defective for DNA-binding (Q221A) retains the ability to inhibit BZLF1 transcriptional effects and DNA-binding. Importantly, shRNA-mediated knockdown of endogenous Oct-2 expression in several EBV-positive Burkitt lymphoma and lymphoblastoid cell lines increases the level of lytic EBV gene expression, while decreasing EBNA1 expression. Moreover, treatments which induce EBV lytic reactivation, such as anti-IgG cross-linking and chemical inducers, also decrease the level of Oct-2 protein expression at the transcriptional level. We conclude that Oct-2 potentiates establishment of EBV latency in B cells. PMID:22346751

  12. Targeting of herpes simplex virus 1 thymidine kinase gene sequences into the OCT4 locus of human induced pluripotent stem cells.

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    Wu Ou

    Full Text Available The in vitro differentiation of human induced pluripotent stem cells (hiPSC to generate specific types of cells is inefficient, and the remaining undifferentiated cells may form teratomas. This raises safety concerns for clinical applications of hiPSC-derived cellular products. To improve the safety of hiPSC, we attempted to site-specifically insert a herpes simplex virus 1 thymidine kinase (HSV1-TK suicide gene at the endogenous OCT4 (POU5F1 locus of hiPSC. Since the endogenous OCT4 promoter is active in undifferentiated cells only, we speculated that the HSV1-TK suicide gene will be transcribed in undifferentiated cells only and that the remaining undifferentiated cells can be depleted by treating them with the prodrug ganciclovir (GCV prior to transplantation. To insert the HSV1-TK gene at the OCT4 locus, we cotransfected hiPSC with a pair of plasmids encoding an OCT4-specific zinc finger nuclease (ZFN and a donor plasmid harboring a promoter-less transgene cassette consisting of HSV1-TK and puromycin resistance gene sequences, flanked by OCT4 gene sequences. Puromycin resistant clones were established and characterized regarding their sensitivity to GCV and the site of integration of the HSV1-TK/puromycin resistance gene cassette. Of the nine puromycin-resistant iPSC clones analyzed, three contained the HSV1-TK transgene at the OCT4 locus, but they were not sensitive to GCV. The other six clones were GCV-sensitive, but the TK gene was located at off-target sites. These TK-expressing hiPSC clones remained GCV sensitive for up to 90 days, indicating that TK transgene expression was stable. Possible reasons for our failed attempt to selectively target the OCT4 locus are discussed.

  13. MRG15 activates the cdc2 promoter via histone acetylation in human cells

    Energy Technology Data Exchange (ETDEWEB)

    Pena, AndreAna N., E-mail: andreana.pena@gmail.com [Sam and Ann Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, San Antonio, TX (United States); Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, TX (United States); Tominaga, Kaoru; Pereira-Smith, Olivia M. [Sam and Ann Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, San Antonio, TX (United States); Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, TX (United States)

    2011-07-01

    Chromatin remodeling is required for transcriptional activation and repression. MRG15 (MORF4L1), a chromatin modulator, is a highly conserved protein and is present in complexes containing histone acetyltransferases (HATs) as well as histone deacetylases (HDACs). Loss of expression of MRG15 in mice and Drosophila results in embryonic lethality and fibroblast and neural stem/progenitor cells cultured from Mrg15 null mouse embryos exhibit marked proliferative defects when compared with wild type cells. To determine the role of MRG15 in cell cycle progression we performed chromatin immunoprecipitation with an antibody to MRG15 on normal human fibroblasts as they entered the cell cycle from a quiescent state, and analyzed various cell cycle gene promoters. The results demonstrated a 3-fold increase in MRG15 occupancy at the cdc2 promoter during S phase of the cell cycle and a concomitant increase in acetylated histone H4. H4 lysine 12 was acetylated at 24 h post-serum stimulation while there was no change in acetylation of lysine 16. HDAC1 and 2 were decreased at this promoter during cell cycle progression. Over-expression of MRG15 in HeLa cells activated a cdc2 promoter-reporter construct in a dose-dependent manner, whereas knockdown of MRG15 resulted in decreased promoter activity. In order to implicate HAT activity, we treated cells with the HAT inhibitor anacardic acid and determined that HAT inhibition results in loss of expression of cdc2 mRNA. Further, chromatin immunoprecipitation with Tip60 localizes the protein to the same 110 bp stretch of the cdc2 promoter pulled down by MRG15. Additionally, we determined that cotransfection of MRG15 with the known associated HAT Tip60 had a cooperative effect in activating the cdc2 promoter. These results suggest that MRG15 is acting in a HAT complex involving Tip60 to modify chromatin via acetylation of histone H4 at the cdc2 promoter to activate transcription.

  14. HAF drives the switch of HIF-1α to HIF-2α by activating the NF-κB pathway, leading to malignant behavior of T24 bladder cancer cells.

    Science.gov (United States)

    Guan, Zhenfeng; Ding, Chen; Du, Yiqing; Zhang, Kai; Zhu, Jian Ning; Zhang, Tingting; He, Dalin; Xu, Shan; Wang, Xinyang; Fan, Jinhai

    2014-02-01

    Hypoxia is a characteristic feature of solid tumors, leading to malignant behavior. During this process, HIF family members (HIFs) and the NF-κB pathway are activated. In addition, the hypoxia-associated factor (HAF) is reported to participate in the regulation of HIFs. However, the precise relationship among HIFs, HAF and the NF-κB pathway in bladder cancer (BC) remains unknown. In the current investigation, T24 BC cells were exposed to hypoxia, or by plasmid transfection to overexpress HAF or RelA (P65) to demonstrate their roles. The results indicate that hypoxia leads to the elevation of HAF plus activation of the NF-κB pathway, accompanied by the switch of HIF-1α to HIF-2α, resulting in the enhanced ability of malignancy in T24 cells. In order to further demonstrate the significance of this switch, HIF-1α and HIF-2α were co-transfected into T24 cells with HIF-β, respectively. The following results indicate that the T24hif-2α/β cells show enhanced ability of malignancy, accompanied by the maintenance of stem-cell markers, but the T24hif-1α/β cells show higher expression of metabolism-related genes. Boyden assays and wound-healing assays indicate the enhanced ability of malignancy for T24hif-2α/β. Thus, we conclude that on the hypoxic microenvironment, the switching of HIF-1α to HIF-2α, which is driven by HAF through activating the NF-κB pathway, contributes to the malignancy of T24 cells, accompanied by the maintenance of stem-cell markers. This provides us an avenue for understanding the progression of bladder cancer.

  15. Seladin-1 is a fundamental mediator of the neuroprotective effects of estrogen in human neuroblast long-term cell cultures.

    Science.gov (United States)

    Luciani, Paola; Deledda, Cristiana; Rosati, Fabiana; Benvenuti, Susanna; Cellai, Ilaria; Dichiara, Francesca; Morello, Matteo; Vannelli, Gabriella Barbara; Danza, Giovanna; Serio, Mario; Peri, Alessandro

    2008-09-01

    Estrogen exerts neuroprotective effects and reduces beta-amyloid accumulation in models of Alzheimer's disease (AD). A few years ago, a new neuroprotective gene, i.e. seladin-1 (for selective AD indicator-1), was identified and found to be down-regulated in AD vulnerable brain regions. Seladin-1 inhibits the activation of caspase-3, a key modulator of apoptosis. In addition, it has been demonstrated that the seladin-1 gene encodes 3beta-hydroxysterol Delta24-reductase, which catalyzes the synthesis of cholesterol from desmosterol. We have demonstrated previously that in fetal neuroepithelial cells, 17beta-estradiol (17betaE2), raloxifene, and tamoxifen exert neuroprotective effects and increase the expression of seladin-1. The aim of the present study was to elucidate whether seladin-1 is directly involved in estrogen-mediated neuroprotection. Using the small interfering RNA methodology, significantly reduced levels of seladin-1 mRNA and protein were obtained in fetal neuroepithelial cells. Seladin-1 silencing determined the loss of the protective effect of 17betaE2 against beta-amyloid and oxidative stress toxicity and caspase-3 activation. A computer-assisted analysis revealed the presence of half-palindromic estrogen responsive elements upstream from the coding region of the seladin-1 gene. A 1490-bp region was cloned in a luciferase reporter vector, which was transiently cotransfected with the estrogen receptor alpha in Chinese hamster ovarian cells. The exposure to 17betaE2, raloxifene, tamoxifen, and the soy isoflavones genistein and zearalenone increased luciferase activity, thus suggesting a functional role for the half-estrogen responsive elements of the seladin-1 gene. Our data provide for the first time a direct demonstration that seladin-1 may be considered a fundamental mediator of the neuroprotective effects of estrogen.

  16. Coexpression of luxA and luxB genes of Vibrio fischeri in NIH3T3 mammalian cells and evaluation of its bioluminescence activities.

    Science.gov (United States)

    Tehrani, Golnaz Asaadi; Mirzaahmadi, Sina; Bandehpour, Mojgan; Kazemi, Bahram

    2014-02-01

    Expression of bacterial luciferase enzyme (lux) in eukaryotic cells would provide a new bioreporter system for in vivo imaging and diagnostics technology. In spite of this, until now only a few efforts have been made to express bacterial luciferase enzyme in eukaryotic cells. We attempted to synthesize an expression construct of luxA and luxB genes from Vibrio fischeri. The luxA and luxB genes were cloned into the MCS of pTZ57R via the 5' kpnI, BamHI and BamHI, EcoRI restriction sites to generate pTZ57R/luxA and pTZ57R/luxB respectively, then newly synthesized constructs were cleaved with the same enzymes and respectively cloned into the pcDNA3.1(+) (Hyg) and pcDNA3.1(+) (Neo) expression vectors to create pcDNA3.1(+) (Hyg)/luxA and pcDNA3.1(+) (neo)/luxB. Recombinant constructs were cotransfected to the NIH3T3 cell line. Gene expression was confirmed by reverse transcription-polymerase chain reaction, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting; in addition, bioluminescence characteristics of transfected NIH3T3 cell lines were evaluated by decanal supplement. In conclusion, in the current research, separate vector systems were constructed, which are composed of bacterial luciferase genes (luxA and luxB) that accordingly have not already been reported. These results hold promise toward the potential development of an autonomous light-generating lux reporter system in eukaryotic cells. Copyright © 2013 John Wiley & Sons, Ltd.

  17. 1alpha,25-dehydroxyvitamin D3 synergism toward transforming growth factor-beta1-induced AP-1 transcriptional activity in mouse osteoblastic cells via its nuclear receptor.

    Science.gov (United States)

    Takeshita, A; Imai, K; Kato, S; Kitano, S; Hanazawa, S

    1998-06-12

    The present study demonstrates 1alpha,25-dehydroxyvitamin D3 (1alpha-25-(OH)2D3) synergism toward transforming growth factor (TGF)-beta1-induced activation protein-1 (AP-1) activity in mouse osteoblastic MC3T3-E1 cells via the nuclear receptor of the vitamin. 1alpha-25-(OH)2D3 synergistically stimulated TGF-beta1-induced expression of the c-jun gene in the cells but not that of the c-fos gene. We actually showed by a gel mobility shift assay 1alpha-25-(OH)2D3 synergism of TGF-beta1-induced AP-1 binding to the 12-(O-tetradecanoylphorbol-13-acetate response element (TRE). 1alpha-25-(OH)2D3 markedly stimulated the transient activity of TGF-beta1-induced AP-1 in the cells transfected with a TRE-chloramphenicol acetyltransferase (CAT) reporter gene. Also, a synergistic increase in TGF-beta1-induced CAT activity was observed in the cells cotransfected with an expression vector encoding vitamin D3 receptor (VDR) and the reporter gene. However, the synergistic CAT activity was inhibited by pretreatment with VDR antisense oligonucleotides. In addition, in a Northern blot assay, we observed 1alpha-25-(OH)2D3 synergism of TGF-beta1-induced expression of the c-jun gene in the cells transfected with the VDR expression vector and also found that the synergistic action was clearly blocked by VDR antisense oligonucleotide pretreatment. The present study strongly suggests a novel positive regulation by 1alpha-25-(OH)2D3 of TGF-beta1-induced AP-1 activity in osteoblasts via "genomic action."

  18. Inhibitory effects of two G protein-coupled receptor kinases on the cell surface expression and signaling of the human adrenomedullin receptor

    Energy Technology Data Exchange (ETDEWEB)

    Kuwasako, Kenji, E-mail: kuwasako@med.miyazaki-u.ac.jp [Frontier Science Research Center, University of Miyazaki, Miyazaki, 889-1692 (Japan); Sekiguchi, Toshio [Noto Marine Laboratory, Division of Marine Environmental Studies, Institute of Nature and Environmental Technology, Kanazawa University, Ishikawa, 927-0553 (Japan); Nagata, Sayaka [Division of Circulatory and Body Fluid Regulation, Faculty of Medicine, University of Miyazaki, Miyazaki, 889-1692 (Japan); Jiang, Danfeng; Hayashi, Hidetaka [Frontier Science Research Center, University of Miyazaki, Miyazaki, 889-1692 (Japan); Murakami, Manabu [Department of Pharmacology, Hirosaki University, Graduate School of Medicine, Hirosaki, 036-8562 (Japan); Hattori, Yuichi [Department of Molecular and Medical Pharmacology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, 930-0194 (Japan); Kitamura, Kazuo [Division of Circulatory and Body Fluid Regulation, Faculty of Medicine, University of Miyazaki, Miyazaki, 889-1692 (Japan); Kato, Johji [Frontier Science Research Center, University of Miyazaki, Miyazaki, 889-1692 (Japan)

    2016-02-19

    Receptor activity-modifying protein 2 (RAMP2) enables the calcitonin receptor-like receptor (CLR, a family B GPCR) to form the type 1 adrenomedullin receptor (AM{sub 1} receptor). Here, we investigated the effects of the five non-visual GPCR kinases (GRKs 2 through 6) on the cell surface expression of the human (h)AM{sub 1} receptor by cotransfecting each of these GRKs into HEK-293 cells that stably expressed hRAMP2. Flow cytometric analysis revealed that when coexpressed with GRK4 or GRK5, the cell surface expression of the AM{sub 1} receptor was markedly decreased prior to stimulation with AM, thereby attenuating both the specific [{sup 125}I]AM binding and AM-induced cAMP production. These inhibitory effects of both GRKs were abolished by the replacement of the cytoplasmic C-terminal tail (C-tail) of CLR with that of the calcitonin receptor (a family B GPCR) or β{sub 2}-adrenergic receptor (a family A GPCR). Among the sequentially truncated CLR C-tail mutants, those lacking the five residues 449–453 (Ser-Phe-Ser-Asn-Ser) abolished the inhibition of the cell surface expression of CLR via the overexpression of GRK4 or GRK5. Thus, we provided new insight into the function of GRKs in agonist-unstimulated GPCR trafficking using a recombinant AM{sub 1} receptor and further determined the region of the CLR C-tail responsible for this GRK function. - Highlights: • We discovered a novel function of GRKs in GPCR trafficking using human CLR/RAMP2. • GRKs 4 and 5 markedly inhibited the cell surface expression of human CLR/RAMP2. • Both GRKs exhibited highly significant receptor signaling inhibition. • Five residues of the C-terminal tail of CLR govern this function of GRKs.

  19. MiR-218 Inhibits Migration and Invasion of Lung Cancer Cell 
by Regulating Robo1 Expression

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    Ping CHEN

    2017-06-01

    Full Text Available Background and objective To explore the function and the potential molecular mechanism of miR-218 in lung cancer cell. Methods The expression of miR-218 mRNA was determined by real-time PCR in lung cancer tissues, adjacent tissues and lung cancer cells. Transwell assay was used to detect the migration and invasion of A549 cell after transfected with Anti-miR-218 or negative control and HC4006 cell after transfected with miR-218 mimics and miR-218 negative control. Targetscan and MiRanda were used to calculate the potential targets of miR-218 and Luciferase reporter assay was performed to identify that the Robo1 was one target genes of miR-218. Transwell assay was used to detect whether miR-218 regulated the invasion of lung cancer cell transfected with anti-miR-218 or negative control via Robo1. Results The expression of miR-218 in the lung cancer tissues was significantly lower than that in the adjacent tissues (P<0.05. Inhibition of miR-218 improved the migration and invasion of A549 cell. Overexpression of miR-218 suppressed the migration and invasion of HCC4006 cell. The co-transfection of anti-miR-218 or miR-218 mimics and the Robo1 3′UTR increased or reduced the luciferase activity of Robo1 compared with the control group (P<0.05. Inhibition of miR-218 and Robo1 recovered the invaded cells of A549. Overexpression of miR-218 and inhibition of Robo1 reduced the number of the invased cells of HCC4006. These results suggested that miR-218 banded Robo1 directly and inhibited lung cancer cell invasion by targeting Robo1. Conclusion MiR-218 inhibited the migration and invasion of lung cancer cells through regulating Robo1 expression.

  20. Stem Cells

    Science.gov (United States)

    Stem cells are cells with the potential to develop into many different types of cells in the body. ... the body. There are two main types of stem cells: embryonic stem cells and adult stem cells. Stem ...

  1. GPCR Interaction: 19 [GRIPDB[Archive

    Lifescience Database Archive (English)

    Full Text Available SY5Y cells results in coaggregation, cointernalization and codesensitization of t...orms hetero complexes. Long term exposure of the hetero complexes to their agonists in D2R-cotransfected SH-

  2. Induction of VMAT-1 and TPH-1 expression induces vesicular accumulation of serotonin and protects cells and tissue from cooling/rewarming injury.

    Directory of Open Access Journals (Sweden)

    Fatemeh Talaei

    Full Text Available DDT₁ MF-2 hamster ductus deferens cells are resistant to hypothermia due to serotonin secretion from secretory vesicles and subsequent cystathionine beta synthase (CBS mediated formation of H₂S. We investigated whether the mechanism promoting resistance to hypothermia may be translationally induced in cells vulnerable to cold storage. Thus, VMAT-1 (vesicular monoamino transferase and TPH-1 (tryptophan hydroxylase were co-transfected in rat aortic smooth muscle cells (SMAC and kidney tissue to create a serotonin-vesicular phenotype (named VTSMAC and VTkidney, respectively. Effects on hypothermic damage were assessed. VTSMAC showed a vesicular phenotype and an 8-fold increase in serotonin content and 5-fold increase in its release upon cooling. Cooled VTSMAC produced up to 10 fold higher concentrations of H₂S, and were protected from hypothermia, as shown by a 50% reduction of caspase 3/7 activity and 4 times higher survival compared to SMAC. Hypothermic resistance was abolished by the inhibition of CBS activity or blockade of serotonin re-uptake. In VTkidney slices, expression of CBS was 3 fold increased in cold preserved kidney tissue, with two-fold increase in H₂S concentration. While cooling induced substantial damage to empty vector transfected kidney as shown by caspase 3/7 activity and loss of FABP1, VTkidney was fully protected and comparable to non-cooled control. Thus, transfection of VMAT-1 and TPH-1 induced vesicular storage of serotonin which is triggered release upon cooling and has protective effects against hypothermia. The vesicular serotonergic phenotype protects against hypothermic damage through re-uptake of serotonin inducing CBS mediated H₂S production both in cells and kidney slices.

  3. Induction of VMAT-1 and TPH-1 expression induces vesicular accumulation of serotonin and protects cells and tissue from cooling/rewarming injury.

    Science.gov (United States)

    Talaei, Fatemeh; Schmidt, Martina; Henning, Robert H

    2012-01-01

    DDT₁ MF-2 hamster ductus deferens cells are resistant to hypothermia due to serotonin secretion from secretory vesicles and subsequent cystathionine beta synthase (CBS) mediated formation of H₂S. We investigated whether the mechanism promoting resistance to hypothermia may be translationally induced in cells vulnerable to cold storage. Thus, VMAT-1 (vesicular monoamino transferase) and TPH-1 (tryptophan hydroxylase) were co-transfected in rat aortic smooth muscle cells (SMAC) and kidney tissue to create a serotonin-vesicular phenotype (named VTSMAC and VTkidney, respectively). Effects on hypothermic damage were assessed. VTSMAC showed a vesicular phenotype and an 8-fold increase in serotonin content and 5-fold increase in its release upon cooling. Cooled VTSMAC produced up to 10 fold higher concentrations of H₂S, and were protected from hypothermia, as shown by a 50% reduction of caspase 3/7 activity and 4 times higher survival compared to SMAC. Hypothermic resistance was abolished by the inhibition of CBS activity or blockade of serotonin re-uptake. In VTkidney slices, expression of CBS was 3 fold increased in cold preserved kidney tissue, with two-fold increase in H₂S concentration. While cooling induced substantial damage to empty vector transfected kidney as shown by caspase 3/7 activity and loss of FABP1, VTkidney was fully protected and comparable to non-cooled control. Thus, transfection of VMAT-1 and TPH-1 induced vesicular storage of serotonin which is triggered release upon cooling and has protective effects against hypothermia. The vesicular serotonergic phenotype protects against hypothermic damage through re-uptake of serotonin inducing CBS mediated H₂S production both in cells and kidney slices.

  4. CRISPR/Cas9, a new approach to successful knockdown of ABCB1/P-glycoprotein and reversal of chemosensitivity in human epithelial ovarian cancer cell line

    Directory of Open Access Journals (Sweden)

    Leyla Norouzi-Barough

    2018-02-01

    Full Text Available Objective(s: Multidrug resistance (MDR is a major obstacle in the successful chemotherapy of ovarian cancer. Inhibition of P-glycoprotein (P-gp, a member of ATP-binding cassette (ABC transporters, is a well-known strategy to overcome MDR in cancer. The aim of this study was to investigate the efficiency and ability of CRISPR/Cas9 genome editing technology to knockdown ABCB1 gene expression in adriamycin resistant (A2780/ADR ovarian cancer cell line and evaluate the sensitivity changes to doxorubicin. Materials and Methods: Three single-guide RNAs (sgRNAs targeting the fourth and fifth exons of human ABCB1 gene were designed in this study. Expression level of ABCB1 was detected using quantitative real time PCR (qRT-PCR after co-transfection of all three sgRNAs into A2780/ADR cell line and subsequent antibiotic selection. Drug sensitivity to doxorubicin was determined by the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay. Results: The results showed that CRISPR/Cas9 system could significantly reduce the expression of P-gp. The dramatic decline in ABCB1 gene expression was associated with increased sensitivity of cells transfected with sgRNAs to doxorubicin. Conclusion: Based on the results of this study, it is concluded that the CRISPR-based systems, used in the present study, effectively down-regulated the target gene and acted as an ideal and cost-effective tool for gene editing of A2780/ADR cell line resulting in restoration of nonmalignant phenotype.

  5. Up-Regulation of CYP2C19 Expression by BuChang NaoXinTong via PXR Activation in HepG2 Cells.

    Directory of Open Access Journals (Sweden)

    Hong Sun

    Full Text Available Cytochrome P450 2C19 (CYP2C19 is an important drug-metabolizing enzyme (DME, which is responsible for the biotransformation of several kinds of drugs such as proton pump inhibitors, platelet aggregation inhibitors and antidepressants. Previous studies showed that Buchang NaoXinTong capsules (NXT increased the CYP2C19 metabolic activity in vitro and enhanced the antiplatelet effect of clopidogrel in vivo. However, the underlying molecular mechanism remained unclear. In the present study, we examined whether Pregnane X receptor (PXR plays a role in NXT-mediated regulation of CYP2C19 expression.We applied luciferase assays, real-time quantitative PCR (qPCR, Western blotting and cell-based analysis of metabolic activity experiments to investigate the NXT regulatory effects on the CYP2C19 promoter activity, the mRNA/ protein expression and the metabolic activity.Our results demonstrated that NXT significantly increased the CYP2C19 promoter activity when co-transfected with PXR in HepG2 cells. Mutations in PXR responsive element abolished the NXT inductive effects on the CYP2C19 promoter transcription. Additionally, NXT incubation (150 and 250μg/mL also markedly up-regulated endogenous CYP2C19 mRNA and protein levels in PXR-transfected HepG2 cells. Correspondingly, NXT leaded to a significant enhancement of the CYP2C19 catalytic activity in PXR-transfected HepG2 cells.In summary, this is the first study to suggest that NXT could induce CYP2C19 expression via PXR activation.

  6. Studying the Effect of Downregulating Autophagy-Related Gene LC3 on TLR3 Apoptotic Pathway Mediated by dsRNA in Hepatocellular Carcinoma Cells.

    Science.gov (United States)

    Wang, Guilan; Zhang, Maona; Li, Yunlong; Zhou, Jiaming; Chen, Li

    2017-01-01

    The purpose of this study is to examine the role of the double-stranded RNA (dsRNA) activated Toll-interleukin-1 receptor domain-containing adaptor inducing interferon β (TRIF) signal pathway in triggering apoptosis in hepatocellular carcinoma (HCC) cells. First, siRNA targeted autophagy-related gene LC3 (pU6H1-LC3 siRNA and siLC3) and a dsRNA used as a Toll-like receptor 3 (TLR3) ligand was constructed and synthesized, respectively. Then, a human HCC cell line was transfected with dsRNA, siLC3, and cotransfected with siLC3 and dsRNA (siLC3+dsRNA), respectively. Finally, quantification real-time polymerase chain reaction, western blotting, and immunofluorescence staining were used in the HCC line (SMMC7721), and MTT assay, flow cytometry, terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling, and transmission electron microscopy were used in an HCC xenograft model of nude mice. Human umbilical vein endothelial cell tube forming assay, color Doppler ultrasonographic flow image examination, and CD34-positive microvessel density were used in vitro and in vivo . Compared with untreated cells, the protein and mRNA expression of TLR3 and TRIF was up-regulated, in order, siLC3+dsRNA, dsRNA, and siLC3. Expression of LC3 was obviously down-regulated and the autophagosomes were significantly decreased in siLC3+dsRNA and siLC3, whereas in dsRNA (p protein, which can promote triggering of apoptosis by the TLR3-TRIF pathway. dsRNA and siLC3 could play anticancer roles in coordination.

  7. Effect of Surfactants on Plasmid DNA Stability and Release from ...

    African Journals Online (AJOL)

    ... diffusion mechanism was found to predominate in DNA release. Conclusion: The microspheres were non-toxic to the neuro-2a cells which suggest they can be potentially used in the gene therapy of neuronal diseases. Keywords: Surfactant, Gene therapy, Microspheres, Polylactic glycolide, Plasmid DNA, Supercoil index, ...

  8. Experiment list: SRX691798 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available SRX691798 mm9 TFs and others Epitope tags Neural Neuro-2a Tissue=Brain|Cell Type=Neuroblast|Disease=Neurobla...stoma 17719999,76.2,13.1,605 GSM1496578: N2A KRAB S1; Mus musculus; ChIP-Seq source

  9. Experiment list: SRX691800 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available SRX691800 mm9 TFs and others Epitope tags Neural Neuro-2a Tissue=Brain|Cell Type=Neuroblast|Disease=Neurobla...stoma 18890141,95.9,18.4,364 GSM1496580: N2A Control EV; Mus musculus; ChIP-Seq sou

  10. Experiment list: SRX691795 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available SRX691795 mm9 TFs and others Epitope tags Neural Neuro-2a Tissue=Brain|Cell Type=Neuroblast|Disease=Neurobla...stoma 11793765,69.6,12.7,161 GSM1496575: N2A noED S1 rep2; Mus musculus; ChIP-Seq s

  11. Experiment list: SRX691796 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available SRX691796 mm9 TFs and others Epitope tags Neural Neuro-2a Tissue=Brain|Cell Type=Neuroblast|Disease=Neurobla...stoma 12696256,79.9,26.4,263 GSM1496576: N2A noED S2 rep1; Mus musculus; ChIP-Seq s

  12. MicroRNA 203 Modulates Glioma Cell Migration via Robo1/ERK/MMP-9 Signaling.

    Science.gov (United States)

    Dontula, Ranadheer; Dinasarapu, Ashok; Chetty, Chandramu; Pannuru, Padmavathi; Herbert, Engelhard; Ozer, Howard; Lakka, Sajani S

    2013-07-01

    Glioblastoma (GBM) is the most common and malignant primary adult brain cancer. Allelic deletion on chromosome 14q plays an important role in the pathogenesis of GBM, and this site was thought to harbor multiple tumor suppressor genes associated with GBM, a region that also encodes microRNA-203 (miR-203). In this study, we sought to identify the role of miR-203 as a tumor suppressor in the pathogenesis of GBM. We analyzed the miR-203 expression data of GBM patients in 10 normal and 495 tumor tissue samples derived from The Cancer Genome Atlas data set. Quantitative real-time PCR and in situ hybridization in 10 high-grade GBM and 10 low-grade anaplastic astrocytoma tumor samples showed decreased levels of miR-203 expression in anaplastic astrocytoma and GBM tissues and cell lines. Exogenous expression of miR-203 using a plasmid expressing miR-203 precursor (pmiR-203) suppressed glioma cell proliferation, migration, and invasion. We determined that one relevant target of miR-203 was Robo1, given that miR-203 expression decreased mRNA and protein levels as determined by RT-PCR and Western blot analysis. Moreover, cotransfection experiments using a luciferase-based transcription reporter assay have shown direct regulation of Robo1 by miR-203. We also show that Robo1 mediates miR-203 mediated antimigratory functions as up-regulation of Robo1 abrogates miR-203 mediated antimigratory effects. We also show that miR-203 expression suppressed ERK phosphorylation and MMP-9 expression in glioma cells. Furthermore, we demonstrate that miR-203 inhibits migration of the glioma cells by disrupting the Robo1/ERK/MMP-9 signaling axis. Taken together, these studies demonstrate that up-regulation of Robo1 in response to the decrease in miR-203 in glioma cells is responsible for glioma tumor cell migration and invasion.

  13. GPCR Interaction: 25 [GRIPDB[Archive

    Lifescience Database Archive (English)

    Full Text Available e of A1R and P2Y1R colocalization was observed in cotransfected cells by double immunofluorescence experiments with confocal laser...2) and Myc-P2Y1R-Rluc was also observed in the co-transfected HEK293T cells by confocal laser microscopy. Th..., 12417330 Double immunofluorescence experiments, confocal laser microscopy Modified BRET NP_001041695.1 ...

  14. GPCR Interaction: 26 [GRIPDB[Archive

    Lifescience Database Archive (English)

    Full Text Available ree of A1R and P2Y1R colocalization was observed in cotransfected cells by double immunofluorescence experiments with confocal laser...P(2) and Myc-P2Y1R-Rluc was also observed in the co-transfected HEK293T cells by confocal laser microscopy. ...75, 12417330 Double immunofluorescence experiments, confocal laser microscopy Modified BRET NP_002554.1 ...

  15. Lichen-derived compounds show potential for central nervous system therapeutics.

    Science.gov (United States)

    Reddy, R Gajendra; Veeraval, Lenin; Maitra, Swati; Chollet-Krugler, Marylène; Tomasi, Sophie; Dévéhat, Françoise Lohézic-Le; Boustie, Joël; Chakravarty, Sumana

    2016-11-15

    Natural products from lichens are widely investigated for their biological properties, yet their potential as central nervous system (CNS) therapeutic agents is less explored. The present study investigated the neuroactive properties of selected lichen compounds (atranorin, perlatolic acid, physodic acid and usnic acid), for their neurotrophic, neurogenic and acetylcholine esterase (AChE) activities. Neurotrophic activity (neurite outgrowth) was determined using murine neuroblastoma Neuro2A cells. A MTT assay was performed to assess the cytotoxicity of compounds at optimum neurotrophic activity. Neuro2A cells treated with neurotrophic lichen compounds were used for RT-PCR to evaluate the induction of genes that code for the neurotrophic markers BDNF and NGF. Immunoblotting was used to assess acetyl H3 and H4 levels, the epigenetic markers associated with neurotrophic and/or neurogenic activity. The neurogenic property of the compounds was determined using murine hippocampal primary cultures. AChE inhibition activity was performed using a modified Ellman's esterase method. Lichen compounds atranorin, perlatolic acid, physodic acid and (+)-usnic acid showed neurotrophic activity in a preliminary cell-based screening based on Neuro2A neurite outgrowth. Except for usnic acid, no cytotoxic effects were observed for the two depsides (atranorin and perlatolic acid) and the alkyl depsidone (physodic acid). Perlatolic acid appears to be promising, as it also exhibited AChE inhibition activity and potent proneurogenic activity. The neurotrophic lichen compounds (atranorin, perlatolic acid, physodic acid) modulated the gene expression of BDNF and NGF. In addition, perlatolic acid showed increased protein levels of acetyl H3 and H4 in Neuro2A cells. These lichen depsides and depsidones showed neuroactive properties in vitro (Neuro2A cells) and ex vivo (primary neural stem or progenitor cells), suggesting their potential to treat CNS disorders. Copyright © 2016 Elsevier Gmb

  16. MicroRNA-223 Enhances Radiation Sensitivity of U87MG Cells In Vitro and In Vivo by Targeting Ataxia Telangiectasia Mutated

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Liping; Zhu, Ji [Departments of Radiation Oncology, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai (China); Zaorsky, Nicholas G. [Department of Radiation Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania (United States); Deng, Yun [Departments of Radiation Oncology, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai (China); Wu, Xingzhong [Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai (China); Liu, Yong [Departments of Radiation Oncology, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai (China); Liu, Fangqi; Cai, Guoxiang; Gu, Weilie [Department of Colorectal Cancer, Fudan University, Shanghai Cancer Center, Shanghai (China); Shen, Lijun [Departments of Radiation Oncology, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai (China); Zhang, Zhen, E-mail: zhenzhang6@hotmail.com [Departments of Radiation Oncology, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai (China)

    2014-03-15

    Purpose: Ataxia telangiectasia mutated (ATM) protein is important in the DNA damage response because it repairs radiation-induced damage in cancers. We examined the effect of microRNA-223 (miR-223), a regulator of ATM expression, on radiation sensitivity of cancer cells. Methods and Materials: Human embryonic kidney 293 T (293T) cells were infected with pLL3.7-miR-223 plasmid to generate the pLL3.7-miR-223 and -empty virus (EV) lentivirus (miR-223 and EV). A dual luciferase assay in which the reporter contained wild-type 3′ untranslated region (UTR) of ATM was performed. U87MG cells were infected with miR-223 or EV to establish the overexpressed stable cell lines (U87-223 or U87-EV, respectively). Cells were irradiated in vitro, and dose enhancement ratios at 2 Gy (DER{sub 2}) were calculated. Hind legs of BALB/c athymic mice were injected with U87-223 or U87-EV cells; after 2 weeks, half of the tumors were irradiated. Tumor volumes were tracked for a total of 5 weeks. Results: The dual luciferase reporter assay showed a significant reduction in luciferase activity of 293T cells cotransfected with miR-223 and the ATM 3′UTR compared to that in EV control. Overexpression of miR-223 in U87MG cells showed that ATM expression was significantly downregulated in the U87-223 cells compared to that in U87-EV (ATM/β-actin mRNA 1.0 vs 1.5, P<.05). U87-223 cells were hypersensitive to radiation compared to U87-EV cells in vitro (DER{sub 2} = 1.32, P<.01). Mice injected with miR-223-expressing tumors had almost the same tumors after 3 weeks (1.5 cm{sup 3} vs 1.7 cm{sup 3}). However, irradiation significantly decreased tumor size in miR-223-expressing tumors compared to those in controls (0.033 cm{sup 3} vs 0.829 cm{sup 3}). Conclusions: miR-223 overexpression downregulates ATM expression and sensitizes U87 cells to radiation in vitro and in vivo. MicroRNA-223 may be a novel cancer-targeting therapy, although its cancer- and patient-specific roles are

  17. Homeostatic regulatory role of Pokemon in NF-κB signaling: stimulating both p65 and IκBα expression in human hepatocellular carcinoma cells.

    Science.gov (United States)

    Zhang, Nan-Nan; Sun, Qin-Sheng; Chen, Zhe; Liu, Feng; Jiang, Yu-Yang

    2013-01-01

    NF-κB consists of p50, p65 (RelA), p52, c-Rel, and RelB, and among them p65 is a representative protein to investigate the regulation and function of this signaling. NF-κB integrates inflammation and carcinogenesis and regulates the expression of a variety of genes in response to immunity, inflammation, and apoptosis. IκBα acts as an inhibitor of NF-κB through forming an inactive NF-κB/IκBα complex. Pokemon is a ubiquitous transcription factor involved in different signaling pathways, playing a pivotal role in cell proliferation, anti-apoptosis, embryonic development, and maintenance. In this study, we found that p65 and IκBα are both novel regulatory targets of Pokemon. Ectopic expression of Pokemon in immortalized liver cells HL7702 enhanced p65 and IκBα expression, whereas silencing of Pokemon in hepatocellular carcinoma cells QGY7703 reduced cellular p65 levels. ChIP assay and targeted mutagenesis revealed that Pokemon directly binds to the element of -434 to -430 bp in p65 promoter and of -453 to -448 bp in IκBα promoter and stimulates luciferase reporter gene expression. Co-transfection of Pokemon with p65 or IκBα promoter-reporter notably enhanced their promoter activity. These data suggest that Pokemon activates the expression of both p65 and IκBα by sequence-specific binding to their promoters and plays a dual role in regulating NF-κB signaling.

  18. Homeodomain-interacting protein kinase 2, a novel autoimmune regulator interaction partner, modulates promiscuous gene expression in medullary thymic epithelial cells.

    Science.gov (United States)

    Rattay, Kristin; Claude, Janine; Rezavandy, Esmail; Matt, Sonja; Hofmann, Thomas G; Kyewski, Bruno; Derbinski, Jens

    2015-02-01

    Promiscuous expression of a plethora of tissue-restricted Ags (TRAs) by medullary thymic epithelial cells (mTECs) plays an essential role in T cell tolerance. Although the cellular mechanisms by which promiscuous gene expression (pGE) imposes T cell tolerance have been well characterized, the underlying molecular mechanisms remain poorly understood. The autoimmune regulator (AIRE) is to date the only validated molecule known to regulate pGE. AIRE is part of higher-order multiprotein complexes, which promote transcription, elongation, and splicing of a wide range of target genes. How AIRE and its partners mediate these various effects at the molecular level is still largely unclear. Using a yeast two-hybrid screen, we searched for novel AIRE-interacting proteins and identified the homeodomain-interacting protein kinase 2 (HIPK2) as a novel partner. HIPK2 partially colocalized with AIRE in nuclear bodies upon cotransfection and in human mTECs in situ. Moreover, HIPK2 phosphorylated AIRE in vitro and suppressed the coactivator activity of AIRE in a kinase-dependent manner. To evaluate the role of Hipk2 in modulating the function of AIRE in vivo, we compared whole-genome gene signatures of purified mTEC subsets from TEC-specific Hipk2 knockout mice with control mice and identified a small set of differentially expressed genes. Unexpectedly, most differentially expressed genes were confined to the CD80(lo) mTEC subset and preferentially included AIRE-independent TRAs. Thus, although it modulates gene expression in mTECs and in addition affects the size of the medullary compartment, TEC-specific HIPK2 deletion only mildly affects AIRE-directed pGE in vivo. Copyright © 2015 by The American Association of Immunologists, Inc.

  19. Directed differentiation of patient-specific induced pluripotent stem cells identifies the transcriptional repression and epigenetic modification of NKX2-5, HAND1, and NOTCH1 in hypoplastic left heart syndrome.

    Directory of Open Access Journals (Sweden)

    Junko Kobayashi

    Full Text Available The genetic basis of hypoplastic left heart syndrome (HLHS remains unknown, and the lack of animal models to reconstitute the cardiac maldevelopment has hampered the study of this disease. This study investigated the altered control of transcriptional and epigenetic programs that may affect the development of HLHS by using disease-specific induced pluripotent stem (iPS cells. Cardiac progenitor cells (CPCs were isolated from patients with congenital heart diseases to generate patient-specific iPS cells. Comparative gene expression analysis of HLHS- and biventricle (BV heart-derived iPS cells was performed to dissect the complex genetic circuits that may promote the disease phenotype. Both HLHS- and BV heart-derived CPCs were reprogrammed to generate disease-specific iPS cells, which showed characteristic human embryonic stem cell signatures, expressed pluripotency markers, and could give rise to cardiomyocytes. However, HLHS-iPS cells exhibited lower cardiomyogenic differentiation potential than BV-iPS cells. Quantitative gene expression analysis demonstrated that HLHS-derived iPS cells showed transcriptional repression of NKX2-5, reduced levels of TBX2 and NOTCH/HEY signaling, and inhibited HAND1/2 transcripts compared with control cells. Although both HLHS-derived CPCs and iPS cells showed reduced SRE and TNNT2 transcriptional activation compared with BV-derived cells, co-transfection of NKX2-5, HAND1, and NOTCH1 into HLHS-derived cells resulted in synergistic restoration of these promoters activation. Notably, gain- and loss-of-function studies revealed that NKX2-5 had a predominant impact on NPPA transcriptional activation. Moreover, differentiated HLHS-derived iPS cells showed reduced H3K4 dimethylation as well as histone H3 acetylation but increased H3K27 trimethylation to inhibit transcriptional activation on the NKX2-5 promoter. These findings suggest that patient-specific iPS cells may provide molecular insights into complex

  20. Effects of RNA interference-mediated NRP-1 silencing on the proliferation and apoptosis of breast cancer cells.

    Science.gov (United States)

    Han, Zhengxiang; Jiang, Guan; Zhang, Yingying; Xu, Jie; Chen, Chong; Zhang, Lansheng; Xu, Zhenyuan; Du, Xiuping

    2015-07-01

    Lentiviral expression vectors carrying human NRP-1 short hairpin RNA (shRNA) were constructed and selected to present highly efficient NRP-1/shRNA interference sequences, in order to investigate the effects of RNA interference (RNAi)-mediated NRP-1 silencing on the biological activities of breast cancer cells. Three pairs of human NRP-1 targeted specific interference sequences and one pair of non-specific control sequences were designed, synthesized and subcloned into pLB lentiviral vectors, which were further identified by polymerase chain reaction (PCR) and sequencing. Recombinant and lentiviral packaging plasmids were co-transfected into 293FT cell lines in order to produce lentiviral particles and to infect breast cancer cells with high NRP-1 expression. Flow cytometry was used to sort green fluorescent protein-positive cells. Fluorescence quantitative-reverse transcription-PCR and western blot analysis were employed to identify the interference silencing sequence with the most efficient silencing profile. A cell counting kit-8 assay and an Annexin V-propidium iodide method in combination with flow cytometry were used to examine the effects of RNA interference-mediated NRP-1 gene silencing on cell proliferation, apoptosis and sensitivity to chemotherapy. The recombinant lentiviral plasmid pLB-NRP-1/shRNA was constructed successfully, as confirmed by PCR and sequencing. After the infection of recombinant lentiviral plasmids, the expression profiles of NRP-1 mRNA, and proteins of MCF-7 and SK-BR-3 cell-specific interference group (pLB-NRP-1/shRNA3) were significantly lower than that of the control group (Pinterference group (pLB-NRP-1/shRNA3) showed lower optical density values and higher apoptotic rates at 48, 72 and 96 h; these differences were statistically significant (Pinterference groups compared with the control group (Pinterference sequences were selected. Furthermore, RNA interference (RNAi)-mediated NRP-1 silencing may induce proliferation suppression

  1. Inhibition of hepatitis B virus and induction of hepatoma cell apoptosis by ASGPR-directed delivery of shRNAs.

    Directory of Open Access Journals (Sweden)

    Jingwei Ma

    Full Text Available Hepatitis B virus (HBV infection is a worldwide liver disease and nearly 25% of chronic HBV infections terminate in hepatocellular carcinoma (HCC. Currently, there is no effective therapy to inhibit HBV replication and to eliminate hepatoma cells, making it highly desired to develop novel therapies for these two stages of the HBV-caused detrimental disease. Recently, short hairpin RNA (shRNA has emerged as a potential therapy for virus-infected disease and cancer. Here, we have generated a shRNA, pGenesil-siHBV4, which effectively inhibits HBV replication in the human hepatoma cell line HepG2.2.15. The inhibitory effects of pGenesil-siHBV4 are manifested by the decrease of both the HBV mRNA level and the protein levels of the secreted HBV surface antigen (HBsAg and HBV e antigen (HBeAg, and by the reduction of secreted HBV DNA. Using mouse hydrodynamic tail vein injection, we demonstrate that pGenesil-siHBV4 is effective in inhibiting HBV replication in vivo. Because survivin plays a key role in cancer cell escape from apoptosis, we further generated pGenesil-siSurvivin, a survivin-silencing shRNA, and showed its effect of triggering apoptosis of HBV-containing hepatoma cells. To develop targeted shRNA therapy, we have identified that as a specific binder of the asialoglycoprotein receptor (ASGPR, jetPEI-Hepatocyte delivers pGenesil-siHBV4 and pGenesil-siSurvivin specifically to hepatocytes, not other types of cells. Finally, co-transfection of pGenesil-siHBV4 and pGenesil-siSurvivin exerts synergistic effects in inducing hepatoma cell apoptosis, a novel approach to eliminate hepatoma by downregulating survivin via multiple mechanisms. The application of these novel shRNAs with the jetPEI-Hepatocyte targeting strategy demonstrates the proof-of-principle for a promising approach to inhibit HBV replication and eliminate hepatoma cells with high specificity.

  2. MicroRNA-184 inhibits neuroblastoma cell survival through targeting the serine/threonine kinase AKT2.

    Science.gov (United States)

    Foley, Niamh H; Bray, Isabella M; Tivnan, Amanda; Bryan, Kenneth; Murphy, Derek M; Buckley, Patrick G; Ryan, Jacqueline; O'Meara, Anne; O'Sullivan, Maureen; Stallings, Raymond L

    2010-04-21

    Neuroblastoma is a paediatric cancer of the sympathetic nervous system. The single most important genetic indicator of poor clinical outcome is amplification of the MYCN transcription factor. One of many down-stream MYCN targets is miR-184, which is either directly or indirectly repressed by this transcription factor, possibly due to its pro-apoptotic effects when ectopically over-expressed in neuroblastoma cells. The purpose of this study was to elucidate the molecular mechanism by which miR-184 conveys pro-apoptotic effects. We demonstrate that the knock-down of endogenous miR-184 has the opposite effect of ectopic up-regulation, leading to enhanced neuroblastoma cell numbers. As a mechanism of how miR-184 causes apoptosis when over-expressed, and increased cell numbers when inhibited, we demonstrate direct targeting and degradation of AKT2, a major downstream effector of the phosphatidylinositol 3-kinase (PI3K) pathway, one of the most potent pro-survival pathways in cancer. The pro-apoptotic effects of miR-184 ectopic over-expression in neuroblastoma cell lines is reproduced by siRNA inhibition of AKT2, while a positive effect on cell numbers similar to that obtained by the knock-down of endogenous miR-184 can be achieved by ectopic up-regulation of AKT2. Moreover, co-transfection of miR-184 with an AKT2 expression vector lacking the miR-184 target site in the 3'UTR rescues cells from the pro-apoptotic effects of miR-184. MYCN contributes to tumorigenesis, in part, by repressing miR-184, leading to increased levels of AKT2, a direct target of miR-184. Thus, two important genes with positive effects on cell growth and survival, MYCN and AKT2, can be linked into a common genetic pathway through the actions of miR-184. As an inhibitor of AKT2, miR-184 could be of potential benefit in miRNA mediated therapeutics of MYCN amplified neuroblastoma and other forms of cancer.

  3. MicroRNA-184 inhibits neuroblastoma cell survival through targeting the serine/threonine kinase AKT2

    Directory of Open Access Journals (Sweden)

    Murphy Derek M

    2010-04-01

    Full Text Available Abstract Background Neuroblastoma is a paediatric cancer of the sympathetic nervous system. The single most important genetic indicator of poor clinical outcome is amplification of the MYCN transcription factor. One of many down-stream MYCN targets is miR-184, which is either directly or indirectly repressed by this transcription factor, possibly due to its pro-apoptotic effects when ectopically over-expressed in neuroblastoma cells. The purpose of this study was to elucidate the molecular mechanism by which miR-184 conveys pro-apoptotic effects. Results We demonstrate that the knock-down of endogenous miR-184 has the opposite effect of ectopic up-regulation, leading to enhanced neuroblastoma cell numbers. As a mechanism of how miR-184 causes apoptosis when over-expressed, and increased cell numbers when inhibited, we demonstrate direct targeting and degradation of AKT2, a major downstream effector of the phosphatidylinositol 3-kinase (PI3K pathway, one of the most potent pro-survival pathways in cancer. The pro-apoptotic effects of miR-184 ectopic over-expression in neuroblastoma cell lines is reproduced by siRNA inhibition of AKT2, while a positive effect on cell numbers similar to that obtained by the knock-down of endogenous miR-184 can be achieved by ectopic up-regulation of AKT2. Moreover, co-transfection of miR-184 with an AKT2 expression vector lacking the miR-184 target site in the 3'UTR rescues cells from the pro-apoptotic effects of miR-184. Conclusions MYCN contributes to tumorigenesis, in part, by repressing miR-184, leading to increased levels of AKT2, a direct target of miR-184. Thus, two important genes with positive effects on cell growth and survival, MYCN and AKT2, can be linked into a common genetic pathway through the actions of miR-184. As an inhibitor of AKT2, miR-184 could be of potential benefit in miRNA mediated therapeutics of MYCN amplified neuroblastoma and other forms of cancer.

  4. In vivo trafficking and immunostimulatory potential of an intranasally-administered primary dendritic cell-based vaccine

    Directory of Open Access Journals (Sweden)

    Shankar Nathan

    2010-12-01

    Full Text Available Abstract Background Coccidioidomycosis or Valley fever is caused by a highly virulent fungal pathogen: Coccidioides posadasii or immitis. Vaccine development against Coccidioides is of contemporary interest because a large number of relapses and clinical failures are reported with antifungal agents. An efficient Th1 response engenders protection. Thus, we have focused on developing a dendritic cell (DC-based vaccine for coccidioidomycosis. In this study, we investigated the immunostimulatory characteristics of an intranasal primary DC-vaccine in BALB/c mouse strain that is most susceptible to coccidioidomycosis. The DCs were transfected nonvirally with Coccidioides-Ag2/PRA-cDNA. Expression of DC-markers, Ag2/PRA and cytokines were studied by flow cytometry, dot-immunoblotting and cytometric bead array methods, respectively. The T cell activation was studied by assessing the upregulation of activation markers in a DC-T cell co-culture assay. For trafficking, the DCs were co-transfected with a plasmid DNA encoding HSV1 thymidine kinase (TK and administered intranasally into syngeneic mice. The trafficking and homing of TK-expressing DCs were monitored with positron emission tomography (PET using 18F-FIAU probe. Based on the PET-probe accumulation in vaccinated mice, selected tissues were studied for antigen-specific response and T cell phenotypes using ELISPOT and flow cytometry, respectively. Results We found that the primary DCs transfected with Coccidioides-Ag2/PRA-cDNA were of immature immunophenotype, expressed Ag2/PRA and activated naïve T cells. In PET images and subsequent biodistribution, intranasally-administered DCs were found to migrate in blood, lung and thymus; lymphocytes showed generation of T effector memory cell population (TEM and IFN-γ release. Conclusions In conclusion, our results demonstrate that the intranasally-administered primary DC vaccine is capable of inducing Ag2/PRA-specific T cell response. Unique approaches

  5. MicroRNA-98 and microRNA-214 post-transcriptionally regulate enhancer of zeste homolog 2 and inhibit migration and invasion in human esophageal squamous cell carcinoma

    Directory of Open Access Journals (Sweden)

    Huang Sheng-Dong

    2012-08-01

    Full Text Available Abstract Background The enhancer of zeste homolog 2 (EZH2 was found to be overexpressed and associated with tumor metastasis in esophageal squamous cell carcinoma (ESCC. On the other hand, it was reported that miR-26a, miR-98, miR-101, miR-124, miR-138 and miR-214 could inhibit the expression of EZH2 in some tumors. However, the role of miRNAs in the regulation of EZH2 expression in human ESCC has not been documented. The aim of this study was to determine the role of these miRNAs in the regulation of tumor metastasis via EZH2 overexpression in human ESCC. Methods and results The expression of these miRNAs and EZH2 mRNA were examined by qPCR and the expression of EZH2 protein was detected by western blot. The role of these miRNAs in migration and invasion was studied in ESCC cell line (Eca109 transfected with miRNA mimics or cotransfected with miRNA mimics and pcDNA-EZH2 plasmid (without the 3’-UTR of EZH2. Through clinical investigation, we found that miR-98 and miR-214 expression was significantly lower in ESCC tissues than in matched normal tissues, and the expression level of miR-98 and miR-214 was inversely correlated to EZH2 protein expression and the clinical features such as pathological grade, tumor stage and lymph node metastasis in ESCC. In Eca109 cells, overexpression of miR-98 and miR-214 significantly inhibited the migration and invasion of ESCC cells, which was reversed by transfection of EZH2. Conclusions These findings suggest that decreased expression of miR-98 and miR-214 might promote metastasis of human ESCC by inducing accumulation of EZH2 protein.

  6. MiR-143 and miR-135 inhibitors treatment induces skeletal myogenic differentiation of human adult dental pulp stem cells.

    Science.gov (United States)

    Li, Dongxia; Deng, Tianzheng; Li, Hongshi; Li, Ying

    2015-11-01

    Dental pulp stem cells (DPSCs) possess pluripotent properties that allow them to differentiate into multiple cell lineages, which can be potentially used in tissue regeneration. The aim of this in vitro study is to explore the effect of miRNAs on the myogenic differentiation of human adult DPSCs and seek for some potential biological factors for stable and feasible application in DPSC myogenic differentiation. Human adult DPSCs were isolated from normal impacted third molars were treated with 5-Aza-2'-deoxycytidine to induce to myogenic differentiation in vitro. During this process the levels of myomiRNAs and myogenic marker genes were detected by real-time qPCR and Western blotting. Then antisense oligonucleotides of miR-143 and miR-135 were transfected into DPSCs to explore their effects on myogenic differentiation. Gene expression detection and MyHC immunofluorescence microscopy analysis were applied to characterize the myogenic differentiation of DPSCs. Expression of miR-135 and miR-143 was markedly decreased in myoblast DPSCs induced by 5-Aza. Part of the DPSCs treated with miR-135 or miR-143 inhibitors showed apparent myocytic properties and eventually fused to form myotubes. Co-transfection of miR-135 and miR-143 inhibitors impelled half of DPSCs to form myotubes. MiR-135 and miR-143 inhibitors could induce myogenic differentiation of DPSCs. Our findings indicated that miRNAs could exert a decisive function in induction of myogenic differentiation of DPSCs. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Depletion of human micro-RNA miR-125b reveals that it is critical for the proliferation of differentiated cells but not for the down-regulation of putative targets during differentiation.

    Science.gov (United States)

    Lee, Yong Sun; Kim, Hak Kyun; Chung, Sangmi; Kim, Kwang-Soo; Dutta, Anindya

    2005-04-29

    Micro-RNAs are small non-coding RNAs that regulate target gene expression post-transcriptionally through base pairing with the target messenger RNA. Functional characterization of micro-RNAs awaits robust experimental methods to knock-down a micro-RNA as well as to assay its function in vivo. In addition to the recently developed method to sequester micro-RNA with 2'-O-methyl antisense oligonucleotide, we report that small interfering RNA against the loop region of a micro-RNA precursor can be used to deplete the micro-RNA. The depletion of miR-125b by this method had a profound effect on the proliferation of adult differentiated cancer cells, and this proliferation defect was rescued by co-transfected mature micro-RNA. This technique has unique advantages over the 2'-O-methyl antisense oligonucleotide and can be used to determine micro-RNA function, assay micro-RNAs in vivo, and identify the contribution of a predicted micro-RNA precursor to the pool of mature micro-RNA in a given cell. miR-125b and let-7 micro-RNAs are induced, whereas their putative targets, lin-28 and lin-41, are decreased during in vitro differentiation of Tera-2 or embryonic stem cells. Experimental increase or decrease of micro-RNA concentrations did not, however, affect the levels of the targets, a finding that is explained by the fact that the down-regulation of the targets appears to be mostly at the transcriptional level in these in vitro differentiation systems. Collectively these results reveal the importance of micro-RNA depletion strategies for directly determining micro-RNA function in vivo.

  8. Human FasL gene is a target of β-catenin/T-cell factor pathway and complex FasL haplotypes alter promoter functions.

    Directory of Open Access Journals (Sweden)

    Jianming Wu

    Full Text Available FasL expression on human immune cells and cancer cells plays important roles in immune homeostasis and in cancer development. Our previous study suggests that polymorphisms in the FasL promoter can significantly affect the gene expression in human cells. In addition to the functional FasL SNP -844C>T (rs763110, three other SNPs (SNP -756A>G or rs2021837, SNP -478A>T or rs41309790, and SNP -205 C>G or rs74124371 exist in the proximal FasL promoter. In the current study, we established three major FasL hyplotypes in humans. Interestingly, a transcription motif search revealed that the FasL promoter possessed two consensus T-cell factor (TCF/LEF1 binding elements (TBEs, which is either polymorphic (SNP -205C>G or close to the functional SNP -844C>T. Subsequently, we demonstrate that both FasL TBEs formed complexes with the TCF-4 and β-catenin transcription factors in vitro and in vivo. Co-transfection of LEF-1 and β-catenin transcription factors significantly increased FasL promoter activities, suggesting that FasL is a target gene of the β-catenin/T-cell factor pathway. More importantly, we found that the rare allele (-205G of the polymorphic FasL TBE (SNP -205C>G failed to bind the TCF-4 transcription factor and that SNP -205 C>G significantly affected the promoter activity. Furthermore, promoter reporter assays revealed that FasL SNP haplotypes influenced promoter activities in human colon cancer cells and in human T cells. Finally, β-catenin knockdown significantly decreased the FasL expression in human SW480 colon cancer cells. Collectively, our data suggest that β-catenin may be involved in FasL gene regulation and that FasL expression is influenced by FasL SNP haplotypes, which may have significant implications in immune response and tumorigenesis.

  9. Coffee and caffeine potentiate the antiamyloidogenic activity of melatonin via inhibition of Aβ oligomerization and modulation of the Tau-mediated pathway in N2a/APP cells

    Directory of Open Access Journals (Sweden)

    Zhang LF

    2014-12-01

    Full Text Available Li-Fang Zhang,1,2 Zhi-Wei Zhou,2 Zhen-Hai Wang,1 Yan-Hui Du,1 Zhi-Xu He,3 Chuanhai Cao,2 Shu-Feng Zhou2,31Department of Neurology, General Hospital of Ningxia Medical University, Yinchuan, Ningxia, People’s Republic of China; 2Department of Pharmaceutical Science, College of Pharmacy, University of South Florida, Tampa, FL, USA; 3Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guiyang Medical University, Guiyang, Guizhou, People’s Republic of ChinaAbstract: There is an increasing prevalence of Alzheimer’s disease (AD, which has become a public health issue. However, the underlying mechanisms for the pathogenesis of AD are not fully understood, and the current therapeutic drugs cannot produce acceptable efficacy in AD patients. Previous animal studies have shown that coffee (Coff, caffeine (Caff, and melatonin (Mel have beneficial effects on AD. Disturbed circadian rhythms are observed in AD, and chronotherapy has shown promising effects on AD. In this study, we examined whether a combination of Coff or Caff plus Mel produced a synergistic/additive effect on amyloid-ß (Aß generation in Neuro-2a (N2a/amyloid precursor protein (APP cells and the possible mechanisms involved. Cells were treated with Coff or Caff, with or without combined Mel, with three different chronological regimens. In regimen 1, cells were treated with Coff or Caff for 12 hours in the day, followed by Mel for 12 hours in the night. For regimen 2, cells were treated with Coff or Caff plus Mel for 24 hours, from 7 am to 7 am the next day. In regimen 3, cells were treated with Coff or Caff plus Mel with regimen 1 or 2 for 5 consecutive days. The extracellular Aβ40/42 and Aβ oligomer levels were determined using enzyme-linked immunosorbent assay (ELISA kits. The expression and/or phosphorylation levels of glycogen synthase kinase 3β (GSK3β, Erk1/2, PI3K

  10. Coffee and caffeine potentiate the antiamyloidogenic activity of melatonin via inhibition of Aβ oligomerization and modulation of the Tau-mediated pathway in N2a/APP cells.

    Science.gov (United States)

    Zhang, Li-Fang; Zhou, Zhi-Wei; Wang, Zhen-Hai; Du, Yan-Hui; He, Zhi-Xu; Cao, Chuanhai; Zhou, Shu-Feng

    2015-01-01

    There is an increasing prevalence of Alzheimer's disease (AD), which has become a public health issue. However, the underlying mechanisms for the pathogenesis of AD are not fully understood, and the current therapeutic drugs cannot produce acceptable efficacy in AD patients. Previous animal studies have shown that coffee (Coff), caffeine (Caff), and melatonin (Mel) have beneficial effects on AD. Disturbed circadian rhythms are observed in AD, and chronotherapy has shown promising effects on AD. In this study, we examined whether a combination of Coff or Caff plus Mel produced a synergistic/additive effect on amyloid-β (Aβ) generation in Neuro-2a (N2a)/amyloid precursor protein (APP) cells and the possible mechanisms involved. Cells were treated with Coff or Caff, with or without combined Mel, with three different chronological regimens. In regimen 1, cells were treated with Coff or Caff for 12 hours in the day, followed by Mel for 12 hours in the night. For regimen 2, cells were treated with Coff or Caff plus Mel for 24 hours, from 7 am to 7 am the next day. In regimen 3, cells were treated with Coff or Caff plus Mel with regimen 1 or 2 for 5 consecutive days. The extracellular Aβ40/42 and Aβ oligomer levels were determined using enzyme-linked immunosorbent assay (ELISA) kits. The expression and/or phosphorylation levels of glycogen synthase kinase 3β (GSK3β), Erk1/2, PI3K, Akt, Tau, Wnt3α, β-catenin, and Nrf2 were detected by Western blot assay. The results showed that regimen 1 produced an additive antiamyloidogenic effect with significantly reduced extracellular levels of Aβ40/42 and Aβ42 oligomers. Regimen 2 did not result in remarkable effects, and regimen 3 showed a less antiamyloidogenic effect compared to regimen 1. Coff or Caff, plus Mel reduced oxidative stress in N2a/APP cells via the Nrf2 pathway. Coff or Caff, plus Mel inhibited GSK3β, Akt, PI3K p55, and Tau phosphorylation but enhanced PI3K p85 and Erk1/2 phosphorylation in N2a/APP cells

  11. [shRNAs targeting high mobility group box-1 lead to inhibition of E-selectin expression via homeobox A9 in human umbilical vein endothelial cells].

    Science.gov (United States)

    Zhang, Xiaojuan; Jiao, Lili; Luan, Zhenggang; Ma, Xiaochun

    2015-08-01

    To approach the regulatory mechanism of high mobility group box-1 ( HMGB1 ) on the expression of E-selectin in human umbilical vein endothelial cell ( HUVEC ). Homeobox A9 ( HOXA9 ) siRNA was transfected to HUVEC at logarithmic phase, real-time fluorescence quantitative polymerase chain reaction ( real-time qPCR ) and Western Blot were used to determine the HOXA9 mRNA expression and protein expressions; a blank control group and a nonsilence negative control group were set. HUVEC stable transfected with pRNA-u6.1/Neo-HMGB1 shRNA plasmids ( HUVEC with low-expression HMGB1 ) was obtained, and HOXA9 and E-selectin mRNA expressions were determined with real-time qPCR; a nonsilence transfection group served as the negative control. The HOXA9 siRNA was transfected to HUVEC with low-expression HMGB1 as co-transfection group, and the E-selectin expressions was determined with real-time qPCR; a HMGB1 shRNA group and a HOXA9 nonsilence group served as control. (1) HOXA9 mRNA ( 2(-Δ ΔCT) ) and protein expression ( integral A value ) in blank control group were 1.094±0.115 and 1.031±0.060. Compared with nonsilence transfection group, HOXA9 siRNA transfection group could significantly reduced mRNA and protein expression of HOXA9 [ HOXA9 mRNA ( 2(-Δ ΔCT) ): 0.257±0.030 vs. 1.035±0.091, t = 14.010, P = 0.002; HOXA9 protein ( integral A value ): 0.278±0.042 vs. 0.975±0.014, t = 27.310, P = 0.002 ]. (2) Compared with nonsilence transfection group, HMGB1 shRNA transfection could up-regulate HOXA9 mRNA expression in HUVEC ( 2(-Δ ΔCT) : 2.519±0.278 vs. 0.856±0.063, t = 10.100, P = 0.001 ), also could down-regulate E-selectin mRNA expression ( 0.311±0.046 vs. 1.080±0.201, t = 7.415, P = 0.000 ). (3) Compared with HOXA9 nonsilence group and HMGB1 shRNA group, HMGB1 shRNA and HOXA9 siRNA co-transfected HUVEC cells could significantly elevate E-selectin mRNA expression ( 2(-Δ ΔCT) : 3.445±0.428 vs. 1.085±0.212, 1.004±0.104, t(1) = 8.507, t(2) = 9.603, both P < 0

  12. Cell Phones

    Science.gov (United States)

    ... Procedures Home, Business, and Entertainment Products Cell Phones Cell Phones Share Tweet Linkedin Pin it More sharing options ... safety of radiation-emitting consumer products such as cell phones and similar wireless devices before they can be ...

  13. Cell microencapsulation.

    Science.gov (United States)

    Lim, Grace J; Zare, Shirin; Van Dyke, Mark; Atala, Anthony

    2010-01-01

    In the past several decades, many attempts have been made to prevent the rejection of transplanted cells by the immune system. Cell encapsulation is primary machinery for cell transplantation and new materials and approaches were developed to encapsulate various types of cells to treat a wide range of diseases. This technology involves placing the transplanted cells within a biocompatible membrane in attempt to isolate the cells from the host immune attack and enhance or prolong their function in vivo. In this chapter, we will review the situation of cell microencapsulation field and discuss its potentials and challenges for cell therapy and regeneration of tissue function.

  14. Pregnane and Xenobiotic Receptor gene expression in liver cells is modulated by Ets-1 in synchrony with transcription factors Pax5, LEF-1 and c-jun

    Energy Technology Data Exchange (ETDEWEB)

    Kumari, Sangeeta; Saradhi, Mallampati; Rana, Manjul; Chatterjee, Swagata [Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi 110067 (India); Aumercier, Marc [IRI, CNRS USR 3078, Université de Lille-Nord de France, Parc CNRS de la Haute Borne, 50 Avenue de Halley, BP 70478, 59658 Villeneuve d’Ascq Cedex (France); Mukhopadhyay, Gauranga [Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi 110067 (India); Tyagi, Rakesh K., E-mail: rktyagi@yahoo.com [Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi 110067 (India)

    2015-01-15

    Nuclear receptor PXR is predominantly expressed in liver and intestine. Expression of PXR is observed to be dysregulated in various metabolic disorders indicating its involvement in disease development. However, information available on mechanisms of PXR self-regulation is fragmentary. The present investigation identifies some of the regulatory elements responsible for its tight regulation and low cellular expression. Here, we report that the PXR-promoter is a target for some key transcription factors like PU.1/Ets-1, Pax5, LEF-1 and c-Jun. Interestingly, we observed that PXR-promoter responsiveness to Pax5, LEF-1 and c-Jun, is considerably enhanced by Ets transcription factors (PU.1 and Ets-1). Co-transfection of cells with Ets-1, LEF-1 and c-Jun increased PXR-promoter activity by 5-fold and also induced expression of endogenous human PXR. Site-directed mutagenesis and transfection studies revealed that two Ets binding sites and two of the three LEF binding sites in the PXR-promoter are functional and have a positive effect on PXR transcription. Results suggest that expression of Ets family members, in conjunction with Pax5, LEF-1 and c-Jun, lead to coordinated up-regulation of PXR gene transcription. Insights obtained on the regulation of PXR gene have relevance in offering important cues towards normal functioning as well as development of several metabolic disorders via PXR signaling. - Highlights: • The study identified cis-regulatory elements in the nuclear receptor PXR promoter. • Several trans-acting factors modulating the PXR-promoter have been identified. • PU.1/Ets-1, Pax5, LEF-1, c-Jun, LyF-VI and NF-1 act as modulators of the PXR-promoter. • Ets-1 in conjunction with LEF-1 and c-Jun exhibit 5-fold activation of the PXR-promoter. • Insights into PXR-regulation have relevance in normal and pathological conditions.

  15. Nucleotide sequences of Herpes Simplex Virus type 1 (HSV-1) affecting virus entry, cell fusion, and production of glycoprotein gB (VP7)

    Energy Technology Data Exchange (ETDEWEB)

    DeLuca, N.; Bzik, D.J.; Bond, V.C.; Person, S.; Snipes, W.

    1982-10-30

    The tsB5 strain of Herpes Simplex Virus type 1 (HSV-1) contains at least two mutations; one mutation specifies the syncytial phenotype and the other confers temperature sensitivity for virus growth. These functions are known to be located between the prototypic map coordinates 0.30 and 0.42. In this study it was demonstrated that tsB5 enters human embryonic lung (HEL) cells more rapidly than KOS, another strain of HSV-1. The EcoRI restriction fragment F from the KOS strain (map coordinates 0.315 to 0.421) was mapped with eight restriction endonucleases, and 16 recombinant plasmids were constructed which contained varying portions of the KOS genome. Recombinant viruses were generated by marker-rescue and marker-transfer cotransfection procedures, using intact DNA from one strain and a recombinant plasmid containing DNA from the other strain. The region of the crossover between the two nonisogenic strains was inferred by the identification of restriction sites in the recombinants that were characteristic of the parental strains. The recombinants were subjected to phenotypic analysis. Syncytium formation, rate of virus entry, and the production of gB were all separable by the crossovers that produced the recombinants. The KOS sequences which rescue the syncytial phenotype of tsB5 were localized to 1.5 kb (map coordinates 0.345 to 0.355), and the temperature-sensitive mutation was localized to 1.2 kb (0.360 to 0.368), giving an average separation between the mutations of 2.5 kb on the 150-kb genome. DNA sequences that specify a functional domain for virus entry were localized to the nucleotide sequences between the two mutations. All three functions could be encoded by the virus gene specifying the gB glycoprotein.

  16. In vitro cytotoxicity of cyanobacteria from water ecosystems of Serbia.

    Science.gov (United States)

    Cetojevic-Simin, D; Svircev, Z; Baltic, V V

    2009-01-01

    The purpose of this study was to investigate whether water samples from water ecosystems of Serbia, unknown so far with regard to cyanotoxin levels, are the source of toxic compounds originating from the biological activity of cyanobacteria. The growth inhibition activity was evaluated using in vitro toxicity assay in Neuro-2a (mouse neuroblastoma) and MRC-5 (human fetal lung) cell lines, after 48 h of exposure time. Cell growth was evaluated by the colorimetric sulforhodamine B (SRB) assay. Our experiments revealed that some of the investigated water samples are toxigenic and alter cell growth of Neuro-2 and MRC-5 cell lines in vitro. Neuro-2a and MRC-5 cell lines responded to the presence of secondary metabolites of cyanobacteria. Significant cytotoxic effects were detected in the samples from lakes (Ludos and Palic), reservoirs (Zobnatica) and rivers (Krivaja).

  17. Estrogen receptor α and aryl hydrocarbon receptor cross-talk in a transfected hepatoma cell line (HepG2 exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin

    Directory of Open Access Journals (Sweden)

    Manuela Göttel

    2014-01-01

    Full Text Available The prototype dioxin congener 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD is known to exert anti-estrogenic effects via activation of the aryl hydrocarbon receptor (AhR by interfering with the regulation of oestrogen homeostasis and the estrogen receptor α (ERα signalling pathway. The AhR/ER cross-talk is considered to play a crucial role in TCDD- and E2-dependent mechanisms of carcinogenesis, though the concerted mechanism of action in the liver is not yet elucidated. The present study investigated TCDD's impact on the transcriptional cross-talk between AhR and ERα and its modulation by 17β-estradiol (E2 in the human hepatoma cell line HepG2, which is AhR-responsive but ERα-negative. Transient transfection assays with co-transfection of hERα and supplementation of receptor antagonists showed anti-estrogenic action of TCDD via down-regulation of E2-induced ERα signaling. In contrast, enhancement of AhR signaling dependent on ERα was observed providing evidence for increased cytochrome P450 (CYP induction to promote E2 metabolism. However, relative mRNA levels of major E2-metabolizing CYP1A1 and 1B1 and the main E2-detoxifying catechol-O-methyltransferase were not affected by the co-treatments. This study provides new evidence of a TCDD-activated AhR-mediated molecular AhR/ERα cross-talk mechanism at transcriptional level via indirect inhibition of ERα and enhanced transcriptional activity of AhR in HepG2 cells.

  18. Mechanism of nuclear factor of activated T-cells mediated FasL expression in corticosterone -treated mouse Leydig tumor cells

    Directory of Open Access Journals (Sweden)

    Wang Qian

    2008-06-01

    Full Text Available Abstract Background Fas and FasL is important mediators of apoptosis. We have previously reported that the stress levels of corticosterone (CORT, glucocorticoid in rat increase expression of Fas/FasL and activate Fas/FasL signal pathway in rat Leydig cells, which consequently leads to apoptosis. Moreover, our another study showed that nuclear factor of activated T-cells (NFAT may play a potential role in up-regulation of FasL during CORT-treated rat Leydig cell. It is not clear yet how NFAT is involved in CORT-induced up-regulation of FasL. The aim of the present study is to investigate the molecular mechanisms of NFAT-mediated FasL expression in CORT-treated Leydig cells. Results Western blot analysis showed that NFAT2 expression is present in mouse Leydig tumor cell (mLTC-1. CORT-induced increase in FasL expression in mLTC-1 was ascertained by Western Blot analysis and CORT-induced increase in apoptotic frequency of mLTC-1 cells was detected by FACS with annexin-V labeling. Confocal imaging of NFAT2-GFP in mLTC-1 showed that high level of CORT stimulated NFAT translocation from the cytoplasm to the nucleus. RNA interference-mediated knockdown of NFAT2 significantly attenuated CORT-induced up-regulation of FasL expression in mLTC. These results corroborated our previous finding that NFAT2 is involved in CORT-induced FasL expression in rat Leydig cells and showed that mLTC-1 is a suitable model for investigating the mechanism of CORT-induced FasL expression. The analysis of reporter constructs revealed that the sequence between -201 and +71 of mouse FasL gene is essential for CORT-induced FasL expression. The mutation analysis demonstrated that CORT-induced FasL expression is mediated via an NFAT binding element located in the -201 to +71 region. Co-transfection studies with an NFAT2 expression vector and reporter construct containing -201 to +71 region of FasL gene showed that NFAT2 confer a strong inducible activity to the FasL promoter at its

  19. TRAF6 inhibits proangiogenic signals in endothelial cells and regulates the expression of vascular endothelial growth factor

    Energy Technology Data Exchange (ETDEWEB)

    Bruneau, Sarah; Datta, Dipak; Flaxenburg, Jesse A.; Pal, Soumitro [Transplantation Research Center, Division of Nephrology, Department of Medicine, Children' s Hospital Boston, Boston, MA (United States); Department of Pediatrics, Harvard Medical School, Boston, MA (United States); Briscoe, David M., E-mail: david.briscoe@childrens.harvard.edu [Transplantation Research Center, Division of Nephrology, Department of Medicine, Children' s Hospital Boston, Boston, MA (United States); Department of Pediatrics, Harvard Medical School, Boston, MA (United States)

    2012-03-02

    Highlights: Black-Right-Pointing-Pointer TNF-receptor associated factors (TRAFs) function in the angiogenesis response. Black-Right-Pointing-Pointer TRAF6 regulates basal and inducible expression of VEGF in endothelial cells (EC). Black-Right-Pointing-Pointer TRAF6 is an endogenous inhibitor of EC proliferation and migration in EC. Black-Right-Pointing-Pointer TRAF6 inhibits VEGF expression in part via its ability to regulate Src signaling. -- Abstract: TNF-family molecules induce the expression Vascular Endothelial Growth Factor (VEGF) in endothelial cells (EC) and elicit signaling responses that result in angiogenesis. However, the role of TNF-receptor associated factors (TRAFs) as upstream regulators of VEGF expression or as mediators of angiogenesis is not known. In this study, HUVEC were cotransfected with a full-length VEGF promoter-luciferase construct and siRNAs to TRAF 1, -2, -3, -5, -6, and promoter activity was measured. Paradoxically, rather than inhibiting VEGF expression, we found that knockdown of TRAF6 resulted in a 4-6-fold increase in basal VEGF promoter activity compared to control siRNA-transfected EC (P < 0.0001). In addition, knockdown of TRAF 1, -2, -3 or -5 resulted in a slight increase or no change in VEGF promoter activation. Using [{sup 3}H]thymidine incorporation assays as well as the in vitro wound healing assay, we also found that basal rates of EC proliferation and migration were increased following TRAF6 knockdown; and this response was inhibited by the addition of a blocking anti-VEGF antibody into cell cultures. Using a limited protein array to gain insight into TRAF6-dependent intermediary signaling responses, we observed that TRAF6 knockdown resulted in an increase in the activity of Src family kinases. In addition, we found that treatment with AZD-0530, a pharmacological Src inhibitor, reduced the regulatory effect of TRAF6 knockdown on VEGF promoter activity. Collectively, these findings define a novel pro-angiogenic signaling

  20. Sequence of the 5'-flanking region and promoter activity of the human mucin gene MUC5B in different phenotypes of colon cancer cells.

    Science.gov (United States)

    Van Seuningen, I; Perrais, M; Pigny, P; Porchet, N; Aubert, J P

    2000-06-15

    site. Finally, in co-transfection experiments a transactivating effect of Sp1 on to MUC5B promoter was seen in LS174T and Caco-2 cells.

  1. Daidzein suppresses pro-inflammatory chemokine Cxcl2 transcription in TNF-α-stimulated murine lung epithelial cells via depressing PARP-1 activity

    Science.gov (United States)

    Li, Hai-yan; Pan, Lang; Ke, Yue-shuang; Batnasan, Enkhzaya; Jin, Xiang-qun; Liu, Zhong-ying; Ba, Xue-qing

    2014-01-01

    Aim: Daidzein (4′,7-dihydroxyisoflavone) is an isoflavone exiting in many herbs that has shown anti-inflammation activity. The aim of this study was to investigate the mechanism underlying its anti-inflammatory action in murine lung epithelial cells. Methods: C57BL/6 mice were intranasally exposed to TNF-α to induce lung inflammation. The mice were injected with daidzein (400 mg/kg, ip) before TNF-α challenge, and sacrificed 12 h after TNF-α challenge, and lung tissues were collected for analyisis. In in vitro studies, murine MLE-12 epithelial cells were treated with TNF-α (20 ng/mL). The expression of pro-inflammatory chemokine Cxcl2 mRNA and NF-κB transcriptional activity were examined using real-time PCR and a dual reporter assay. Protein poly-adenosine diphosphate-ribosylation (PARylation) was detecyed using Western blotting and immunoprecipitation assays. Results: Pretreatment of the mice with daidzein markedly attenuated TNF-α-induced lung inflammation, and inhibited Cxcl2 expression in lung tissues. Furthermore, daidzein (10 μmol/L) prevented TNF-α-induced increases in Cxcl2 expression and activity and NF-κB transcriptional activity, and markedly inhibited TNF-α-induced protein PARylation in MLE-12 cells in vitro. In MLE-12 cells co-transfected with the PARP-1 expression plasmid and NF-κB-luc (or Cxcl2-luc) reporter plasmid, TNF-α markedly increased NF-κB (or Cxcl2) activation, which were significantly attenuated in the presence of daidzein (or the protein PARylation inhibitor PJ 34). PARP-1 activity assay showed that daidzein (10 μmol/L) reduced the activity of PARP-1 by ∼75%. Conclusion: The anti-inflammatory action of daidzein in murine lung epithelial cells seems to be mediated via a direct interaction with PARP-1, which inhibits RelA/p65 protein PARylation required for the transcriptional modulation of pro-inflammatory chemokines such as Cxcl2. PMID:24632845

  2. Cell Motility

    CERN Document Server

    Lenz, Peter

    2008-01-01

    Cell motility is a fascinating example of cell behavior which is fundamentally important to a number of biological and pathological processes. It is based on a complex self-organized mechano-chemical machine consisting of cytoskeletal filaments and molecular motors. In general, the cytoskeleton is responsible for the movement of the entire cell and for movements within the cell. The main challenge in the field of cell motility is to develop a complete physical description on how and why cells move. For this purpose new ways of modeling the properties of biological cells have to be found. This long term goal can only be achieved if new experimental techniques are developed to extract physical information from these living systems and if theoretical models are found which bridge the gap between molecular and mesoscopic length scales. Cell Motility gives an authoritative overview of the fundamental biological facts, theoretical models, and current experimental developments in this fascinating area.

  3. Photovoltaic Cells

    National Research Council Canada - National Science Library

    Karolis Kiela

    2012-01-01

    The article deals with an overview of photovoltaic cells that are currently manufactured and those being developed, including one or several p-n junction, organic and dye-sensitized cells using quantum dots...

  4. Electrochemical Cell

    DEFF Research Database (Denmark)

    1999-01-01

    The invention relates to a rechargeable electrochemical cell comprising a negative electrode, an electrolyte and a positive electrode in which the positive electrode structure comprises a lithium cobalt manganese oxide of the composition Li¿2?Co¿y?Mn¿2-y?O¿4? where 0 ... for capacity losses in lithium ion cells and lithium-alloy cells....

  5. Challenges in using cultured primary rodent hepatocytes or cell lines to study hepatic HDL receptor SR-BI regulation by its cytoplasmic adaptor PDZK1.

    Directory of Open Access Journals (Sweden)

    Kosuke Tsukamoto

    Full Text Available BACKGROUND: PDZK1 is a four PDZ-domain containing cytoplasmic protein that binds to a variety of membrane proteins via their C-termini and can influence the abundance, localization and/or function of its target proteins. One of these targets in hepatocytes in vivo is the HDL receptor SR-BI. Normal hepatic expression of SR-BI protein requires PDZK1 - <5% of normal hepatic SR-BI is seen in the livers of PDZK1 knockout mice. Progress has been made in identifying features of PDZK1 required to control hepatic SR-BI in vivo using hepatic expression of wild-type and mutant forms of PDZK1 in wild-type and PDZK1 KO transgenic mice. Such in vivo studies are time consuming and expensive, and cannot readily be used to explore many features of the underlying molecular and cellular mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: Here we have explored the potential to use either primary rodent hepatocytes in culture using 2D collagen gels with newly developed optimized conditions or PDZK1/SR-BI co-transfected cultured cell lines (COS, HEK293 for such studies. SR-BI and PDZK1 protein and mRNA expression levels fell rapidly in primary hepatocyte cultures, indicating this system does not adequately mimic hepatocytes in vivo for analysis of the PDZK1 dependence of SR-BI. Although PDZK1 did alter SR-BI protein expression in the cell lines, its influence was independent of SR-BI's C-terminus, and thus is not likely to occur via the same mechanism as that which occurs in hepatocytes in vivo. CONCLUSIONS/SIGNIFICANCE: Caution must be exercised in using primary hepatocytes or cultured cell lines when studying the mechanism underlying the regulation of hepatic SR-BI by PDZK1. It may be possible to use SR-BI and PDZK1 expression as sensitive markers for the in vivo-like state of hepatocytes to further improve primary hepatocyte cell culture conditions.

  6. [Regulation of [12Asp]K-ras4B on transcriptional activity of estrogen receptor in endometrial carcinoma HEC-1A cell lines].

    Science.gov (United States)

    Gui, Li-ming; Wei, Li-hui; Xu, Ming-xu; Wang, Jian-liu; Zhong, Ying-cheng; Li, Xiao-ping; Tu, Zheng; Sun, Peng-ming; Ma, Da-long

    2004-01-01

    To investigate the effect of mutant-type [(12)Asp]K-ras4B gene on the expression of estrogen receptor (ER) alpha and beta and their transcriptional activity as a transcription factor in endometrial carcinoma HEC-1A cell line. (1) Effect of [(12)Asp]K-ras4B on the expression of ER alpha and beta were determined using Western blot assay. (2) Eukaryotic expression plasmid pGL3-luciferase-ERE containing luciferase report gene and estrogen receptor element (ERE) was constructed, and co-transfected into NIH3T3 and HEC-1A cell lines with pEGFP-N1 to examine the effect of [(12)Asp]K-ras4B on ER transcription that is regulated by estradiol. In addition, they were transfected into pSV5-HER0 (containing full length wide type ERalpha cDNA) and pCMV-rafS621A (inhibiting raf kinase) plasmids to test the effect of [(12)Asp]K-ras4B/raf signal pathway on transcriptional activity of ER proteins. (1) Protein level of ERs expressed in pcDI transfected control cells was low while it was increased for 3.6-fold (97 +/- 25, 349 +/- 67, P ras4B NIH3T3 cells after transfection. (2) In pcDI-[(12)Asp]K-ras4B NIH3T3 cells, the ratios for ERalpha and and ERbeta levels before transfection of rafS621A plasmids to that after the transfection, were 2.4:1 (724 +/- 45, 310 +/- 46, P HEC-1A cells, these ratios were 2.1:1 (566 +/- 22, 279 +/- 30, P ras4B NIH3T3 cells, 19-fold (141 +/- 39, 2644 +/- 331, P HEC-1A cells, respectively, when compared with those in the absence of E(2). (4) In pSV5-HER0 transfected pcDI-[(12)Asp] K-ras4B NIH3T3 cells and HEC-1A cells, compared to the untransfected cells, the ER transcriptional activity in the transfected cells increased markedly. The luciferase activity was increased for 8-fold (1048 +/- 91, 8099 +/- 452, P HEC-1A cells and pcDI-[(12)Asp]K-ras4B NIH3T3 cells. The ratio of luciferase activities in pcDI-[(12)Asp]K-ras4B NIH3T3 and HEC-1A cells, before and after transfection was 7.8:1 (1184 +/- 168, 152 +/- 27, P ras4B can enhance the expression of ERalpha and

  7. Stem Cells

    Directory of Open Access Journals (Sweden)

    Madhukar Thakur

    2015-02-01

    Full Text Available Objective: The objective of this presentation is to create awareness of stem cell applications in the ISORBE community and to foster a strategy of how the ISORBE community can disseminate information and promote the use of radiolabeled stem cells in biomedical applications. Methods: The continued excitement in Stem Cells, in many branches of basic and applied biomedical science, stems from the remarkable ability of stem cells to divide and develop into different types of cells in the body. Often called as Magic Seeds, stem cells are produced in bone marrow and circulate in blood, albeit at a relatively low concentration. These virtues together with the ability of stem cells to grow in tissue culture have paved the way for their applications to generate new and healthy tissues and to replace diseased or injured human organs. Although possibilities of stem cell applications are many, much remains yet to be understood of these remarkable magic seeds. Conclusion: This presentation shall briefly cover the origin of stem cells, the pros and cons of their growth and division, their potential application, and shall outline some examples of the contributions of radiolabeled stem cells, in this rapidly growing branch of biomedical science

  8. Types of Stem Cells

    Science.gov (United States)

    ... Stem Cell Glossary Search Toggle Nav Types of Stem Cells Stem cells are the foundation from which all ... Learn About Stem Cells > Types of Stem Cells Stem cells Stem cells are the foundation for every organ ...

  9. Fuel Cells

    DEFF Research Database (Denmark)

    Smith, Anders; Pedersen, Allan Schrøder

    2014-01-01

    Fuel cells have been the subject of intense research and development efforts for the past decades. Even so, the technology has not had its commercial breakthrough yet. This entry gives an overview of the technological challenges and status of fuel cells and discusses the most promising applications...... of the different types of fuel cells. Finally, their role in a future energy supply with a large share of fluctuating sustainable power sources, e.g., solar or wind, is surveyed....

  10. Cell Chauvinism

    Science.gov (United States)

    Keller, Dolores Elaine

    1972-01-01

    Indicates that biological terminology, such as mother cell'' and labels of sex factors in bacteria, reflect discrimination against females by reinforcing perpetuation of stereotyped gender roles. (AL)

  11. In vitro detection of cardiotoxins or neurotoxins affecting ion channels or pumps using beating cardiomyocytes as alternative for animal testing.

    Science.gov (United States)

    Nicolas, Jonathan; Hendriksen, Peter J M; de Haan, Laura H J; Koning, Rosella; Rietjens, Ivonne M C M; Bovee, Toine F H

    2015-03-01

    The present study investigated if and to what extent murine stem cell-derived beating cardiomyocytes within embryoid bodies can be used as a broad screening in vitro assay for neurotoxicity testing, replacing for example in vivo tests for marine neurotoxins. Effect of nine model compounds, acting on either the Na(+), K(+), or Ca(2+) channels or the Na(+)/K(+) ATP-ase pump, on the beating was assessed. Diphenhydramine, veratridine, isradipine, verapamil and ouabain induced specific beating arrests that were reversible and none of the concentrations tested induced cytotoxicity. Three K(+) channel blockers, amiodarone, clofilium and sematilide, and the Na(+)/K(+) ATPase pump inhibitor digoxin had no specific effect on the beating. In addition, two marine neurotoxins i.e. saxitoxin and tetrodotoxin elicited specific beating arrests in cardiomyocytes. Comparison of the results obtained with cardiomyocytes to those obtained with the neuroblastoma neuro-2a assay revealed that the cardiomyocytes were generally somewhat more sensitive for the model compounds affecting Na(+) and Ca(2+) channels, but less sensitive for the compounds affecting K(+) channels. The stem cell-derived cardiomyocytes were not as sensitive as the neuroblastoma neuro-2a assay for saxitoxin and tetrodotoxin. It is concluded that the murine stem cell-derived beating cardiomyocytes provide a sensitive model for detection of specific neurotoxins and that the neuroblastoma neuro-2a assay may be a more promising cell-based assay for the screening of marine biotoxins. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. A new bis-seco-abietane diterpenoid from Hyptis crenata Pohl ex Benth.

    Science.gov (United States)

    Yun, Young Sook; Fukaya, Haruhiko; Nakane, Takahisa; Takano, Akihito; Takahashi, Shigeru; Takahashi, Yuji; Inoue, Hideshi

    2014-12-05

    A new, highly oxidized, bis-seco-abietane diterpenoid named hyptisolide A (1) was isolated from Hyptis crenata Pohl ex Benth. Its structure and stereochemistry were elucidated on the basis of data obtained by HRESIMS, NMR, and X-ray diffraction analyses, and its absolute configuration was determined with vibrational circular dichroism spectroscopy. By reporter gene assay, 1 was demonstrated to induce cAMP-responsive element-dependent transcription in Neuro2A cells.

  13. miR-15a-3p and miR-16-1-3p Negatively Regulate Twist1 to Repress Gastric Cancer Cell Invasion and Metastasis

    Science.gov (United States)

    Wang, Tao; Hou, Jingjing; Li, Zengpeng; Zheng, Zihan; Wei, Jie; Song, Dan; Hu, Tao; Wu, Qiao; Yang, James Y.; Cai, Jian-chun

    2017-01-01

    MicroRNAs are a novel class of gene regulators that function as oncogenes or tumor suppressors. In our current study, we investigated the role of miR-15a-3p and miR-16-1-3p in the regulation of Twist1 expression and EMT process. Our bioinformatics analysis suggested that on the 3' UTR of Twist1, there are two conserved miRNA recognition sites for miR-15a-3p and miR-16-1-3p respectively. Interestingly, overexpression of miR-15a-3p and miR-16-1-3p significantly suppressed the activity of luciferase reporter containing Twist1-3' UTR, reduced mRNA and protein level of EMT related genes such as TWIST1, N-cadherin, α-SMA and Fibronectin, and repressed MMP9 and MMP2 activity, as well as cell migration and invasion. Conversely, inhibition of miR-15a-3p and miR-16-1-3p significantly increased TWIST1, N-cadherin, α-SMA and Fibronectin protein expression. In addition, Twist1 co-transfection significantly ameliorated the loss of cell migration and invasion. Moreover, overexpression of miR-15a-3p and miR-16-1-3p dramatically suppressed the ability of BGC823 cells to form colonies in vitro and develop tumors in vivo in nude mice. Finally, qPCR and Western blot analysis showed that miR-15a-3p and miR-16-1-3p were significantly reduced in clinical gastric cancer tissue, whereas Twist1 mRNA and protein were significantly up-regulated, suggesting that this aberrant down-regulation of miR-15a-3p and miR-16-1-3p might be associated with the abnormal regulation of Twist1 and the EMT process in gastric cancer development. Our results help to elucidate a novel and important mechanism for the regulation of Twist1 in the development of cancer. PMID:28123352

  14. Fuel Cells

    Science.gov (United States)

    Hawkins, M. D.

    1973-01-01

    Discusses the theories, construction, operation, types, and advantages of fuel cells developed by the American space programs. Indicates that the cell is an ideal small-scale power source characterized by its compactness, high efficiency, reliability, and freedom from polluting fumes. (CC)

  15. Stem Cells

    DEFF Research Database (Denmark)

    Sommerlund, Julie

    2004-01-01

    '. This paper is about tech-noscience, and about the proliferation of connections and interdependencies created by it.More specifically, the paper is about stem cells. Biotechnology in general has the power to capture the imagination. Within the field of biotechnology nothing seems more provocative...... and tantalizing than stem cells, in research, in medicine, or as products....

  16. Solar cell

    Energy Technology Data Exchange (ETDEWEB)

    Tsukamoto, Moriaki; Hayashibara, Mitsuo

    1988-08-18

    Concerning the exsisting solar cell utilizing wavelength transition, the area of the solar cell element necessary for unit electric power output can be made small, but transition efficiency of the solar cell as a whole including a plastic plate with phosphor is not high. This invention concerns a solar cell which is appropriate for transferring the light within a wide spectrum range of the sunlight to electricilty efficiently, utilizes wavelength transition and has high efficiency per unit area. In other words, the solar cell of this invention has the feature of providing in parallel with a photoelectric transfer layer a layer of wavelength transitioning material (phosphor) which absorbs the light within the range of wavelength of low photoelectric transfer efficiency at the photoelectric transfer layer and emits the light within the range of wavelength in which the photoelectric transfer rate is high on the light incident side of the photoelectric transfer layer. (5 figs)

  17. Microarray analysis of Foxl2 mediated gene regulation in the mouse ovary derived KK1 granulosa cell line: Over-expression of Foxl2 leads to activation of the gonadotropin releasing hormone receptor gene promoter

    Science.gov (United States)

    2010-01-01

    Background The Foxl2 transcription factor is required for ovarian function during follicular development. The mechanism of Foxl2 regulation of this process has not been elucidated. Our approach to begin to understand Foxl2 function is through the identification of Foxl2 regulated genes in the ovary. Methods Transiently transfected KK1 mouse granulosa cells were used to identify genes that are potentially regulated by Foxl2. KK1 cells were transfected in three groups (mock, activated, and repressed) and twenty-four hours later RNA was isolated and submitted for Affymetrix microarray analysis. Genesifter software was used to carry out analysis of microarray data. One identified target, the gonadotropin releasing hormone receptor (GnRHR) gene, was chosen for further study and validation of Foxl2 responsiveness. Transient transfection analyses were carried out to study the effect of Foxl2 over-expression on GnRHR gene promoter-luciferase fusion activity. Data generated was analyzed with GraphPad Prism software. Results Microarray analysis identified 996 genes of known function that are potentially regulated by Foxl2 in mouse KK1 granulosa cells. The steroidogenic acute regulatory protein (StAR) gene that has been identified as Foxl2 responsive by others was identified in this study also, thereby supporting the effectiveness of our strategy. The GnRHR gene was chosen for further study because it is known to be expressed in the ovary and the results of previous work has indicated that Foxl2 may regulate GnRHR gene expression. Cellular levels of Foxl2 were increased via transient co-transfection of KK1 cells using a Foxl2 expression vector and a GnRHR promoter-luciferase fusion reporter vector. The results of these analyses indicate that over-expression of Foxl2 resulted in a significant increase in GnRHR promoter activity. Therefore, these transfection data validate the microarray data which suggest that Foxl2 regulates GnRHR and demonstrate that Foxl2 acts as an

  18. Microarray analysis of Foxl2 mediated gene regulation in the mouse ovary derived KK1 granulosa cell line: Over-expression of Foxl2 leads to activation of the gonadotropin releasing hormone receptor gene promoter

    Directory of Open Access Journals (Sweden)

    Escudero Jean M

    2010-02-01

    Full Text Available Abstract Background The Foxl2 transcription factor is required for ovarian function during follicular development. The mechanism of Foxl2 regulation of this process has not been elucidated. Our approach to begin to understand Foxl2 function is through the identification of Foxl2 regulated genes in the ovary. Methods Transiently transfected KK1 mouse granulosa cells were used to identify genes that are potentially regulated by Foxl2. KK1 cells were transfected in three groups (mock, activated, and repressed and twenty-four hours later RNA was isolated and submitted for Affymetrix microarray analysis. Genesifter software was used to carry out analysis of microarray data. One identified target, the gonadotropin releasing hormone receptor (GnRHR gene, was chosen for further study and validation of Foxl2 responsiveness. Transient transfection analyses were carried out to study the effect of Foxl2 over-expression on GnRHR gene promoter-luciferase fusion activity. Data generated was analyzed with GraphPad Prism software. Results Microarray analysis identified 996 genes of known function that are potentially regulated by Foxl2 in mouse KK1 granulosa cells. The steroidogenic acute regulatory protein (StAR gene that has been identified as Foxl2 responsive by others was identified in this study also, thereby supporting the effectiveness of our strategy. The GnRHR gene was chosen for further study because it is known to be expressed in the ovary and the results of previous work has indicated that Foxl2 may regulate GnRHR gene expression. Cellular levels of Foxl2 were increased via transient co-transfection of KK1 cells using a Foxl2 expression vector and a GnRHR promoter-luciferase fusion reporter vector. The results of these analyses indicate that over-expression of Foxl2 resulted in a significant increase in GnRHR promoter activity. Therefore, these transfection data validate the microarray data which suggest that Foxl2 regulates GnRHR and demonstrate

  19. Regulation of glucose phosphate isomerase by the 3'UTR-specific miRNAs miR-302b and miR-17-5p in chicken primordial germ cells.

    Science.gov (United States)

    Rengaraj, Deivendran; Park, Tae Sub; Lee, Sang In; Lee, Bo Ram; Han, Beom Ku; Song, Gwonhwa; Han, Jae Yong

    2013-08-01

    Glucose phosphate isomerase (GPI) involves in the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate in glucose pathways. Because glucose metabolism is crucial for the proliferation and differentiation of embryonic stem and germ cells, reducing GPI expression may affect the characteristic features of these cells. MicroRNAs (miRNAs) have been shown to regulate genes. In the present study, we investigated the regulation of chicken GPI by its predicted miRNAs. We determined the expression patterns of seven GPI 3'-untranslated region (3'UTR)-targeting miRNAs, including the gga-miR-302 cluster, gga-miR-106, gga-miR-17-5p, and gga-miR-20 cluster in chicken primordial germ cells (PGCs), compared with GPI mRNA. Among the miRNAs, gga-miR-302b, gga-miR-302d, and gga-miR-17-5p were expressed at lower levels than GPI mRNA. The remaining four miRNAs-gga-miR-302c, gga-miR-106, gga-miR-20a, and gga-miR-20b-were expressed at higher levels than the expression of GPI mRNA. Next, we cotransfected four candidate miRNAs-gga-miR-302b, gga-miR-106, gga-miR-17-5p, and gga-miR-20a-with GPI 3'UTR into 293FT cells by dual fluorescence reporter assay. Overexpression of gga-miR-302b and gga-miR-17-5p miRNAs in 293FT cells significantly downregulated GPI expression, whereas the other two miRNAs had no effect. Then, knockdown and overexpression of these four candidate miRNAs were performed by RNA interference assay to regulate GPI in PGCs. In the RNA interference assay, the expression of GPI was greatly regulated by gga-miR-302b and gga-miR-17-5p. Finally, we examined the effects of GPI regulation on PGC proliferation and migration. Our results suggested that the regulation of GPI by gga-miR-302b and gga-miR-17-5p affected PGCs proliferation. However, regulation of GPI using these two miRNAs did not affect the migration of PGCs into embryonic gonads.

  20. Functional processing of nuclear Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP-N): evidence for a critical role of proteolytic processing in the regulation of its catalytic activity, subcellular localization and substrate targeting in vivo.

    Science.gov (United States)

    Sueyoshi, Noriyuki; Nimura, Takaki; Onouchi, Takashi; Baba, Hiromi; Takenaka, Shinobu; Ishida, Atsuhiko; Kameshita, Isamu

    2012-01-01

    Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) and its nuclear homolog CaMKP-N are Ser/Thr protein phosphatases that belong to the PPM family. These phosphatases are highly specific for multifunctional CaM kinases and negatively regulate their activities. CaMKP-N is only expressed in the brain and specifically localized in the nucleus. In this study, we found that zebrafish CaMKP-N (zCaMKP-N) underwent proteolytic processing in both the zebrafish brain and Neuro2a cells. In Neuro2a cells, the proteolytic processing was effectively inhibited by the proteasome inhibitors MG-132, Epoxomicin, and Lactacystin, suggesting that the ubiquitin-proteasome pathway was involved in this processing. Using MG-132, we found that the proteolytic processing changed the subcellular localization of zCaMKP-N from the nucleus to the cytosol. Accompanying this change, the cellular targets of zCaMKP-N in Neuro2a cells were significantly altered. Furthermore, we obtained evidence that the zCaMKP-N activity was markedly activated when the C-terminal domain was removed by the processing. Thus, the proteolytic processing of zCaMKP-N at the C-terminal region regulates its catalytic activity, subcellular localization and substrate targeting in vivo. Copyright © 2011 Elsevier Inc. All rights reserved.

  1. Study of Mechanisms of an Anti-Apoptotic Proteins, BI-1 in Prostate Cancer

    Science.gov (United States)

    2006-12-01

    cotransfected with potential target proteins in HEK293 T cells. None of them was shown to have altered protein level when coexpressed with BAR-full... HEK293T cells were transfected with BAR, BARΔDED, BARdR, BARdRmt with or without Bap31. Cells were treated with 25 μM MG132 for 12 hours compared with the

  2. Bi-Cell Unit for Fuel Cell.

    Science.gov (United States)

    The patent concerns a bi-cell unit for a fuel cell . The bi-cell unit is comprised of two electrode packs. Each of the electrode packs includes an...invention relates in general to a bi-cell unit for a fuel cell and in particular, to a bi-cell unit for a hydrazine-air fuel cell .

  3. Cell, cell, cell: fuel cell applications moving ahead

    Energy Technology Data Exchange (ETDEWEB)

    Ross, E.

    2001-11-01

    Developments in fuel cell technology within the last decade, such as the targeting by major automakers of non-polluting fuel cells as an alternative to the internal combustion engine, are reviewed. For example, Ballard Power Systems of Vancouver is the exclusive supplier to both DaimlerCrysler and the Ford Motor Company of the fuel cell stacks that produce the power in fuel cell systems. Ballard plans the commercial launch of transit bus engines in 2002 and automotive products between 2003 and 2005. The company also sees huge opportunities for fuel cells in stationary and portable power applications. At the same time, the Calgary-based fuel cell division of Energy Ventures Inc. is developing a direct methanol fuel cell that eliminates the intermediate step of 'reforming' methanol into hydrogen that is required in the Ballard process. Energy Ventures targets small niche markets such as small utility vehicles for its direct methanol fuel cell. A completely self-contained fuel cell of this type is expected to be ready in 2002. Solid oxide fuel cells for off-grid remote power units as well as for home heat and power is yet another field of development that will be particularly attractive to operations in remote areas where reliable grid electricity is expensive and hard to obtain. A prototype 2.3 kW residential power system using natural gas was made available by Global Thermoelectric Inc in June 2001; field testing is planned for 2002, with commercial production in late 2003 or 2004. The Calgary-based Snow Leopard Resources Inc plans to use pure hydrogen sulphide obtained from sour natural gas as a hydrogen source. The prime focus of Snow Leopard is on gas plants looking for ways to increase their efficiency, obtain carbon dioxide credits and generate electricity on site. This type of fuel cell also could be of interest to companies with shut-in sour gas since these companies could use the stationary fuel cell system to generate electricity.

  4. Learn About Stem Cells

    Science.gov (United States)

    ... Patient Handbook Stem Cell Glossary Search Toggle Nav Stem Cell Basics Stem cells are the foundation from which ... original cell’s DNA, cytoplasm and cell membrane. About stem cells Stem cells are the foundation of development in ...

  5. T Cells

    Science.gov (United States)

    ... cells) against some component of central nervous system myelin (the fatty sheath that surrounds and insulates nerve fibers). Demyelination — the destruction of myelin — causes nerve impulses to be slowed or halted ...

  6. Mast cells

    National Research Council Canada - National Science Library

    D. D. Metcalfe; D. Baram; Y. A. Mekori

    1997-01-01

    Mast cells are found resident in tissues throughout the body, particularly in association with structures such as blood vessels and nerves, and in proximity to surfaces that interface the external environment...

  7. CELL ZAPPER

    National Research Council Canada - National Science Library

    Thomas Grose

    2017-01-01

      Investigators at Britain's University of Warwick recently found a new organo-metal compound, Organo-Osmium FY26, which destroys cancer cells from the inside, finding and attacking their weakest point...

  8. Dry cell battery poisoning

    Science.gov (United States)

    Batteries - dry cell ... Acidic dry cell batteries contain: Manganese dioxide Ammonium chloride Alkaline dry cell batteries contain: Sodium hydroxide Potassium hydroxide Lithium dioxide dry cell batteries ...

  9. The alternative Epac/cAMP pathway and the MAPK pathway mediate hCG induction of leptin in placental cells.

    Directory of Open Access Journals (Sweden)

    Julieta Lorena Maymó

    Full Text Available Pleiotropic effects of leptin have been identified in reproduction and pregnancy, particularly in the placenta, where it works as an autocrine hormone. In this work, we demonstrated that human chorionic gonadotropin (hCG added to JEG-3 cell line or to placental explants induces endogenous leptin expression. We also found that hCG increased cAMP intracellular levels in BeWo cells in a dose-dependent manner, stimulated cAMP response element (CRE activity and the cotransfection with an expression plasmid of a dominant negative mutant of CREB caused a significant inhibition of hCG stimulation of leptin promoter activity. These results demonstrate that hCG indeed activates cAMP/PKA pathway, and that this pathway is involved in leptin expression. Nevertheless, we found leptin induction by hCG is dependent on cAMP levels. Treatment with (Bu(2cAMP in combination with low and non stimulatory hCG concentrations led to an increase in leptin expression, whereas stimulatory concentrations showed the opposite effect. We found that specific PKA inhibition by H89 caused a significant increase of hCG leptin induction, suggesting that probably high cAMP levels might inhibit hCG effect. It was found that hCG enhancement of leptin mRNA expression involved the MAPK pathway. In this work, we demonstrated that hCG leptin induction through the MAPK signaling pathway is inhibited by PKA. We observed that ERK1/2 phosphorylation increased when hCG treatment was combined with H89. In view of these results, the involvement of the alternative cAMP/Epac signaling pathway was studied. We observed that a cAMP analogue that specifically activates Epac (CPT-OMe stimulated leptin expression by hCG. In addition, the overexpression of Epac and Rap1 proteins increased leptin promoter activity and enhanced hCG. In conclusion, we provide evidence suggesting that hCG induction of leptin gene expression in placenta is mediated not only by activation of the MAPK signaling pathway but also

  10. The Alternative Epac/cAMP Pathway and the MAPK Pathway Mediate hCG Induction of Leptin in Placental Cells

    Science.gov (United States)

    Maymó, Julieta Lorena; Pérez Pérez, Antonio; Maskin, Bernardo; Dueñas, José Luis; Calvo, Juan Carlos; Sánchez Margalet, Víctor; Varone, Cecilia Laura

    2012-01-01

    Pleiotropic effects of leptin have been identified in reproduction and pregnancy, particularly in the placenta, where it works as an autocrine hormone. In this work, we demonstrated that human chorionic gonadotropin (hCG) added to JEG-3 cell line or to placental explants induces endogenous leptin expression. We also found that hCG increased cAMP intracellular levels in BeWo cells in a dose-dependent manner, stimulated cAMP response element (CRE) activity and the cotransfection with an expression plasmid of a dominant negative mutant of CREB caused a significant inhibition of hCG stimulation of leptin promoter activity. These results demonstrate that hCG indeed activates cAMP/PKA pathway, and that this pathway is involved in leptin expression. Nevertheless, we found leptin induction by hCG is dependent on cAMP levels. Treatment with (Bu)2cAMP in combination with low and non stimulatory hCG concentrations led to an increase in leptin expression, whereas stimulatory concentrations showed the opposite effect. We found that specific PKA inhibition by H89 caused a significant increase of hCG leptin induction, suggesting that probably high cAMP levels might inhibit hCG effect. It was found that hCG enhancement of leptin mRNA expression involved the MAPK pathway. In this work, we demonstrated that hCG leptin induction through the MAPK signaling pathway is inhibited by PKA. We observed that ERK1/2 phosphorylation increased when hCG treatment was combined with H89. In view of these results, the involvement of the alternative cAMP/Epac signaling pathway was studied. We observed that a cAMP analogue that specifically activates Epac (CPT-OMe) stimulated leptin expression by hCG. In addition, the overexpression of Epac and Rap1 proteins increased leptin promoter activity and enhanced hCG. In conclusion, we provide evidence suggesting that hCG induction of leptin gene expression in placenta is mediated not only by activation of the MAPK signaling pathway but also by the

  11. The dyad palindromic glutathione transferase P enhancer binds multiple factors including AP1.

    OpenAIRE

    Diccianni, M B; Imagawa, M; Muramatsu, M

    1992-01-01

    Glutathione Transferase P (GST-P) gene expression is dominantly regulated by an upstream enhancer (GPEI) consisting of a dyad of palindromically oriented imperfect TPA (12-O-tetradecanoyl-phorbol-13-acetate)-responsive elements (TRE). GPEI is active in AP1-lacking F9 cells as well in AP1-containing HeLa cells. Despite GPEI's similarity to a TRE, c-jun co-transfection has only a minimal effect on transactivation. Antisense c-jun and c-fos co-transfection experiments further demonstrate the lac...

  12. Electrochemical cell

    Science.gov (United States)

    Nagy, Zoltan; Yonco, Robert M.; You, Hoydoo; Melendres, Carlos A.

    1992-01-01

    An electrochemical cell has a layer-type or sandwich configuration with a Teflon center section that houses working, reference and counter electrodes and defines a relatively narrow electrolyte cavity. The center section is surrounded on both sides with thin Teflon membranes. The membranes are pressed in place by a pair of Teflon inner frames which are in turn supported by a pair of outer metal frames. The pair of inner and outer frames are provided with corresponding, appropriately shaped slits that are in plane generally transverse to the plane of the working electrode and permit X-ray beams to enter and exit the cell through the Teflon membranes that cover the slits so that the interface between the working electrode and the electrolyte within the cell may be analyzed by transmission geometry. In one embodiment, the center section consists of two parts, one on top of the other. Alternatively, the center section of the electrochemical cell may consist of two intersliding pieces or may be made of a single piece of Teflon sheet material. The electrolyte cavity is shaped so that the electrochemical cell can be rotated 90.degree. in either direction while maintaining the working and counter electrodes submerged in the electrolyte.

  13. Cell Phones

    Science.gov (United States)

    Sansone, Lori A.

    2013-01-01

    Cell phones are a relatively novel and evolving technology. While the potential benefits of this technology continue to emerge, so do the potential psychosocial risks. For example, one psychosocial risk is user stress, which appears to be related to feeling compelled to promptly respond to cell-phone activity in order to maintain spontaneity and access with others. Other potential psychosocial risks include disruptions in sleep; the user’s risk of exposure to cyberbullying, particularly the unwanted exposure of photographs and/or videos of the victim; and overuse, particularly among adolescents. With regard to the latter phenomenon, the boundaries among overuse, misuse, dependence, and addiction are not scientifically clear. Therefore, while cell phones are a convenient and expedient technology, they are not without their potential psychosocial hazards. PMID:23439568

  14. Solar cell

    Energy Technology Data Exchange (ETDEWEB)

    Sakai, Yoshiyuki.

    1989-07-06

    In this invention, in a solar cell which has an electrode consisting of a superconductor, the superconductor electrode is partly or entirely covered with a metal or light reflecting material. In the above, the pattern on the substrate at the junction of the electrode and the semiconductor is the same as that of a comb-type electrode formed at the top of the semiconductor. By this, a solar cell was provided wherein a superconductive electrode which is not subject to degradation of the superconductive characteristics even in the light of high intensity, operating stably at high efficiency, indicating very high practical effect. In addition to the use of amorphous silicon as a semiconductor of the soalr cell, such other material as Si-single crystal, Ge and Ge/As can be used. For the superconductor electrode, such other material as YBaCuO can also be used. 2 figs.

  15. [Effect of CCR1 gene overexpression on the migration of bone marrow - derived mesenchymal stem cells towards hepatocellular carcinoma].

    Science.gov (United States)

    Gao, Y; Huang, X L; Zhang, L; Deng, L; Yin, A H; Sun, B C; Lu, S

    2017-05-20

    Objective: To evaluate the effect of human CCR1 (hCCR1) gene overexpression on the migration of human bone marrow-derived mesenchymal stem cells (hMSCs) towards hepatocellular carcinoma (HCC), and to examine the application prospects of MSCs as gene delivery vectors in the treatment of HCC. Methods: The hCCR1 gene was subcloned into a lentiviral vector to generate the recombinant plasmid pLV-hCCR1. The pLV-hCCR1 plasmid and two other packaging plasmids were co-transfected into 293T cells using calcium phosphate, and the virus-containing supernatant was collected. hMSCs were then infected with the recombinant lentivirus, and the expression of hCCR1 mRNA and protein was analyzed by RT-PCR and Western blot, respectively. The effect of CCR1 gene overexpression on the in vitro migration of hMSCs was examined using the Transwell migration assay. Orthotopic nude mice models of HCC were established using the MHCC-97H-GFP cell line, and the mice were divided into two groups ( n = 8 per group). hMSCs were then intravenously injected via the tail vein into the tumor-bearing nude mice to examine the effect of hCCR1 overexpression on the in vivo migration of hMSCs towards HCC. Unpaired Student's t-test was used for two-group comparisons, and one-way ANOVA was used for multi-group comparisons. Results: Restriction enzyme digestion and DNA sequencing demonstrated that the recombinant plasmid pLV-hCCR1 was constructed successfully. The LV-hCCR1 lentivirus packaged by 293T cells has high infection efficiency in hMSCs, and hCCR1 was overexpressed in hMSCs after LV-hCCR1 infection. Transwell migration assay showed that hCCR1-transfected hMSCs had significantly enhanced migration towards HCC cell line-derived condition medium (CM) compared with the control RFP-hMSCs [(134.8±15.7)/LPF vs (83.5±10.9)/LPF, t = 10.40, P migration experiment also demonstrated that there was significantly higher number of hCCR1-hMSCs localized within the MHCC-97H-GFP xenografts than hMSCs-RFP on day 14

  16. Tuning Collective Cell Migration by Cell-Cell Junction Regulation

    NARCIS (Netherlands)

    Friedl, P.; Mayor, R.

    2017-01-01

    Collective cell migration critically depends on cell-cell interactions coupled to a dynamic actin cytoskeleton. Important cell-cell adhesion receptor systems implicated in controlling collective movements include cadherins, immunoglobulin superfamily members (L1CAM, NCAM, ALCAM), Ephrin/Eph

  17. Energy storage cells

    Energy Technology Data Exchange (ETDEWEB)

    Gulia, N.V.

    1980-01-01

    The book deals with the characteristics and potentialities of energy storage cells of various types. Attention is given to electrical energy storage cells (electrochemical, electrostatic, and electrodynamic cells), mechanical energy storage cells (mechanical flywheel storage cells), and hybrid storage systems.

  18. Sickle Cell Anemia

    Science.gov (United States)

    Sickle cell anemia is a disease in which your body produces abnormally shaped red blood cells. The cells are shaped like ... normal, round red blood cells. This leads to anemia. The sickle cells also get stuck in blood ...

  19. Photovoltaic cell

    Science.gov (United States)

    Gordon, Roy G.; Kurtz, Sarah

    1984-11-27

    In a photovoltaic cell structure containing a visibly transparent, electrically conductive first layer of metal oxide, and a light-absorbing semiconductive photovoltaic second layer, the improvement comprising a thin layer of transition metal nitride, carbide or boride interposed between said first and second layers.

  20. Fuel cells:

    DEFF Research Database (Denmark)

    Sørensen, Bent

    2013-01-01

    A brief overview of the progress in fuel cell applications and basic technology development is presented, as a backdrop for discussing readiness for penetration into the marketplace as a solution to problems of depletion, safety, climate or environmental impact from currently used fossil...... and nuclear fuel-based energy technologies....

  1. Potent Cells

    Science.gov (United States)

    Liu, Dennis

    2007-01-01

    It seems hard to believe that Dolly the cloned sheep was born 10 years ago, kindling furious arguments over the prospects and ethics of cloning a human. Today, the controversy over cloning is entwined, often confused, with concerns over the use of human embryonic stem cells. Most people are unclear what cloning is, and they know even less when it…

  2. Generation of anti-TLR2 intrabody mediating inhibition of macrophage surface TLR2 expression and TLR2-driven cell activation

    Directory of Open Access Journals (Sweden)

    Lindenmaier Werner

    2010-04-01

    Full Text Available Abstract Background Toll-like receptor (TLR 2 is a component of the innate immune system and senses specific pathogen associated molecular patterns (PAMPs of both microbial and viral origin. Cell activation via TLR2 and other pattern recognition receptors (PRRs contributes to sepsis pathology and chronic inflammation both relying on overamplification of an immune response. Intracellular antibodies expressed and retained inside the endoplasmatic reticulum (ER-intrabodies are applied to block translocation of secreted and cell surface molecules from the ER to the cell surface resulting in functional inhibition of the target protein. Here we describe generation and application of a functional anti-TLR2 ER intrabody (αT2ib which was generated from an antagonistic monoclonal antibody (mAb towards human and murine TLR2 (T2.5 to inhibit the function of TLR2. αT2ib is a scFv fragment comprising the variable domain of the heavy chain and the variable domain of the light chain of mAb T2.5 linked together by a synthetic (Gly4Ser3 amino acid sequence. Results Coexpression of αT2ib and mouse TLR2 in HEK293 cells led to efficient retention and accumulation of TLR2 inside the ER compartment. Co-immunoprecipitation of human TLR2 with αT2ib indicated interaction of αT2ib with its cognate antigen within cells. αT2ib inhibited NF-κB driven reporter gene activation via TLR2 but not through TLR3, TLR4, or TLR9 if coexpressed in HEK293 cells. Co-transfection of human TLR2 with increasing amounts of the expression plasmid encoding αT2ib into HEK293 cells demonstrated high efficiency of the TLR2-αT2ib interaction. The αT2ib open reading frame was integrated into an adenoviral cosmid vector for production of recombinant adenovirus (AdV-αT2ib. Transduction with AdVαT2ib specifically inhibited TLR2 surface expression of murine RAW264.7 and primary macrophages derived from bone marrow (BMM. Furthermore, TLR2 activation dependent TNFα mRNA accumulation, as well

  3. Repression of let-7 by transforming growth factor-β1-induced Lin28 upregulates collagen expression in glomerular mesangial cells under diabetic conditions

    Science.gov (United States)

    Park, Jung Tak; Kato, Mitsuo; Lanting, Linda; Castro, Nancy; Nam, Bo Young; Wang, Mei; Kang, Shin-Wook

    2014-01-01

    Accumulation of mesangial extracellular matrix (ECM) proteins such as collagen type 1-α2 (Col1a2) and collagen type 4-α1 (Col4a1) is a key feature of diabetic nephropathy (DN). Transforming growth factor (TGF)-β1 plays important roles in ECM accumulation in DN, and evidence shows a mediatory role for microRNAs. In the present study, we found that microRNA let-7 family members (let-7b/c/d/g/i) were downregulated in TGF-β-treated mouse mesangial cells (MMCs) along with upregulation of Col1a2 and Col4a1. Ectopic expression of let-7b in TGF-β-treated MMCs attenuated Col1a2 and Col4a1 upregulation. Conversely, let-7b inhibitors increased Col1a2 and Col4a1 levels. Cotransfection of MMCs with mouse Col1a2 or Col4a1 3′-untranslated region luciferase constructs and let-7b inhibitors increased luciferase activity. However, constructs with let-7 target site mutations were unresponsive to TGF-β. TGF-β-induced 3′-untranslated region activity was attenuated by let-7b mimics, suggesting that Col1a2 and Col4a1 are direct targets of let-7b. In addition, Lin28b, a negative regulator of let-7 biogenesis, was upregulated in TGF-β-treated MMCs. Luciferase assays showed that the Lin28b promoter containing the Smad-binding element (SBE) responded to TGF-β, which was abolished in constructs without SBE. Chromatin immunoprecipitation assays showed TGF-β-induced enrichment of Smad2/3 at the Lin28b promoter, together suggesting that Lin28b is transcriptionally induced by TGF-β through SBE. Furthermore, let-7b levels were decreased, whereas Lin28b, Col1a2, and Col4a1 levels were increased, in glomeruli of diabetic mice compared with nondiabetic control mice, demonstrating the in vivo relevance of this Lin28/let-7/collagen axis. These results identify Lin28 as a new TGF-β target gene and suggest a novel role for the Lin28/let-7 pathway in controlling TGF-β-induced collagen accumulation in DN. PMID:25354942

  4. Identification of novel microRNAs in post-transcriptional control of Nrf2 expression and redox homeostasis in neuronal, SH-SY5Y cells.

    Directory of Open Access Journals (Sweden)

    Madhusudhanan Narasimhan

    Full Text Available Nuclear factor-erythroid 2-related factor 2 (Nrf2/NFE2L2, a redox-sensitive transcription factor plays a critical role in adaptation to cellular stress and affords cellular defense by initiating transcription of antioxidative and detoxification genes. While a protein can be regulated at multiple levels, control of Nrf2 has been largely studied at post-translational regulation points by Keap1. Importantly, post-transcriptional/translational based regulation of Nrf2 is less understood and to date there are no reports on such mechanisms in neuronal systems. In this context, studies involving the role of microRNAs (miRs which are normally considered as fine tuning regulators of protein production through translation repression and/or post-transcriptional alterations, are in place. In the current study, based on in-silico analysis followed by immunoblotting and real time analysis, we have identified and validated for the first time that human NFE2L2 could be targeted by miR153/miR27a/miR142-5p/miR144 in neuronal, SH-SY5Y cells. Co-transfection studies with individual miR mimics along with either WT 3' UTR of human Nrf2 or mutated miRNA targeting seed sequence within Nrf2 3' UTR, demonstrated that Nrf2 is a direct regulatory target of these miRs. In addition, ectopic expression of miR153/miR27a/miR142-5p/miR144 affected Nrf2 mRNA abundance and nucleo-cytoplasmic concentration of Nrf2 in a Keap1 independent manner resulting in inefficient transactivating ability of Nrf2. Furthermore, forced expression of miRs diminished GCLC and GSR expression resulting in alteration of Nrf2 dependent redox homeostasis. Finally, bioinformatics based miRNA-disease network analysis (MDN along with extended computational network analysis of Nrf2 associated pathologic processes suggests that if in a particular cellular scenario where any of these miR153/miR27a/miR142-5p/miR144 either individually or as a group is altered, it could affect Nrf2 thus triggering and

  5. Fuel cell

    Energy Technology Data Exchange (ETDEWEB)

    Enomoto, Hirofumi.

    1989-05-22

    This invention aims to maintain a long-term operation with stable cell output characteristics by uniformly supplying an electrolyte from the reserver to the matrix layer over the entire matrix layer, and further to prevent the excessive wetting of the catalyst layer by smoothly absorbing the volume change of the electrolyte, caused by the repeated stop/start-up of the fuel cell, within the reserver system. For this purpose, in this invention, an electrolyte transport layer, which connects with an electrolyte reservor formed at the electrode end, is partly formed between the electrode material and the catalyst layer; a catalyst layer, which faces the electrolyte transport layer, has through-holes, which connect to the matrix, dispersely distributed. The electrolyte-transport layer is a thin sheet of a hydrophilic fibers which are non-wovens of such fibers as carbon, silicon carbide, silicon nitride or inorganic oxides. 11 figs.

  6. Development of CRTEIL and CETRIZ, Cre-loxP-Based Systems, Which Allow Change of Expression of Red to Green or Green to Red Fluorescence upon Transfection with a Cre-Expression Vector

    OpenAIRE

    Masato Ohtsuka; Takayuki Warita; Takayuki Sakurai; Satoshi Watanabe; Hidetoshi Inoko; Masahiro Sato

    2009-01-01

    We developed Cre-loxP-based systems, termed CRTEIL and CETRIZ, which allow gene switching in a noninvasive manner. Single transfection with pCRTEIL resulted in predominant expression of red fluorescence. Cotransfection with pCRTEIL and Cre-expression plasmid (pCAG/NCre) caused switching from red to green fluorescence. Similarly, cotransfection with pCETRIZ and pCAG/NCre resulted in change of green to red fluorescence. These noninvasive systems will be useful in cell lineage analysis, since de...

  7. Eukaryotic cells and their cell bodies: Cell Theory revised.

    Science.gov (United States)

    Baluska, Frantisek; Volkmann, Dieter; Barlow, Peter W

    2004-07-01

    Cell Theory, also known as cell doctrine, states that all eukaryotic organisms are composed of cells, and that cells are the smallest independent units of life. This Cell Theory has been influential in shaping the biological sciences ever since, in 1838/1839, the botanist Matthias Schleiden and the zoologist Theodore Schwann stated the principle that cells represent the elements from which all plant and animal tissues are constructed. Some 20 years later, in a famous aphorism Omnis cellula e cellula, Rudolf Virchow annunciated that all cells arise only from pre-existing cells. General acceptance of Cell Theory was finally possible only when the cellular nature of brain tissues was confirmed at the end of the 20th century. Cell Theory then rapidly turned into a more dogmatic cell doctrine, and in this form survives up to the present day. In its current version, however, the generalized Cell Theory developed for both animals and plants is unable to accommodate the supracellular nature of higher plants, which is founded upon a super-symplasm of interconnected cells into which is woven apoplasm, symplasm and super-apoplasm. Furthermore, there are numerous examples of multinucleate coenocytes and syncytia found throughout the eukaryote superkingdom posing serious problems for the current version of Cell Theory. To cope with these problems, we here review data which conform to the original proposal of Daniel Mazia that the eukaryotic cell is composed of an elemental Cell Body whose structure is smaller than the cell and which is endowed with all the basic attributes of a living entity. A complement to the Cell Body is the Cell Periphery Apparatus, which consists of the plasma membrane associated with other periphery structures. Importantly, boundary structures of the Cell Periphery Apparatus, although capable of some self-assembly, are largely produced and maintained by Cell Body activities and can be produced from it de novo. These boundary structures serve not only as

  8. NKT Cell Responses to B Cell Lymphoma

    OpenAIRE

    Junxin Li; Wenji Sun; Subrahmanyam, Priyanka B.; Carly Page; Younger, Kenisha M.; Tiper, Irina V.; Matthew Frieman; Kimball, Amy S.; Webb, Tonya J

    2014-01-01

    Natural killer T (NKT) cells are a unique subset of CD1d-restricted T lymphocytes that express characteristics of both T cells and natural killer cells. NKT cells mediate tumor immune-surveillance; however, NKT cells are numerically reduced and functionally impaired in lymphoma patients. Many hematologic malignancies express CD1d molecules and co-stimulatory proteins needed to induce anti-tumor immunity by NKT cells, yet most tumors are poorly immunogenic. In this study, we sought to investig...

  9. Differential effect of shear stress on extracellular signal-regulated kinase and N-terminal Jun kinase in endothelial cells. Gi2- and Gbeta/gamma-dependent signaling pathways.

    Science.gov (United States)

    Jo, H; Sipos, K; Go, Y M; Law, R; Rong, J; McDonald, J M

    1997-01-10

    Shear stress differentially regulates production of many vasoactive factors at the level of gene expression in endothelial cells that may be mediated by mitogen-activated protein kinases, including extracellular signal-regulated kinase (ERK) and N-terminal Jun kinase (JNK). Here we show, using bovine aortic endothelial cells (BAEC), that shear stress differentially regulates ERK and JNK by mechanisms involving Gi2 and pertussis toxin (PTx)-insensitive G-protein-dependent pathways, respectively. Shear activated ERK with a rapid, biphasic time course (maximum by 5 min and basal by 30-min shear exposure) and force dependence (minimum and maximum at 1 and 10 dyn/cm2 shear stress, respectively). PTx treatment prevented shear-dependent activation of ERK1/2, consistent with a Gi-dependent mechanism. In contrast, JNK activity was maximally turned on by a threshold level of shear force (0.5 dyn/cm2 or higher) with a much slower and prolonged time course (requiring at least 30 min to 4 h) than that of ERK. Also, PTx had no effect on shear-dependent activation of JNK. To further define the shear-sensitive ERK and JNK pathways, vectors expressing hemagglutinin epitope-tagged ERK (HA-ERK) or HA-JNK were co-transfected with other vectors by using adenovirus-polylysine in BAEC. Expression of the mutant (alpha)i2(G203), antisense G(alpha)i2 and a dominant negative Ras (N17Ras) prevented shear-dependent activation of HA-ERK, while that of (alpha)i2(G204) and antisense (alpha)i3 did not. Expression of a Gbeta/gamma scavenger, the carboxyl terminus of beta-adrenergic receptor kinase (betaARK-ct), and N17Ras inhibited shear-dependent activation of HA-JNK. Treatment of BAEC with genistein prevented shear-dependent activation of ERK and JNK, indicating the essential role of tyrosine kinase(s) in both ERK and JNK pathways. These results provide evidence that 1) Gi2-protein, Ras, and tyrosine kinase(s) are upstream regulators of shear-dependent activation of ERK and 2) that shear

  10. Antiparietal cell antibody test

    Science.gov (United States)

    APCA; Anti-gastric parietal cell antibody; Atrophic gastritis - anti-gastric parietal cell antibody; Gastric ulcer - anti-gastric parietal cell antibody; Pernicious anemia - anti-gastric parietal cell antibody; ...

  11. Electrorefining cell evaluation

    Energy Technology Data Exchange (ETDEWEB)

    Bronson, M.C.; Thomas, R.L. (ed.)

    1989-04-14

    Operational characteristics of the LANL electrorefining cell, a modified LANL electrorefining cell, and an advanced electrorefining cell (known as the CRAC cell) were determined. Average process yields achieved were: 75% for the LANL cell, 82% for the modified LANL cell, and 86% for the CRAC cell. All product metal from the LANL and modified LANL cells was within foundry specifications. Metal from one run in the CRAC cell exceeded foundry specifications for tantalum. The LANL and modified LANL cells were simple in design and operation, but product separation was more labor intensive than with the CRAC cell. The CRAC cell was more complicated in design but remained relatively simple in operation. A decision analysis concluded that the modified LANL cell was the preferred cell. It was recommended that the modified LANL cell be implemented by the Plutonium Recovery Project at Rocky Flats and that development of the CRAC cell continue. 8 refs., 22 figs., 12 tabs.

  12. Stem Cell Basics

    Science.gov (United States)

    ... Tips Info Center Research Topics Federal Policy Glossary Stem Cell Information General Information Clinical Trials Funding Information Current ... Basics » Stem Cell Basics I. Back to top Stem Cell Basics I. Introduction: What are stem cells, and ...

  13. Potency of Stem Cells

    Indian Academy of Sciences (India)

    First page Back Continue Last page Overview Graphics. Potency of Stem Cells. Totipotent Stem Cells (Zygote + first 2 divisions). -Can form placenta, embryo, and any cell of the body. Pluripotent (Embryonic Stem Cells). -Can form any cell of the body but can not form placenta, hence no embryo. Multipotent (Adult stem cells).

  14. DNA-cell conjugates

    Science.gov (United States)

    Hsiao, Shih-Chia; Francis, Matthew B.; Bertozzi, Carolyn; Mathies, Richard; Chandra, Ravi; Douglas, Erik; Twite, Amy; Toriello, Nicholas; Onoe, Hiroaki

    2016-05-03

    The present invention provides conjugates of DNA and cells by linking the DNA to a native functional group on the cell surface. The cells can be without cell walls or can have cell walls. The modified cells can be linked to a substrate surface and used in assay or bioreactors.

  15. Mechanisms Of Cell Aging in Cell Culture

    OpenAIRE

    Feit, Julia; Gorzelańczyk, Edward Jacek

    2013-01-01

    A key element in the life of cells in culture is the number of cell divisions, not their life time in culture. Serially in vivo transplanted cells also exhibit a finite lifetime, which means that the cell aging is not unique only to a cell culture. There are theories suggesting that the aging of cells in culture may be associated with the aging of the organism from which they were obtained. Cells may stop dividing because of replicative aging, which is the result of telomere shortening. The a...

  16. Skin Stem Cells in Skin Cell Therapy

    Directory of Open Access Journals (Sweden)

    Mollapour Sisakht

    2015-12-01

    Full Text Available Context Preclinical and clinical research has shown that stem cell therapy is a promising therapeutic option for many diseases. This article describes skin stem cells sources and their therapeutic applications. Evidence Acquisition Compared with conventional methods, cell therapy reduces the surgical burden for patients because it is simple and less time-consuming. Skin cell therapy has been developed for variety of diseases. By isolation of the skin stem cell from the niche, in vitro expansion and transplantation of cells offers a surprising healing capacity profile. Results Stem cells located in skin cells have shown interesting properties such as plasticity, transdifferentiation, and specificity. Mesenchymal cells of the dermis, hypodermis, and other sources are currently being investigated to promote regeneration. Conclusions Because skin stem cells are highly accessible from autologous sources and their immunological profile is unique, they are ideal for therapeutic approaches. Optimization of administrative routes requires more investigation own to the lack of a standard protocol.

  17. Structural and functional organization of the HF.10 human zinc finger gene (ZNF35) located on chromosome 3p21-p22

    DEFF Research Database (Denmark)

    Lanfrancone, L; Pengue, G; Pandolfi, P P

    1992-01-01

    the last 3' exon. The genomic region surrounding HF.10 exon 1 contains a CpG island and acts as a promoter in vitro. Using transient CAT assay in cotransfection experiments in cultured cells, we have determined that the HF.10 finger protein is a transcriptional transactivator. Restriction enzyme mapping...

  18. Characterization of the effects of four HTR3B polymorphisms on human 5-HT3AB receptor expression and signalling

    DEFF Research Database (Denmark)

    Krzywkowski, Karen Margrethe; Davies, Paul A.; Irving, Andrew J.

    2008-01-01

    -HT3AB receptors. METHODS AND RESULTS: 5-HT3AB receptor signalling was studied in a fluorescence-based cell membrane potential assay and by electrophysiology. Expression levels of cotransfected epitope-tagged 5-HT3A and 5-HT3B subunits were determined using enzyme-linked immunosorbent assay...

  19. CHO glyco-engineering using CRISPR/Cas9 multiplexing for protein production with homogeneous N-glycan profiles

    DEFF Research Database (Denmark)

    Amann, Thomas; Hansen, Anders Holmgaard; Pristovsek, Nusa

    Combining the chinese hamster ovary (CHO) - K1 draft genome1,2, identified CHO glycosyltransferases3 and the power of multiplexing gene knock-outs with CRISPR/Cas94 via co-transfection of Cas9 and one single guiding RNA (sgRNA) per target, we generated 20 Rituximab expressing CHO-S cell lines...

  20. Study of Mechanisms of an Anti-Apoptotic Protein, BI-1, in Prostate Cancer

    Science.gov (United States)

    2007-12-01

    alter the protein level of selected protein targets. BAR full length, BARdR, or pcDNA was cotransfected with potential target proteins in HEK293 T...pDED domain of BAR may be involved in regulating the protein stability in a proteosome- dependent pathway. HEK293T cells were transfected with BAR

  1. GPCR Interaction: 29 [GRIPDB[Archive

    Lifescience Database Archive (English)

    Full Text Available observed by Western blotting of immunoprecipitates of either Flag-A2AR or hemagglutinin-mGluR5 in membrane preparation...s from cotransfected HEK-293 cells and of native A2AR and mGluR5 in rat striatal membrane preparation

  2. GPCR Interaction: 28 [GRIPDB[Archive

    Lifescience Database Archive (English)

    Full Text Available served by Western blotting of immunoprecipitates of either Flag-A2AR or hemagglutinin-mGluR5 in membrane preparation...s from cotransfected HEK-293 cells and of native A2AR and mGluR5 in rat striatal membrane preparation

  3. Environmental polycyclic aromatic hydrocarbons affect androgen receptor activation in vitro

    DEFF Research Database (Denmark)

    Vinggaard, Anne Marie; Hnida, Christina; Larsen, John Christian

    2000-01-01

    Nine structurally different polycyclic aromatic hydrocarbons (PAHs) were tested for their ability to either agonize or antagonize the human androgen receptor (hAR) in a sensitive reporter gene assay based on CHO cells transiently cotransfected with a hAR vector and an MMTV-LUC vector. Benz...

  4. Different activities of the adenovirus types 5 and 12 E1A regions in transformation with the EJ Ha-ras oncogene

    NARCIS (Netherlands)

    Jochemsen, A.G.; Bernards, R.A.; Kranen, H.J. van; Houweling, A.; Bos, J.L.; Eb, A.J. van der

    1986-01-01

    We have compared the capacities of the E1A regions of nononcogenic adenovirus type 5 (Ad5) and highly oncogenic Ad12 to cooperate with the EJ bladder carcinoma Ha-ras-1 oncogene in the transformation of primary baby rat kidney cells. Both E1A regions, when cotransfected with the Ha-ras oncogene,

  5. GPCR Interaction: 24 [GRIPDB[Archive

    Lifescience Database Archive (English)

    Full Text Available pe 1 mGluR1 Experiment Co-immunoprecipitation experiments showed a close and subtype-specific interaction be...tween mGluR1alpha and A1R in both rat cerebellar synaptosomes and co-transfected HEK-293 cells. ... 11278325 Immunoprecipitation NP_001041695.1 ...

  6. GPCR Interaction: 23 [GRIPDB[Archive

    Lifescience Database Archive (English)

    Full Text Available enosine A1 A1R Experiment Co-immunoprecipitation experiments showed a close and subtype-specific interaction... between mGluR1alpha and A1R in both rat cerebellar synaptosomes and co-transfected HEK-293 cells. ... 11278325 Immunoprecipitation NP_001107801.1 ...

  7. Functional effects of KCNE3 mutation and its role in the development of Brugada syndrome

    DEFF Research Database (Denmark)

    Delpón, Eva; Cordeiro, Jonathan M; Núñez, Lucía

    2008-01-01

    KCND3 or KCNQ1. Whole-cell patch clamp studies were performed after 48 hours. Interactions between Kv4.3 and KCNE3 were analyzed in co-immunoprecipitation experiments in human atrial samples. Co-transfection of R99H-KCNE3 with KCNQ1 produced no alteration in current magnitude or kinetics. However, co...

  8. Rescue of ligand binding of a mutant IGF-I receptor by complementation

    DEFF Research Database (Denmark)

    Chakravarty, Arjun Anders; Hinrichsen, Jane; Whittaker, Linda

    2005-01-01

    from a wild-type receptor monomer and a mutant receptor monomer devoid of binding activity. Receptor hybrids were generated by transient co-transfection of cDNAs encoding wild-type and mutant receptors with unique epitope tags. Hybrid receptors were purified from transfected cells by sequential immuno...

  9. Integrated circuit cell library

    Science.gov (United States)

    Whitaker, Sterling R. (Inventor); Miles, Lowell H. (Inventor)

    2005-01-01

    According to the invention, an ASIC cell library for use in creation of custom integrated circuits is disclosed. The ASIC cell library includes some first cells and some second cells. Each of the second cells includes two or more kernel cells. The ASIC cell library is at least 5% comprised of second cells. In various embodiments, the ASIC cell library could be 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more comprised of second cells.

  10. Modeling cell-in-cell structure into its biological significance

    OpenAIRE

    He, M-f; Wang, S; Wang, Y.; Wang, X-n

    2013-01-01

    Although cell-in-cell structure was noted 100 years ago, the molecular mechanisms of ?entering' and the destination of cell-in-cell remain largely unclear. It takes place among the same type of cells (homotypic cell-in-cell) or different types of cells (heterotypic cell-in-cell). Cell-in-cell formation affects both effector cells and their host cells in multiple aspects, while cell-in-cell death is under more intensive investigation. Given that cell-in-cell has an important role in maintainin...

  11. Automated Cell-Cutting for Cell Cloning

    Science.gov (United States)

    Ichikawa, Akihiko; Tanikawa, Tamio; Matsukawa, Kazutsugu; Takahashi, Seiya; Ohba, Kohtaro

    We develop an automated cell-cutting technique for cell cloning. Animal cells softened by the cytochalasin treatment are injected into a microfluidic chip. The microfluidic chip contains two orthogonal channels: one microchannel is wide, used to transport cells, and generates the cutting flow; the other is thin and used for aspiration, fixing, and stretching of the cell. The injected cell is aspirated and stretched in the thin microchannel. Simultaneously, the volumes of the cell before and after aspiration are calculated; the volumes are used to calculate the fluid flow required to aspirate half the volume of the cell into the thin microchannel. Finally, we apply a high-speed flow in the orthogonal microchannel to bisect the cell. This paper reports the cutting process, the cutting system, and the results of the experiment.

  12. Modeling cell-in-cell structure into its biological significance.

    Science.gov (United States)

    He, M-f; Wang, S; Wang, Y; Wang, X-n

    2013-05-16

    Although cell-in-cell structure was noted 100 years ago, the molecular mechanisms of 'entering' and the destination of cell-in-cell remain largely unclear. It takes place among the same type of cells (homotypic cell-in-cell) or different types of cells (heterotypic cell-in-cell). Cell-in-cell formation affects both effector cells and their host cells in multiple aspects, while cell-in-cell death is under more intensive investigation. Given that cell-in-cell has an important role in maintaining homeostasis, aberrant cell-in-cell process contributes to the etiopathology in humans. Indeed, cell-in-cell is observed in many pathological processes of human diseases. In this review, we intend to discuss the biological models of cell-in-cell structures under physiological and pathological status.

  13. Cell culture purity issues and DFAT cells

    Energy Technology Data Exchange (ETDEWEB)

    Wei, Shengjuan [College of Animal Science and Technology, Northwest A and F University, Yangling, Shaanxi Province 712100 (China); Department of Animal Sciences, Washington State University, Pullman, WA 99164 (United States); Bergen, Werner G. [Program in Cellular and Molecular Biosciences/Department of Animal Sciences, Auburn University, Auburn, AL 36849 (United States); Hausman, Gary J. [Animal Science Department, University of Georgia, Athens, GA 30602-2771 (United States); Zan, Linsen, E-mail: zanls@yahoo.com.cn [College of Animal Science and Technology, Northwest A and F University, Yangling, Shaanxi Province 712100 (China); Dodson, Michael V., E-mail: dodson@wsu.edu [Department of Animal Sciences, Washington State University, Pullman, WA 99164 (United States)

    2013-04-12

    Highlights: •DFAT cells are progeny cells derived from dedifferentiated mature adipocytes. •Common problems in this research is potential cell contamination of initial cultures. •The initial cell culture purity is crucial in DFAT cell research field. -- Abstract: Dedifferentiation of mature adipocytes, in vitro, has been pursued/documented for over forty years. The subsequent progeny cells are named dedifferentiated adipocyte-derived progeny cells (DFAT cells). DFAT cells are proliferative and likely to possess mutilineage potential. As a consequence, DFAT cells and their progeny/daughter cells may be useful as a potential tool for various aspects of tissue engineering and as potential vectors for the alleviation of several disease states. Publications in this area have been increasing annually, but the purity of the initial culture of mature adipocytes has seldom been documented. Consequently, it is not always clear whether DFAT cells are derived from dedifferentiated mature (lipid filled) adipocytes or from contaminating cells that reside in an impure culture.

  14. In silico characterization of cell-cell interactions using a cellular automata model of cell culture.

    Science.gov (United States)

    Kihara, Takanori; Kashitani, Kosuke; Miyake, Jun

    2017-07-14

    Cell proliferation is a key characteristic of eukaryotic cells. During cell proliferation, cells interact with each other. In this study, we developed a cellular automata model to estimate cell-cell interactions using experimentally obtained images of cultured cells. We used four types of cells; HeLa cells, human osteosarcoma (HOS) cells, rat mesenchymal stem cells (MSCs), and rat smooth muscle A7r5 cells. These cells were cultured and stained daily. The obtained cell images were binarized and clipped into squares containing about 104 cells. These cells showed characteristic cell proliferation patterns. The growth curves of these cells were generated from the cell proliferation images and we determined the doubling time of these cells from the growth curves. We developed a simple cellular automata system with an easily accessible graphical user interface. This system has five variable parameters, namely, initial cell number, doubling time, motility, cell-cell adhesion, and cell-cell contact inhibition (of proliferation). Within these parameters, we obtained initial cell numbers and doubling times experimentally. We set the motility at a constant value because the effect of the parameter for our simulation was restricted. Therefore, we simulated cell proliferation behavior with cell-cell adhesion and cell-cell contact inhibition as variables. By comparing growth curves and proliferation cell images, we succeeded in determining the cell-cell interaction properties of each cell. Simulated HeLa and HOS cells exhibited low cell-cell adhesion and weak cell-cell contact inhibition. Simulated MSCs exhibited high cell-cell adhesion and positive cell-cell contact inhibition. Simulated A7r5 cells exhibited low cell-cell adhesion and strong cell-cell contact inhibition. These simulated results correlated with the experimental growth curves and proliferation images. Our simulation approach is an easy method for evaluating the cell-cell interaction properties of cells.

  15. Fuel cell-fuel cell hybrid system

    Science.gov (United States)

    Geisbrecht, Rodney A.; Williams, Mark C.

    2003-09-23

    A device for converting chemical energy to electricity is provided, the device comprising a high temperature fuel cell with the ability for partially oxidizing and completely reforming fuel, and a low temperature fuel cell juxtaposed to said high temperature fuel cell so as to utilize remaining reformed fuel from the high temperature fuel cell. Also provided is a method for producing electricity comprising directing fuel to a first fuel cell, completely oxidizing a first portion of the fuel and partially oxidizing a second portion of the fuel, directing the second fuel portion to a second fuel cell, allowing the first fuel cell to utilize the first portion of the fuel to produce electricity; and allowing the second fuel cell to utilize the second portion of the fuel to produce electricity.

  16. Red blood cells, sickle cell (image)

    Science.gov (United States)

    ... is an inherited blood disease in which the red blood cells produce abnormal pigment (hemoglobin). The abnormal hemoglobin causes deformity of the red blood cells into crescent or sickle-shapes, as ...

  17. Stem Cell Information: Glossary

    Science.gov (United States)

    ... it is called a fetus . Embryoid bodies - Rounded collections of cells that arise when embryonic stem cells ... dividing without differentiating for a prolonged period in culture, and are known to develop into cells and ...

  18. NK cells and T cells: mirror images?

    NARCIS (Netherlands)

    Versteeg, R.

    1992-01-01

    The expression of MHC class I molecules protects cells against lysis by natural killer (NK) cells. It is possible that NK cells are 'educated' to recognize self MHC class I molecules and that the combination of self peptide and MHC class I molecule blocks NK-mediated lysis. Here, Rogier Versteeg

  19. Snail modulates cell metabolism in MDCK cells

    Energy Technology Data Exchange (ETDEWEB)

    Haraguchi, Misako, E-mail: haraguci@m3.kufm.kagoshima-u.ac.jp [Department of Biochemistry and Molecular Biology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Indo, Hiroko P. [Department of Maxillofacial Radiology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Iwasaki, Yasumasa [Health Care Center, Kochi University, Kochi 780-8520 (Japan); Iwashita, Yoichiro [Department of Maxillofacial Radiology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Fukushige, Tomoko [Department of Dermatology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Majima, Hideyuki J. [Department of Maxillofacial Radiology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Izumo, Kimiko; Horiuchi, Masahisa [Department of Environmental Medicine, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Kanekura, Takuro [Department of Dermatology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Furukawa, Tatsuhiko [Department of Molecular Oncology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Ozawa, Masayuki [Department of Biochemistry and Molecular Biology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan)

    2013-03-22

    Highlights: ► MDCK/snail cells were more sensitive to glucose deprivation than MDCK/neo cells. ► MDCK/snail cells had decreased oxidative phosphorylation, O{sub 2} consumption and ATP content. ► TCA cycle enzyme activity, but not expression, was lower in MDCK/snail cells. ► MDCK/snail cells showed reduced PDH activity and increased PDK1 expression. ► MDCK/snail cells showed reduced expression of GLS2 and ACLY. -- Abstract: Snail, a repressor of E-cadherin gene transcription, induces epithelial-to-mesenchymal transition and is involved in tumor progression. Snail also mediates resistance to cell death induced by serum depletion. By contrast, we observed that snail-expressing MDCK (MDCK/snail) cells undergo cell death at a higher rate than control (MDCK/neo) cells in low-glucose medium. Therefore, we investigated whether snail expression influences cell metabolism in MDCK cells. Although gylcolysis was not affected in MDCK/snail cells, they did exhibit reduced pyruvate dehydrogenase (PDH) activity, which controls pyruvate entry into the tricarboxylic acid (TCA) cycle. Indeed, the activity of multiple enzymes involved in the TCA cycle was decreased in MDCK/snail cells, including that of mitochondrial NADP{sup +}-dependent isocitrate dehydrogenase (IDH2), succinate dehydrogenase (SDH), and electron transport Complex II and Complex IV. Consequently, lower ATP content, lower oxygen consumption and increased survival under hypoxic conditions was also observed in MDCK/snail cells compared to MDCK/neo cells. In addition, the expression and promoter activity of pyruvate dehydrogenase kinase 1 (PDK1), which phosphorylates and inhibits the activity of PDH, was increased in MDCK/snail cells, while expression levels of glutaminase 2 (GLS2) and ATP-citrate lyase (ACLY), which are involved in glutaminolysis and fatty acid synthesis, were decreased in MDCK/snail cells. These results suggest that snail modulates cell metabolism by altering the expression and activity of

  20. Fish Stem Cell Cultures

    OpenAIRE

    Hong, Ni; Li, Zhendong; Hong, Yunhan

    2011-01-01

    Stem cells have the potential for self-renewal and differentiation. First stem cell cultures were derived 30 years ago from early developing mouse embryos. These are pluripotent embryonic stem (ES) cells. Efforts towards ES cell derivation have been attempted in other mammalian and non-mammalian species. Work with stem cell culture in fish started 20 years ago. Laboratory fish species, in particular zebrafish and medaka, have been the focus of research towards stem cell cultures. Medaka is th...

  1. Cell control report

    CERN Document Server

    2013-01-01

    Please note this is a Short Discount publication. This extensive report provides an essential overview of cells and their use as factory automation building blocks. The following issues are discussed in depth: Cell integration Cell software and standards Future technologies applied to cells Plus Cell control applications including: - rotary parts manufacturing - diesel engine component development - general cell control development at the General Electric Corporation - a vendor list.

  2. Lung cancer - small cell

    Science.gov (United States)

    ... carcinoma Small cell carcinoma Squamous cell carcinoma Secondhand smoke and lung cancer Normal lungs and alveoli Respiratory system Smoking hazards Bronchoscope References Horn L, Eisenberg R, ...

  3. GSPEL - Fuel Cell Laboratory

    Data.gov (United States)

    Federal Laboratory Consortium — The Fuel Cell Lab (FCL)Established to investigate, integrate, testand verifyperformance and technology readiness offuel cell systems and fuel reformers for use with...

  4. Epithelial cell polarity, stem cells and cancer

    DEFF Research Database (Denmark)

    Martin-Belmonte, Fernando; Perez-Moreno, Mirna

    2011-01-01

    , deregulation of adhesion and polarity proteins can cause misoriented cell divisions and increased self-renewal of adult epithelial stem cells. In this Review, we highlight some advances in the understanding of how loss of epithelial cell polarity contributes to tumorigenesis.......After years of extensive scientific discovery much has been learned about the networks that regulate epithelial homeostasis. Loss of expression or functional activity of cell adhesion and cell polarity proteins (including the PAR, crumbs (CRB) and scribble (SCRIB) complexes) is intricately related...

  5. Human memory B cells.

    Science.gov (United States)

    Seifert, M; Küppers, R

    2016-12-01

    A key feature of the adaptive immune system is the generation of memory B and T cells and long-lived plasma cells, providing protective immunity against recurring infectious agents. Memory B cells are generated in germinal center (GC) reactions in the course of T cell-dependent immune responses and are distinguished from naive B cells by an increased lifespan, faster and stronger response to stimulation and expression of somatically mutated and affinity matured immunoglobulin (Ig) genes. Approximately 40% of human B cells in adults are memory B cells, and several subsets were identified. Besides IgG(+) and IgA(+) memory B cells, ∼50% of peripheral blood memory B cells express IgM with or without IgD. Further smaller subpopulations have additionally been described. These various subsets share typical memory B cell features, but likely also fulfill distinct functions. IgM memory B cells appear to have the propensity for refined adaptation upon restimulation in additional GC reactions, whereas reactivated IgG B cells rather differentiate directly into plasma cells. The human memory B-cell pool is characterized by (sometimes amazingly large) clonal expansions, often showing extensive intraclonal IgV gene diversity. Moreover, memory B-cell clones are frequently composed of members of various subsets, showing that from a single GC B-cell clone a variety of memory B cells with distinct functions is generated. Thus, the human memory B-cell compartment is highly diverse and flexible. Several B-cell malignancies display features suggesting a derivation from memory B cells. This includes a subset of chronic lymphocytic leukemia, hairy cell leukemia and marginal zone lymphomas. The exposure of memory B cells to oncogenic events during their generation in the GC, the longevity of these B cells and the ease to activate them may be key determinants for their malignant transformation.

  6. Stem cell evolutionary paradigm and cell engineering.

    Science.gov (United States)

    Ivanovic, Z

    2017-09-01

    Studying hematopoietic and mesenchymal stem cells for almost three decades revealed some similarities between the stem cell entity and the single-celled eukaryotes exhibiting the anaerobic/facultative aerobic metabolic features. A careful analysis of nowadays knowledge concerning the early eukaryotic evolution allowed us to reveal some analogies between stem cells in the metazoan tissues and the single-celled eukaryotes which existed during the first phase of eukaryotes evolution in mid-Proterozoic era. In fact, it is possible to trace the principle of the self-renewal back to the first eukaryotic common ancestor, the first undifferentiated nucleated cell possessing the primitive, mostly anaerobically-respiring mitochondria and a capacity to reproduction by a simple cell division "à l'identique". Similarly, the diversification of these single-cell eukaryotes and acquiring of complex life cycle allowed/conditioned by the increase of O2 in atmosphere (and consequently in the water environment) represents a prototype for the phenomenon of commitment/differentiation. This point of view allowed to predict the ex-vivo behavior of stem cells with respect to the O2 availability and metabolic profile which enabled to conceive the successful protocols of stem cell expansion and ex vivo conditioning based on "respecting" this relationship between the anaerobiosis and stemness. In this review, the basic elements of this paradigm and a possible application in cell engineering were discussed. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  7. Cell mechanics: a dialogue

    Science.gov (United States)

    Tao, Jiaxiang; Li, Yizeng; Vig, Dhruv K.; Sun, Sean X.

    2017-03-01

    Under the microscope, eukaryotic animal cells can adopt a variety of different shapes and sizes. These cells also move and deform, and the physical mechanisms driving these movements and shape changes are important in fundamental cell biology, tissue mechanics, as well as disease biology. This article reviews some of the basic mechanical concepts in cells, emphasizing continuum mechanics description of cytoskeletal networks and hydrodynamic flows across the cell membrane. We discuss how cells can generate movement and shape changes by controlling mass fluxes at the cell boundary. These mass fluxes can come from polymerization/depolymerization of actin cytoskeleton, as well as osmotic and hydraulic pressure-driven flow of water across the cell membrane. By combining hydraulic pressure control with force balance conditions at the cell surface, we discuss a quantitative mechanism of cell shape and volume control. The broad consequences of this model on cell mechanosensation and tissue mechanics are outlined.

  8. 8-Alkylcoumarins from the Fruits of Cnidium monnieri Protect against Hydrogen Peroxide Induced Oxidative Stress Damage

    Directory of Open Access Journals (Sweden)

    Chi-I Chang

    2014-03-01

    Full Text Available Three new 8-alkylcoumarins, 7-O-methylphellodenol-B (1, 7-methoxy-8-(3-methyl- 2,3-epoxy-1-oxobutylchromen-2-one (2, and 3'-O-methylvaginol (3, together with seven known compounds (4–10 were isolated from the fruits of Cnidium monnieri. Their structures were determined by detailed analysis of spectroscopic data and comparison with the data of known analogues. All the isolates were evaluated the cytoprotective activity by MTS cell proliferation assay and the results showed that all the three new 8-alkylcoumarins exhibited cytoprotective effect on Neuro-2a neuroblastoma cells injured by hydrogen peroxide.

  9. 8-Alkylcoumarins from the fruits of Cnidium monnieri protect against hydrogen peroxide induced oxidative stress damage.

    Science.gov (United States)

    Chang, Chi-I; Hu, Wan-Chiao; Shen, Che-Piao; Hsu, Ban-Dar; Lin, Wei-Yong; Sung, Ping-Jyun; Wang, Wei-Hsien; Wu, Jin-Bin; Kuo, Yueh-Hsiung

    2014-03-17

    Three new 8-alkylcoumarins, 7-O-methylphellodenol-B (1), 7-methoxy-8-(3-methyl- 2,3-epoxy-1-oxobutyl)chromen-2-one (2), and 3'-O-methylvaginol (3), together with seven known compounds (4-10) were isolated from the fruits of Cnidium monnieri. Their structures were determined by detailed analysis of spectroscopic data and comparison with the data of known analogues. All the isolates were evaluated the cytoprotective activity by MTS cell proliferation assay and the results showed that all the three new 8-alkylcoumarins exhibited cytoprotective effect on Neuro-2a neuroblastoma cells injured by hydrogen peroxide.

  10. Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Felthaus, O. [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany); Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Ettl, T.; Gosau, M.; Driemel, O. [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Brockhoff, G. [Department of Gynecology and Obstetrics, University of Regensburg (Germany); Reck, A. [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Zeitler, K. [Institute of Pathology, University of Regensburg (Germany); Hautmann, M. [Department of Radiotherapy, University of Regensburg (Germany); Reichert, T.E. [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Schmalz, G. [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany); Morsczeck, C., E-mail: christian.morsczeck@klinik.uni-regensburg.de [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany)

    2011-04-01

    Research highlights: {yields} Four oral squamous cancer cell lines (OSCCL) were analyzed for cancer stem cells (CSCs). {yields} Single cell derived colonies of OSCCL express CSC-marker CD133 differentially. {yields} Monoclonal cell lines showed reduced sensitivity for Paclitaxel. {yields} In situ CD133{sup +} cells are slow cycling (Ki67-) indicating a reduced drug sensitivity. {yields} CD133{sup +} and CSC-like cells can be obtained from single colony forming cells of OSCCL. -- Abstract: Resistance of oral squamous cell carcinomas (OSCC) to conventional chemotherapy or radiation therapy might be due to cancer stem cells (CSCs). The development of novel anticancer drugs requires a simple method for the enrichment of CSCs. CSCs can be enriched from OSCC cell lines, for example, after cultivation in serum-free cell culture medium (SFM). In our study, we analyzed four OSCC cell lines for the presence of CSCs. CSC-like cells could not be enriched with SFM. However, cell lines obtained from holoclone colonies showed CSC-like properties such as a reduced rate of cell proliferation and a reduced sensitivity to Paclitaxel in comparison to cells from the parental lineage. Moreover, these cell lines differentially expressed the CSC-marker CD133, which is also upregulated in OSCC tissues. Interestingly, CD133{sup +} cells in OSCC tissues expressed little to no Ki67, the cell proliferation marker that also indicates reduced drug sensitivity. Our study shows a method for the isolation of CSC-like cell lines from OSCC cell lines. These CSC-like cell lines could be new targets for the development of anticancer drugs under in vitro conditions.

  11. Specific cell cycle synchronization with butyrate and cell cycle analysis

    Science.gov (United States)

    Synchronized cells have been invaluable for many kinds of cell cycle and cell proliferation studies. Butyrate induces cell cycle arrest and apoptosis in MDBK cells. To explore the possibility of using butyrate-blocked cells to obtain synchronized cells, we investigated the property of the cell cyc...

  12. Insect Cell Culture

    NARCIS (Netherlands)

    Oers, van M.M.; Lynn, D.E.

    2010-01-01

    Insect cell cultures are widely used in studies on insect cell physiology, developmental biology and microbial pathology. In particular, insect cell culture is an indispensable tool for the study of insect viruses. The first continuously growing insect cell cultures were established from

  13. Tracking adult stem cells

    NARCIS (Netherlands)

    Snippert, H.J.G.; Clevers, H.

    2011-01-01

    The maintenance of stem-cell-driven tissue homeostasis requires a balance between the generation and loss of cell mass. Adult stem cells have a close relationship with the surrounding tissue--known as their niche--and thus, stem-cell studies should preferably be performed in a physiological context,

  14. Nanocomposite Photoelectrochemical Cells

    Science.gov (United States)

    Narayan, Sri R.; Kindler, Andrew; Whitacre, Jay F.

    2007-01-01

    Improved, solid-state photoelectrochemical cells for converting solar radiation to electricity have been proposed. (In general, photoelectrochemical cells convert incident light to electricity through electrochemical reactions.) It is predicted that in comparison with state-of-the-art photoelectrochemical cells, these cells will be found to operate with greater solar-to-electric energy-conversion efficiencies.

  15. Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth

    Energy Technology Data Exchange (ETDEWEB)

    Varga, Nora [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary); Vereb, Zoltan; Rajnavoelgyi, Eva [Department of Immunology, Medical and Health Science Centre, University of Debrecen, Debrecen (Hungary); Nemet, Katalin; Uher, Ferenc; Sarkadi, Balazs [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary); Apati, Agota, E-mail: apati@kkk.org.hu [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary)

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer MSC like cells were derived from hESC by a simple and reproducible method. Black-Right-Pointing-Pointer Differentiation and immunosuppressive features of MSCl cells were similar to bmMSC. Black-Right-Pointing-Pointer MSCl cells as feeder cells support the undifferentiated growth of hESC. -- Abstract: Mesenchymal stem cell like (MSCl) cells were generated from human embryonic stem cells (hESC) through embryoid body formation, and isolated by adherence to plastic surface. MSCl cell lines could be propagated without changes in morphological or functional characteristics for more than 15 passages. These cells, as well as their fluorescent protein expressing stable derivatives, efficiently supported the growth of undifferentiated human embryonic stem cells as feeder cells. The MSCl cells did not express the embryonic (Oct4, Nanog, ABCG2, PODXL, or SSEA4), or hematopoietic (CD34, CD45, CD14, CD133, HLA-DR) stem cell markers, while were positive for the characteristic cell surface markers of MSCs (CD44, CD73, CD90, CD105). MSCl cells could be differentiated toward osteogenic, chondrogenic or adipogenic directions and exhibited significant inhibition of mitogen-activated lymphocyte proliferation, and thus presented immunosuppressive features. We suggest that cultured MSCl cells can properly model human MSCs and be applied as efficient feeders in hESC cultures.

  16. T-Cell Lymphoma

    Science.gov (United States)

    Getting the Facts T-Cell Lymphoma Overview Lymphoma is the most common blood cancer. The two main forms of lymphoma are Hodgkin lymphoma ... develop into lymphomas: B-lymphocytes (B-cells) and T-lymphocytes (T-cells). T-cell lymphomas account for ...

  17. Molten carbonate fuel cell

    Science.gov (United States)

    Kaun, T.D.; Smith, J.L.

    1986-07-08

    A molten electrolyte fuel cell is disclosed with an array of stacked cells and cell enclosures isolating each cell except for access to gas manifolds for the supply of fuel or oxidant gas or the removal of waste gas. The cell enclosures collectively provide an enclosure for the array and effectively avoid the problems of electrolyte migration and the previous need for compression of stack components. The fuel cell further includes an inner housing about and in cooperation with the array enclosure to provide a manifold system with isolated chambers for the supply and removal of gases. An external insulated housing about the inner housing provides thermal isolation to the cell components.

  18. NK Cell Exhaustion

    Science.gov (United States)

    Bi, Jiacheng; Tian, Zhigang

    2017-01-01

    Natural killer cells are important effector lymphocytes of the innate immune system, playing critical roles in antitumor and anti-infection host defense. Tumor progression or chronic infections, however, usually leads to exhaustion of NK cells, thus limiting the antitumor/infection potential of NK cells. In many tumors or chronic infections, multiple mechanisms might contribute to the exhaustion of NK cells, such as dysregulated NK cell receptors signaling, as well as suppressive effects by regulatory cells or soluble factors within the microenvironment. Better understanding of the characteristics, as well as the underlying mechanisms of NK cell exhaustion, not only should increase our understanding of the basic biology of NK cells but also could reveal novel NK cell-based antitumor/infection targets. Here, we provide an overview of our current knowledge on NK cell exhaustion in tumors, and in chronic infections. PMID:28702032

  19. Fuel cells seminar

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1996-12-01

    This year`s meeting highlights the fact that fuel cells for both stationary and transportation applications have reached the dawn of commercialization. Sales of stationary fuel cells have grown steadily over the past 2 years. Phosphoric acid fuel cell buses have been demonstrated in urban areas. Proton-exchange membrane fuel cells are on the verge of revolutionizing the transportation industry. These activities and many more are discussed during this seminar, which provides a forum for people from the international fuel cell community engaged in a wide spectrum of fuel cell activities. Discussions addressing R&D of fuel cell technologies, manufacturing and marketing of fuel cells, and experiences of fuel cell users took place through oral and poster presentations. For the first time, the seminar included commercial exhibits, further evidence that commercial fuel cell technology has arrived. A total of 205 papers is included in this volume.

  20. Cellular function of neuropathy target esterase in lysophosphatidylcholine action.

    Science.gov (United States)

    Vose, Sarah C; Fujioka, Kazutoshi; Gulevich, Alex G; Lin, Amy Y; Holland, Nina T; Casida, John E

    2008-11-01

    Neuropathy target esterase (NTE) plays critical roles in embryonic development and maintenance of peripheral axons. It is a secondary target of some organophosphorus toxicants including analogs of insecticides and chemical warfare agents. Although the mechanistic role of NTE in vivo is poorly defined, it is known to hydrolyze lysophosphatidylcholine (LPC) in vitro and may protect cell membranes from cytotoxic accumulation of LPC. To determine the cellular function of NTE, Neuro-2a and COS-7 cells were transfected with a full-length human NTE-containing plasmid yielding recombinant NTE (rNTE). We find the same inhibitor sensitivity and specificity profiles for rNTE assayed with LPC or phenyl valerate (a standard NTE substrate) and that this correlation extends to the LPC hydrolases of human brain, lymphocytes and erythrocytes. All of these LPC hydrolases are therefore very similar to each other in respect to a conserved inhibitor binding site conformation. NTE is expressed in brain and lymphocytes and contributes to LPC hydrolase activities in these tissues. The enzyme or enzymes responsible for erythrocyte LPC hydrolase activity remain to be identified. We also show that rNTE protects Neuro-2a and COS-7 cells from exogenous LPC cytotoxicity. Expression of rNTE in Neuro-2a cells alters their phospholipid balance (analyzed by liquid chromatography-mass spectrometry with single ion monitoring) by lowering LPC-16:0 and LPC-18:0 and elevating glycerophosphocholine without a change in phosphatidylcholine-16:0/18:1 or 16:0/18:2. NTE therefore serves an important function in LPC homeostasis and action.

  1. Stem Cell Networks

    OpenAIRE

    Werner, Eric

    2016-01-01

    We present a general computational theory of stem cell networks and their developmental dynamics. Stem cell networks are special cases of developmental control networks. Our theory generates a natural classification of all possible stem cell networks based on their network architecture. Each stem cell network has a unique topology and semantics and developmental dynamics that result in distinct phenotypes. We show that the ideal growth dynamics of multicellular systems generated by stem cell ...

  2. Hybrid Fuel Cell Systems

    OpenAIRE

    Brouwer, J.; Samuelsen, GS

    2001-01-01

    Examples of hybrid fuel cell power generation cycles are the combine high-temperature fuel cells and gas turbines, reciprocating engines, or another fuel cell. These represent the hybrid power plants of the future. The conceptual systems have the potential to achieve efficiencies greater than 70 percent and be commercially ready by year 2010 or sooner. The hybrid fuel cell/turbine (FC/T) power plant will combine a high-temperature, conventional molten carbonate fuel cell (MCFC)...

  3. The cell cycle as a brake for ?-cell regeneration from embryonic stem cells

    OpenAIRE

    El-Badawy, Ahmed; El-Badri, Nagwa

    2016-01-01

    The generation of insulin-producing ? cells from stem cells in vitro provides a promising source of cells for cell transplantation therapy in diabetes. However, insulin-producing cells generated from human stem cells show deficiency in many functional characteristics compared with pancreatic ? cells. Recent reports have shown molecular ties between the cell cycle and the differentiation mechanism of embryonic stem (ES) cells, assuming that cell fate decisions are controlled by the cell cycle ...

  4. Efficient edition of the bovine PRNP prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system.

    Science.gov (United States)

    Bevacqua, R J; Fernandez-Martín, R; Savy, V; Canel, N G; Gismondi, M I; Kues, W A; Carlson, D F; Fahrenkrug, S C; Niemann, H; Taboga, O A; Ferraris, S; Salamone, D F

    2016-11-01

    The recently developed engineered nucleases, such as zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease (Cas) 9, provide new opportunities for gene editing in a straightforward manner. However, few reports are available regarding CRISPR application and efficiency in cattle. Here, the CRISPR/Cas9 system was used with the aim of inducing knockout and knock-in alleles of the bovine PRNP gene, responsible for mad cow disease, both in bovine fetal fibroblasts and in IVF embryos. Five single-guide RNAs were designed to target 875 bp of PRNP exon 3, and all five were codelivered with Cas9. The feasibility of inducing homologous recombination (HR) was evaluated with a reporter vector carrying EGFP flanked by 1 kbp PRNP regions (pHRegfp). For somatic cells, plasmids coding for Cas9 and for each of the five single-guide RNAs (pCMVCas9 and pSPgRNAs) were transfected under two different conditions (1X and 2X). For IVF zygotes, cytoplasmic injection was conducted with either plasmids or mRNA. For plasmid injection groups, 1 pg pCMVCas9 + 0.1 pg of each pSPgRNA (DNA2X) was used per zygote. In the case of RNA, two amounts (RNA1X and RNA2X) were compared. To assess the occurrence of HR, a group additionally cotransfected or coinjected with pHRegfp plasmid was included. Somatic cell lysates were analyzed by polymerase chain reaction and surveyor assay. In the case of embryos, the in vitro development and the genotype of blastocysts were evaluated by polymerase chain reaction and sequencing. In somatic cells, 2X transfection resulted in indels and large deletions of the targeted PRNP region. Regarding embryo injection, higher blastocyst rates were obtained for RNA injected groups (46/103 [44.6%] and 55/116 [47.4%] for RNA1X and RNA2X) than for the DNA2X group (26/140 [18.6%], P < 0.05). In 46% (26/56) of the total sequenced blastocysts, specific gene editing was

  5. Quantitative Characterization of Cell Behaviors through Cell Cycle Progression via Automated Cell Tracking

    Science.gov (United States)

    Wang, Yuliang; Jeong, Younkoo; Jhiang, Sissy M.; Yu, Lianbo; Menq, Chia-Hsiang

    2014-01-01

    Cell behaviors are reflections of intracellular tension dynamics and play important roles in many cellular processes. In this study, temporal variations in cell geometry and cell motion through cell cycle progression were quantitatively characterized via automated cell tracking for MCF-10A non-transformed breast cells, MCF-7 non-invasive breast cancer cells, and MDA-MB-231 highly metastatic breast cancer cells. A new cell segmentation method, which combines the threshold method and our modified edge based active contour method, was applied to optimize cell boundary detection for all cells in the field-of-view. An automated cell-tracking program was implemented to conduct live cell tracking over 40 hours for the three cell lines. The cell boundary and location information was measured and aligned with cell cycle progression with constructed cell lineage trees. Cell behaviors were studied in terms of cell geometry and cell motion. For cell geometry, cell area and cell axis ratio were investigated. For cell motion, instantaneous migration speed, cell motion type, as well as cell motion range were analyzed. We applied a cell-based approach that allows us to examine and compare temporal variations of cell behavior along with cell cycle progression at a single cell level. Cell body geometry along with distribution of peripheral protrusion structures appears to be associated with cell motion features. Migration speed together with motion type and motion ranges are required to distinguish the three cell-lines examined. We found that cells dividing or overlapping vertically are unique features of cell malignancy for both MCF-7 and MDA-MB-231 cells, whereas abrupt changes in cell body geometry and cell motion during mitosis are unique to highly metastatic MDA-MB-231 cells. Taken together, our live cell tracking system serves as an invaluable tool to identify cell behaviors that are unique to malignant and/or highly metastatic breast cancer cells. PMID:24911281

  6. Plant stem cell niches.

    Science.gov (United States)

    Aichinger, Ernst; Kornet, Noortje; Friedrich, Thomas; Laux, Thomas

    2012-01-01

    Multicellular organisms possess pluripotent stem cells to form new organs, replenish the daily loss of cells, or regenerate organs after injury. Stem cells are maintained in specific environments, the stem cell niches, that provide signals to block differentiation. In plants, stem cell niches are situated in the shoot, root, and vascular meristems-self-perpetuating units of organ formation. Plants' lifelong activity-which, as in the case of trees, can extend over more than a thousand years-requires that a robust regulatory network keep the balance between pluripotent stem cells and differentiating descendants. In this review, we focus on current models in plant stem cell research elaborated during the past two decades, mainly in the model plant Arabidopsis thaliana. We address the roles of mobile signals on transcriptional modules involved in balancing cell fates. In addition, we discuss shared features of and differences between the distinct stem cell niches of Arabidopsis.

  7. What are Stem Cells?

    Directory of Open Access Journals (Sweden)

    Ahmadshah Farhat

    2014-05-01

    Full Text Available   Stem cells are undifferentiated self regenerating multi potential cells. There are three types of stem cells categories by the ability to form after cells and correlated with the body’s development process. Totipotent: these stem cells can form an entire organism such as fertilized egg. Ploripotent: ploripotent cells are those that can form any cell in the body but cannot form an entire organism such as developing embryo’s totipotent cells become ploripotent  Multipotent: Multi potent stem cells are those that can only form specific cells in the body such as blood cells based. Based on the sources of stem cells we have three types of these cells: Autologous: Sources of the patient own cells are (Autologous either the cells from patient own body or his or her cord blood. For this type of transplant the physician now usually collects the periphery rather than morrow because the procedure is easier on like a bane morrow harvest it take place outside of an operating room, and the patient does not to be under general unsetting . Allogenic: Sources of stem cells from another donore are primarily relatives (familial allogenic or completely unrelated donors. Xenogenic: In these stem cells from different species are transplanted e .g striatal porcine fetal mesan cephalic (FVM xenotransplants for Parkinson’s disease. On sites of isolation such as embryo, umbilical cord and other body tissues stem cells are named embnyonic, cord blood, and adult stem cells. The scope of results and clinical application of stem cells are such as: Neurodegenerative conditions (MS,ALS, Parkinson’s, Stroke, Ocular disorders- Glaucoma, retinitis Pigmentosa (RP, Auto Immune Conditions (Lupus, MS,R. arthritis, Diabetes, etc, Viral Conditions (Hepatitis C and AIDS, Heart Disease, Adrenal Disorders, Injury(Nerve, Brain, etc, Anti aging (hair, skin, weight control, overall well being/preventive, Emotional disorders, Organ / Tissue Cancers, Blood cancers, Blood diseases

  8. Production and characterization of a fusion peptide derived from the rabies virus glycoprotein (RVG29).

    Science.gov (United States)

    Yang, Yu-Jiao; Zhao, Ping-Sen; Wu, Hong-Xia; Wang, Hua-Lei; Zhao, Li-Li; Xue, Xiang-Hong; Gai, Wei-Wei; Gao, Yu-Wei; Yang, Song-Tao; Xia, Xian-Zhu

    2014-12-01

    Gene therapy targeting the brain holds great promise in curing nervous system degenerative diseases in clinical applications. With this in mind, in a previous study a 29 amino-acid peptide derived from the rabies virus glycoprotein (RVG29) with a nonamer stretch of arginine residues (RVG29-9R) at its carboxy-terminus was exploited as a ligand for brain-targeting gene delivery. Importantly, the report demonstrated that the RVG29-9R vector was able to cross the blood-brain barrier. RVG29-9R is currently synthesized by commercial companies with high associated costs. In this study, in order to reduce the costs of producing RVG29-9R, we have expressed and purified 6mg of a recombinant peptide (RVG29-9R-6His) from 0.4g of cultured Escherichia coli. We assessed the physiochemical properties of RVG29-9R-6His, its cytotoxicity, and the in vitro transfection efficiency in Neuro 2a cells (which express the acetylcholine receptor). Our results reveal that the RVG29-9R-6His peptide recognized Neuro 2a cells in a dose-dependent manner and it was also able to bind plasmid DNA and deliver it into the Neuro 2a cells effectively. Therefore, our study has demonstrated that the recombinant RVG29-9R-6His peptide retains the functions of RVG29-9R and so may provide an economically viable and alternative production method for the manufacture of RVG29-9R. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Pluripotent Stem Cells for Schwann Cell Engineering

    NARCIS (Netherlands)

    Ma, Ming-San; Boddeke, Erik; Copray, Sjef

    Tissue engineering of Schwann cells (SCs) can serve a number of purposes, such as in vitro SC-related disease modeling, treatment of peripheral nerve diseases or peripheral nerve injury, and, potentially, treatment of CNS diseases. SCs can be generated from autologous stem cells in vitro by

  10. Induction of Functional Hair-Cell-Like Cells from Mouse Cochlear Multipotent Cells

    Directory of Open Access Journals (Sweden)

    Quanwen Liu

    2016-01-01

    Full Text Available In this paper, we developed a two-step-induction method of generating functional hair cells from inner ear multipotent cells. Multipotent cells from the inner ear were established and induced initially into progenitor cells committed to the inner ear cell lineage on the poly-L-lysine substratum. Subsequently, the committed progenitor cells were cultured on the mitotically inactivated chicken utricle stromal cells and induced into hair-cell-like cells containing characteristic stereocilia bundles. The hair-cell-like cells exhibited rapid permeation of FM1-43FX. The whole-cell patch-clamp technique was used to measure the membrane currents of cells differentiated for 7 days on chicken utricle stromal cells and analyze the biophysical properties of the hair-cell-like cells by recording membrane properties of cells. The results suggested that the hair-cell-like cells derived from inner ear multipotent cells were functional following differentiation in an enabling environment.

  11. GPCR Interaction: 20 [GRIPDB[Archive

    Lifescience Database Archive (English)

    Full Text Available modified BRET signal in a time-dependent manner in the cells expressing HA-A1R-GFP(2) or Myc-P2Y1R-Rluc, which...was also observed in the co-transfected HEK293T cells by confocal laser microscopy. Therefore, A1R and...to form constitutive hetero-oligomers in living cells and this process is promoted by the simultaneous

  12. Identification of direct target genes of miR-7, miR-9, miR-96, and miR-182 in the human breast cancer cell lines MCF-7 and MDA-MB-231.

    Science.gov (United States)

    Moazzeni, Hamidreza; Najafi, Ali; Khani, Marzieh

    2017-08-01

    Some microRNAs have carcinogenic or tumor suppressive effects in breast cancer, which is the most common cancer in women worldwide. MiR-7 and miR-9 are tumor suppressor microRNAs, which induce apoptosis and inhibit proliferation in breast cancer cells. Moreover, miR-96 and miR-182 are onco-microRNAs that increase proliferation, migration, and tumorigenesis in breast cancer cells. This study aimed to identify the direct target genes of these four microRNAs in the human breast cancer cell lines MCF-7 and MDA-MB-231. Initially, bioinformatics tools were used to identify the target genes that have binding sites for miR-7, MiR-9, MiR-96, and miR-182 and are also associated with breast cancer. Subsequently, the findings of the bioinformatics analysis relating to the effects of these four microRNAs on the 3'-UTR activity of the potential target genes were confirmed using the dual luciferase assay in MCF-7 and MDA-MB-231 cells co-transfected with the vectors containing 3'-UTR segments of the target genes downstream of a luciferase coding gene and each of the microRNAs. Finally, the effects of microRNAs on the endogenous expression of potential target genes were assessed by the overexpression of each of the four microRNAs in MCF-7 and MDA-MB-231 cells. Respectively, three, three, three, and seven genes were found to have binding sites for miR-7, miR-9, miR-96, and miR-182 and were associated with breast cancer. The results of empirical studies including dual luciferase assays and real-time PCR confirmed that miR-7 regulates the expression of BRCA1 and LASP1; MiR-9 regulates the expression of AR; miR-96 regulates the expression of ABCA1; and miR-182 regulates the expression of NBN, TOX3, and LASP1. Taken together, our results suggest that the tumor suppressive effects of miR-7 may be mediated partly by regulating the expression of BRCA1 as a tumor suppressor gene in breast cancer. In addition, this microRNA and miR-182 may have effects on the nodal-positivity and tumor

  13. Epidermal Stem Cells

    Directory of Open Access Journals (Sweden)

    Osman Köse

    2015-03-01

    Full Text Available The epidermis is the outermost layer of the human skin and comprises a multilayered epithelium, the interfollicular epidermis, with associated hair follicles, sebaceous glands, and eccrine sweat glands. There are many origins of stem cells in the skin and skin appendages. These stem cells are localized in different part of the pilosebaseous units and also express many different genes. Epidermal stem cells in the pilosebaseous units not only ensure the maintenance of epidermal homeostasis and hair regeneration, but also contribute to repair of the epidermis after injury. In recent years, human induced pluripotent skin stem cells are produced from the epidermal cells such as keratinocytes, fibroblasts and melanocytes. These cells can be transdifferentiated to embriyonic stem cells. Human induced pluripotent stem cells have potential applications in cell replacement therapy and regenerative medicine. These cells provide a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. In this review, we aimed an overview of epidermal stem cells for better understanding their functions in the skin. Skin will be main organ for using the epidermal cells for regenerative medicine in near future.

  14. Apoptosis and cell cycle

    Directory of Open Access Journals (Sweden)

    Petrović Marija

    2014-01-01

    Full Text Available Apoptosis, a form of programmed cell death, is used to eliminate individual cells surrounded by normal cell population. It is a controlled way of cell death in which the cell actively participates by conducting precise, gene-regulated program of self-destruction, that is, cell 'suicide.' Active synthesis of macromolecules is necessary during this process. Death of individual cells is necessary to maintain a balance in living systems, so the process of apoptosis is continuously present in the body, which allows normal development, tissue homeostasis, and many other physiological processes. The molecular mechanisms that regulate apoptosis are functionally linked to other cellular mechanisms, such as control of the cell cycle, cell proliferation and differentiation, genomic stability and cellular metabolism. Damage to the DNA molecule, caused both spontaneously and under the influence of various chemical and physical agents, leads to the cell cycle arrest and activation of mechanisms that repair the damage. Depending on the type and extent of the damage, the cell either continues progression through the cell cycle, or activates the mechanisms that lead to apoptosis. Disturbances in the regulation of apoptosis and cell cycle present the molecular and biological basis of many diseases. Because of the importance of these processes during the development and progression of tumors, their use as biological markers is one of the main strategies in the formation of therapeutic approaches for the treatment of cancer.

  15. Photoacoustic molecular imaging of ferritin as a reporter gene

    Science.gov (United States)

    Ha, S.; Carson, A.; Kim, K.

    2012-02-01

    Spectral analysis of photoacoustic (PA) molecular imaging (PMI) of ferritin expressed in human melanoma cells (SK-24) was performed in vitro. Ferritin is a ubiquitously expressed protein which stores iron that can be detected by PA imaging, allowing ferritin to act as a reporter gene. To over-express ferritin, SK-24 cells were co-transfected with plasmid expressing Heavy chain ferritin (H-FT) and plasmid expressing enhanced green fluorescent protein (pEGFP-C1) using LipofectamineTM 2000. Non-transfected SK-24 cells served as a negative control. Fluorescent imaging of EGFP confirmed transfection and transgene expression in co-transfected cells. To detect iron accumulation in SK-24 cells, a focused high frequency ultrasonic transducer (60 MHz, f/1.5), synchronized to a pulsed laser (molecular imaging and basic science research.

  16. The cell cycle as a brake for β-cell regeneration from embryonic stem cells.

    Science.gov (United States)

    El-Badawy, Ahmed; El-Badri, Nagwa

    2016-01-13

    The generation of insulin-producing β cells from stem cells in vitro provides a promising source of cells for cell transplantation therapy in diabetes. However, insulin-producing cells generated from human stem cells show deficiency in many functional characteristics compared with pancreatic β cells. Recent reports have shown molecular ties between the cell cycle and the differentiation mechanism of embryonic stem (ES) cells, assuming that cell fate decisions are controlled by the cell cycle machinery. Both β cells and ES cells possess unique cell cycle machinery yet with significant contrasts. In this review, we compare the cell cycle control mechanisms in both ES cells and β cells, and highlight the fundamental differences between pluripotent cells of embryonic origin and differentiated β cells. Through critical analysis of the differences of the cell cycle between these two cell types, we propose that the cell cycle of ES cells may act as a brake for β-cell regeneration. Based on these differences, we discuss the potential of modulating the cell cycle of ES cells for the large-scale generation of functionally mature β cells in vitro. Further understanding of the factors that modulate the ES cell cycle will lead to new approaches to enhance the production of functional mature insulin-producing cells, and yield a reliable system to generate bona fide β cells in vitro.

  17. NK Cells and Psoriasis

    Directory of Open Access Journals (Sweden)

    Sinéad Dunphy

    2011-01-01

    Full Text Available Psoriasis is a chronic condition of the skin characterised by distinctive scaly plaques. The immune system is now thought to play a major role in the development and pathogenesis of psoriasis with immune cells and cytokines influencing keratinocyte function. Keratinocytes in turn, can activate and recruit immune cells leading to a positive feedback loop in disease. Natural Killer (NK cells are lymphocytes that are best known for killing virally infected and cancer cells. However, evidence is emerging to support a role for NK cells in psoriasis. NK cells are found in the inflammatory infiltrate in psoriatic skin lesions. They can produce a range of inflammatory cytokines, many of which are important in the pathogenesis of psoriasis. Recent genetic studies have identified a range of potential molecules relating to NK cell biology that are known to be important in psoriasis. This paper will discuss the evidence, both cellular and genetic, for NK cell involvement in psoriasis.

  18. Liquid fuel cells

    National Research Council Canada - National Science Library

    Soloveichik, Grigorii L

    2014-01-01

    The advantages of liquid fuel cells (LFCs) over conventional hydrogen-oxygen fuel cells include a higher theoretical energy density and efficiency, a more convenient handling of the streams, and enhanced safety...

  19. NIA Aging Cell Repository

    Data.gov (United States)

    Federal Laboratory Consortium — To facilitate aging research on cells in culture, the NIA provides support for the NIA Aging Cell Repository, located at the Coriell Institute for Medical Research...

  20. Islet Cell Transplantation

    Science.gov (United States)

    ... the body use glucose for energy. Islet cell transplantation transfers cells from an organ donor into the ... to make and release insulin. Researchers hope islet transplantation will help people with type 1 diabetes live ...

  1. Separators for electrochemical cells

    Energy Technology Data Exchange (ETDEWEB)

    Carlson, Steven Allen; Anakor, Ifenna Kingsley; Farrell, Greg Robert

    2018-01-16

    Provided are separators for use in an electrochemical cell comprising (a) an inorganic oxide and (b) an organic polymer, wherein the inorganic oxide comprises organic substituents. Also provided are electrochemical cells comprising such separators.

  2. Stem Cell Transplant

    Science.gov (United States)

    ... transplant is a procedure that infuses healthy blood stem cells into your body to replace your damaged or ... A bone marrow transplant is also called a stem cell transplant. A bone marrow transplant may be necessary ...

  3. What Are Islet Cells?

    Science.gov (United States)

    ... and address the challenge of foreign tissue rejection. Engineering a Safe Cell Supply The issue of safety ... stem cell (hPSc)-based therapies. To address this problem, DRI researchers set out to engineer hPSc with " ...

  4. Mast Cell Proteoglycans

    National Research Council Canada - National Science Library

    Rönnberg, Elin; Melo, Fabio R; Pejler, Gunnar

    2012-01-01

    Mast cells are versatile effector cells of the immune system, contributing to both innate and adaptive immunity toward pathogens but also having profound detrimental activities in the context of inflammatory disease...

  5. Giant Cell Arteritis

    Science.gov (United States)

    Giant cell arteritis is a disorder that causes inflammation of your arteries, usually in the scalp, neck, and arms. ... arteries, which keeps blood from flowing well. Giant cell arteritis often occurs with another disorder called polymyalgia ...

  6. Leydig cell tumor

    Science.gov (United States)

    Tumor - Leydig cell; Testicular tumor - Leydig; Testicular neoplasm ... The cause of this tumor is unknown. There are no known risk factors for this tumor. Unlike germ cell tumors of the testicles, this tumor ...

  7. Stem Cell Transplant

    Science.gov (United States)

    ... Graft-versus-host disease: A potential risk when stem cells come from donors If you receive a transplant ... medications and blood products into your body. Collecting stem cells for transplant If a transplant using your own ...

  8. Border cell release

    DEFF Research Database (Denmark)

    Mravec, Jozef

    2017-01-01

    Plant border cells are specialised cells derived from the root cap with roles in the biomechanics of root growth and in forming a barrier against pathogens. The mechanism of highly localised cell separation which is essential for their release to the environment is little understood. Here I present...... in situ analysis of Brachypodium distachyon, a model organism for grasses which possess type II primary cell walls poor in pectin content. Results suggest similarity in spatial dynamics of pectic homogalacturonan during dicot and monocot border cell release. Integration of observations from different...... species leads to the hypothesis that this process most likely does not involve degradation of cell wall material but rather employs unique cell wall structural and compositional means enabling both the rigidity of the root cap as well as detachability of given cells on its surface....

  9. Sickle Cell Disease

    Science.gov (United States)

    ... possible. Basics Facts, signs, diagnosis, treatment, and pregnancy. Sickle Cell Trait Facts, complications, and diagnosis. Tips for Healthy Living ... you to join us in this series. More SICKLE CELL TRAIT TOOLKIT CDC, together with the American Society of ...

  10. FUEL CELL ELECTRODE MATERIALS

    Science.gov (United States)

    FUEL CELL ELECTRODE MATERIALS. RAW MATERIAL SELECTION INFLUENCES POLARIZATION BUT IS NOT A SINGLE CONTROLLING FACTOR. AVAILABLE...DATA INDICATES THAT AN INTERRELATIONSHIP OF POROSITY, AVERAGE PORE VOLUME, AND PERMEABILITY CONTRIBUTES TO ELECTRODE FUEL CELL BEHAVIOR.

  11. Design, Synthesis and Evaluation of Novel Tacrine-Ferulic Acid Hybrids as Multifunctional Drug Candidates against Alzheimer's Disease.

    Science.gov (United States)

    Fu, Yingbo; Mu, Yu; Lei, Hui; Wang, Pu; Li, Xin; Leng, Qiao; Han, Li; Qu, Xiaodan; Wang, Zhanyou; Huang, Xueshi

    2016-10-11

    Five novel tacrine-ferulic acid hybrid compounds (8a-e) were synthesized and their structures were identified on the basis of a detailed spectroscopic analysis. The activities of inhibiting acetyl cholinesterase (AChE) and butyryl cholinesterase (BuChE), reducing self-induced β-amyloid (Aβ) aggregation and chelating Cu(2+) were evaluated in vitro. Among them, 8c and 8d displayed the higher selectivity in inhibiting AChE over BuChE. Moreover, 8d also showed dramatic inhibition of self-Aβ aggregation, activity of chelating Cu(2+) and activity against Aβ-induced neurotoxicity in Neuro-2A cells.

  12. Design, Synthesis and Evaluation of Novel Tacrine-Ferulic Acid Hybrids as Multifunctional Drug Candidates against Alzheimer’s Disease

    Directory of Open Access Journals (Sweden)

    Yingbo Fu

    2016-10-01

    Full Text Available Five novel tacrine-ferulic acid hybrid compounds (8a–e were synthesized and their structures were identified on the basis of a detailed spectroscopic analysis. The activities of inhibiting acetyl cholinesterase (AChE and butyryl cholinesterase (BuChE, reducing self-induced β-amyloid (Aβ aggregation and chelating Cu2+ were evaluated in vitro. Among them, 8c and 8d displayed the higher selectivity in inhibiting AChE over BuChE. Moreover, 8d also showed dramatic inhibition of self-Aβ aggregation, activity of chelating Cu2+ and activity against Aβ-induced neurotoxicity in Neuro-2A cells.

  13. Diagram of Cell to Cell Communication

    Science.gov (United States)

    2002-01-01

    Diagram depicts the importance of cell-cell communication as central to the understanding of cancer growth and progression, the focus of the NASA bioreactor demonstration system (BDS-05) investigation. Microgravity studies will allow us to unravel the signaling and communication between these cells with the host and potential development of therapies for the treatment of cancer metastasis. The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators. Credit: Emory University.

  14. Germ Cell Differentiation from Pluripotent Cells

    Science.gov (United States)

    Medrano, Jose V.; Pera, Renee A. Reijo; Simón, Carlos

    2014-01-01

    Infertility is a medical condition with an increasing impact in Western societies with causes linked to toxins, genetics, and aging (primarily delay of motherhood). Within the different pathologies that can lead to infertility, poor quality or reduced quantity of gametes plays an important role. Gamete donation and therefore demand on donated sperm and eggs in fertility clinics is increasing. It is hoped that a better understanding of the conditions related to poor gamete quality may allow scientists to design rational treatments. However, to date, relatively little is known about human germ cell development in large part due to the inaccessibility of human development to molecular genetic analysis. It is hoped that pluripotent human embryonic stem cells and induced pluripotent stem cells may provide an accessible in vitro model to study germline development; these cells are able to differentiate to cells of all three primary embryonic germ layers, as well as to germ cells in vitro. We review the state of the art in germline differentiation from pluripotent stem cells. PMID:23329632

  15. Fish Stem Cell Cultures

    Science.gov (United States)

    Hong, Ni; Li, Zhendong; Hong, Yunhan

    2011-01-01

    Stem cells have the potential for self-renewal and differentiation. First stem cell cultures were derived 30 years ago from early developing mouse embryos. These are pluripotent embryonic stem (ES) cells. Efforts towards ES cell derivation have been attempted in other mammalian and non-mammalian species. Work with stem cell culture in fish started 20 years ago. Laboratory fish species, in particular zebrafish and medaka, have been the focus of research towards stem cell cultures. Medaka is the second organism that generated ES cells and the first that gave rise to a spermatogonial stem cell line capable of test-tube sperm production. Most recently, the first haploid stem cells capable of producing whole animals have also been generated from medaka. ES-like cells have been reported also in zebrafish and several marine species. Attempts for germline transmission of ES cell cultures and gene targeting have been reported in zebrafish. Recent years have witnessed the progress in markers and procedures for ES cell characterization. These include the identification of fish homologs/paralogs of mammalian pluripotency genes and parameters for optimal chimera formation. In addition, fish germ cell cultures and transplantation have attracted considerable interest for germline transmission and surrogate production. Haploid ES cell nuclear transfer has proven in medaka the feasibility of semi-cloning as a novel assisted reproductive technology. In this special issue on “Fish Stem Cells and Nuclear Transfer”, we will focus our review on medaka to illustrate the current status and perspective of fish stem cells in research and application. We will also mention semi-cloning as a new development to conventional nuclear transfer. PMID:21547056

  16. Fluorescence Live Cell Imaging

    OpenAIRE

    Ettinger, Andreas; Wittmann, Torsten

    2014-01-01

    Fluorescence microscopy of live cells has become an integral part of modern cell biology. Fluorescent protein (FP) tags, live cell dyes, and other methods to fluorescently label proteins of interest provide a range of tools to investigate virtually any cellular process under the microscope. The two main experimental challenges in collecting meaningful live cell microscopy data are to minimize photodamage while retaining a useful signal-to-noise ratio and to provide a suitable environment for ...

  17. Fish stem cell cultures.

    Science.gov (United States)

    Hong, Ni; Li, Zhendong; Hong, Yunhan

    2011-04-13

    Stem cells have the potential for self-renewal and differentiation. First stem cell cultures were derived 30 years ago from early developing mouse embryos. These are pluripotent embryonic stem (ES) cells. Efforts towards ES cell derivation have been attempted in other mammalian and non-mammalian species. Work with stem cell culture in fish started 20 years ago. Laboratory fish species, in particular zebrafish and medaka, have been the focus of research towards stem cell cultures. Medaka is the second organism that generated ES cells and the first that gave rise to a spermatogonial stem cell line capable of test-tube sperm production. Most recently, the first haploid stem cells capable of producing whole animals have also been generated from medaka. ES-like cells have been reported also in zebrafish and several marine species. Attempts for germline transmission of ES cell cultures and gene targeting have been reported in zebrafish. Recent years have witnessed the progress in markers and procedures for ES cell characterization. These include the identification of fish homologs/paralogs of mammalian pluripotency genes and parameters for optimal chimera formation. In addition, fish germ cell cultures and transplantation have attracted considerable interest for germline transmission and surrogate production. Haploid ES cell nuclear transfer has proven in medaka the feasibility of semi-cloning as a novel assisted reproductive technology. In this special issue on "Fish Stem Cells and Nuclear Transfer", we will focus our review on medaka to illustrate the current status and perspective of fish stem cells in research and application. We will also mention semi-cloning as a new development to conventional nuclear transfer.

  18. Skeletal (stromal) stem cells

    DEFF Research Database (Denmark)

    Abdallah, Basem M; Kermani, Abbas Jafari; Zaher, Walid

    2015-01-01

    Skeletal (marrow stromal) stem cells (BMSCs) are a group of multipotent cells that reside in the bone marrow stroma and can differentiate into osteoblasts, chondrocytes and adipocytes. Studying signaling pathways that regulate BMSC differentiation into osteoblastic cells is a strategy....../preadipocyte factor 1 (Dlk1/Pref-1), the Wnt co-receptor Lrp5 and intracellular kinases. This article is part of a Special Issue entitled: Stem Cells and Bone....

  19. Anterior Horn Cell Diseases

    Directory of Open Access Journals (Sweden)

    Merve Firinciogullari

    2016-09-01

    Full Text Available The anterior horn cells control all voluntary movement. Motor activity, respiratory, speech, and swallowing functions are dependent upon signals from the anterior horn cells. Diseases that damage the anterior horn cells, therefore, have a profound impact. Symptoms of anterior horn cell loss (weakness, falling, choking lead patients to seek medical attention. In this article, anterior horn diseases were reviewed, diagnostic criteria and management were discussed in detail. [Archives Medical Review Journal 2016; 25(3.000: 269-303

  20. Increased voltage photovoltaic cell

    Science.gov (United States)

    Ross, B.; Bickler, D. B.; Gallagher, B. D. (Inventor)

    1985-01-01

    A photovoltaic cell, such as a solar cell, is provided which has a higher output voltage than prior cells. The improved cell includes a substrate of doped silicon, a first layer of silicon disposed on the substrate and having opposite doping, and a second layer of silicon carbide disposed on the first layer. The silicon carbide preferably has the same type of doping as the first layer.

  1. Cell Factory Engineering

    DEFF Research Database (Denmark)

    Davy, Anne Mathilde; Kildegaard, Helene Faustrup; Andersen, Mikael Rørdam

    2017-01-01

    focused on individual strategies or cell types, but collectively they fall under the broad umbrella of a growing field known as cell factory engineering. Here we condense >130 reviews and key studies in the art into a meta-review of cell factory engineering. We identified 33 generic strategies......-review provides general strategy guides for the broad range of applications of rational engineering of cell factories....

  2. Adventures with Cell Phones

    Science.gov (United States)

    Kolb, Liz

    2011-01-01

    Teachers are finding creative ways to turn the basic cell phone from a digital distraction into a versatile learning tool. In this article, the author explains why cell phones are important in learning and suggests rather than banning them that they be integrated into learning. She presents activities that can be done on a basic cell phone with a…

  3. Cell phones and cancer

    Science.gov (United States)

    Cancer and cell phones; Do cell phones cause cancer? ... Several major studies show no link between cell phones and cancer at this time. However, since the information available is based on short-term studies, the impact of many years of exposure ...

  4. Cell Culture Made Easy.

    Science.gov (United States)

    Dye, Frank J.

    1985-01-01

    Outlines steps to generate cell samples for observation and experimentation. The procedures (which use ordinary laboratory equipment) will establish a short-term primary culture of normal mammalian cells. Information on culture vessels and cell division and a list of questions to generate student interest and involvement in the topics are…

  5. Criticality in cell differentiation

    Indian Academy of Sciences (India)

    Indrani Bose

    2017-11-09

    Nov 9, 2017 ... Cell differentiation is an important process in living organisms. Differentiation is mostly based on binary decisions with the progenitor cells choosing between two specific lineages. The differentiation dynamics have both deterministic and stochastic components. Several theoretical studies suggest that cell ...

  6. Nanostructured Organic Solar Cells

    DEFF Research Database (Denmark)

    Radziwon, Michal Jędrzej; Rubahn, Horst-Günter; Madsen, Morten

    Recent forecasts for alternative energy generation predict emerging importance of supporting state of art photovoltaic solar cells with their organic equivalents. Despite their significantly lower efficiency, number of application niches are suitable for organic solar cells. This work reveals...... the principles of bulk heterojunction organic solar cells fabrication as well as summarises major differences in physics of their operation....

  7. sickle cell disease

    African Journals Online (AJOL)

    Summary. Background: Biochemical abnormalities have been associated with sickle cell disease. Studies on phosphorus and magnesium in sickle cell disease have been conflicting. The re is paucity of information on the role of these ions in the pathogenesis and management of sickle cell disease. This study was set out to ...

  8. Dazlin' pluripotent stem cells

    NARCIS (Netherlands)

    Welling, M.A.

    2014-01-01

    Pluripotent embryonic stem cells (ESCs) can be isolated from the inner cell mass (ICM) of blastocyst embryos and differentiate into all three germ layers in vitro. However, despite their similar origin, mouse embryonic stem cells represent a more naïve ICM-like pluripotent state whereas human

  9. Biomarkers of cell senescence

    Science.gov (United States)

    Dirmi, Goberdhan P.; Campisi, Judith; Peacocke, Monica

    1996-01-01

    The present invention provides a biomarker system for the in vivo and in vitro assessment of cell senescence. In the method of the present invention, .beta.-galactosidase activity is utilized as a means by which cell senescence may be assessed either in in vitro cell cultures or in vivo.

  10. Cell damage after shock.

    Science.gov (United States)

    Barber, A E; Shires, G T

    1996-05-01

    Hypoperfusion of tissue results in cell membrane dysfunction. Normally, the cell membrane serves to preserve the milieu interior through the maintenance of a negative charge or membrane potential. Maintenance of a negative membrane potential across the cell membrane serves as a semipermeable barrier, preserving the balance of intra- and extracellular electrolytes and water.

  11. Aneuploidy in stem cells

    NARCIS (Netherlands)

    Garcia-Martinez, Jorge; Bakker, Bjorn; Schukken, Klaske M; Simon, Judith E; Foijer, Floris

    2016-01-01

    Stem cells hold enormous promise for regenerative medicine as well as for engineering of model systems to study diseases and develop new drugs. The discovery of protocols that allow for generating induced pluripotent stem cells (IPSCs) from somatic cells has brought this promise steps closer to

  12. Embryonic Stem Cell Markers

    Directory of Open Access Journals (Sweden)

    Lan Ma

    2012-05-01

    Full Text Available Embryonic stem cell (ESC markers are molecules specifically expressed in ES cells. Understanding of the functions of these markers is critical for characterization and elucidation for the mechanism of ESC pluripotent maintenance and self-renewal, therefore helping to accelerate the clinical application of ES cells. Unfortunately, different cell types can share single or sometimes multiple markers; thus the main obstacle in the clinical application of ESC is to purify ES cells from other types of cells, especially tumor cells. Currently, the marker-based flow cytometry (FCM technique and magnetic cell sorting (MACS are the most effective cell isolating methods, and a detailed maker list will help to initially identify, as well as isolate ESCs using these methods. In the current review, we discuss a wide range of cell surface and generic molecular markers that are indicative of the undifferentiated ESCs. Other types of molecules, such as lectins and peptides, which bind to ESC via affinity and specificity, are also summarized. In addition, we review several markers that overlap with tumor stem cells (TSCs, which suggest that uncertainty still exists regarding the benefits of using these markers alone or in various combinations when identifying and isolating cells.

  13. Stem cell heterogeneity revealed

    DEFF Research Database (Denmark)

    Andersen, Marianne S; Jensen, Kim B

    2016-01-01

    The skin forms a protective, water-impermeable barrier consisting of heavily crosslinked epithelial cells. However, the specific role of stem cells in sustaining this barrier remains a contentious issue. A detailed analysis of the interfollicular epidermis now proposes a model for how a composite...... of cells with different properties are involved in its maintenance....

  14. Solar Photovoltaic Cells.

    Science.gov (United States)

    Mickey, Charles D.

    1981-01-01

    Reviews information on solar radiation as an energy source. Discusses these topics: the key photovoltaic material; the bank theory of solids; conductors, semiconductors, and insulators; impurity semiconductors; solid-state photovoltaic cell operation; limitations on solar cell efficiency; silicon solar cells; cadmium sulfide/copper (I) sulfide…

  15. Mesenchymal Stem Cells Contribute to Tumor Cell Proliferation by Direct Cell-Cell Contact Interactions

    NARCIS (Netherlands)

    Roorda, Berber D.; ter Elst, Arja; Meeuwsen-de Boer, Tiny G. J.; Kamps, Willem A.; de Bont, Eveline S. J. M.

    Bone marrow (BM)-derived mesenchymal stem cells (MSCs) have been implicated in tumor progression, making MSCs important targets for anti-cancer strategies. In this study, we show that MSCs promote tumor growth in vivo in a lymphoma xenograft model. We show that MSCs provide direct cell-cell contact

  16. Live-cell imaging: The cell's perspective

    OpenAIRE

    Cole, Richard

    2014-01-01

    It would be hard to argue that live-cell imaging has not changed our view of biology. The past 10 years have seen an explosion of interest in imaging cellular processes, down to the molecular level. There are now many advanced techniques being applied to live cell imaging. However, cellular health is often under appreciated. For many researchers, if the cell at the end of the experiment has not gone into apoptosis or is blebbed beyond recognition, than all is well. This is simply incorrect. T...

  17. Cancer stem cell-like side population cells in clear cell renal cell carcinoma cell line 769P.

    Science.gov (United States)

    Huang, Bin; Huang, Yi Jun; Yao, Zhi Jun; Chen, Xu; Guo, Sheng Jie; Mao, Xiao Peng; Wang, Dao Hu; Chen, Jun Xing; Qiu, Shao Peng

    2013-01-01

    Although cancers are widely considered to be maintained by stem cells, the existence of stem cells in renal cell carcinoma (RCC) has seldom been reported, in part due to the lack of unique surface markers. We here identified cancer stem cell-like cells with side population (SP) phenotype in five human RCC cell lines. Flow cytometry analysis revealed that 769P, a human clear cell RCC cell line, contained the largest amount of SP cells as compared with other four cell lines. These 769P SP cells possessed characteristics of proliferation, self-renewal, and differentiation, as well as strong resistance to chemotherapy and radiotherapy that were possibly related to the ABCB1 transporter. In vivo experiments with serial tumor transplantation in mice also showed that 769P SP cells formed tumors in NOD/SCID mice. Taken together, these results indicate that 769P SP cells have the properties of cancer stem cells, which may play important roles in tumorigenesis and therapy-resistance of RCC.

  18. Place Cells, Grid Cells, Attractors, and Remapping

    Directory of Open Access Journals (Sweden)

    Kathryn J. Jeffery

    2011-01-01

    Full Text Available Place and grid cells are thought to use a mixture of external sensory information and internal attractor dynamics to organize their activity. Attractor dynamics may explain both why neurons react coherently following sufficiently large changes to the environment (discrete attractors and how firing patterns move smoothly from one representation to the next as an animal moves through space (continuous attractors. However, some features of place cell behavior, such as the sometimes independent responsiveness of place cells to environmental change (called “remapping”, seem hard to reconcile with attractor dynamics. This paper suggests that the explanation may be found in an anatomical separation of the two attractor systems coupled with a dynamic contextual modulation of the connection matrix between the two systems, with new learning being back-propagated into the matrix. Such a scheme could explain how place cells sometimes behave coherently and sometimes independently.

  19. Transparent ultraviolet photovoltaic cells.

    Science.gov (United States)

    Yang, Xun; Shan, Chong-Xin; Lu, Ying-Jie; Xie, Xiu-Hua; Li, Bing-Hui; Wang, Shuang-Peng; Jiang, Ming-Ming; Shen, De-Zhen

    2016-02-15

    Photovoltaic cells have been fabricated from p-GaN/MgO/n-ZnO structures. The photovoltaic cells are transparent to visible light and can transform ultraviolet irradiation into electrical signals. The efficiency of the photovoltaic cells is 0.025% under simulated AM 1.5 illumination conditions, while it can reach 0.46% under UV illumination. By connecting several such photovoltaic cells in a series, light-emitting devices can be lighting. The photovoltaic cells reported in this Letter may promise the applications in glass of buildings to prevent UV irradiation and produce power for household appliances in the future.

  20. Skeletal muscle satellite cells

    Science.gov (United States)

    Schultz, E.; McCormick, K. M.

    1994-01-01

    Evidence now suggests that satellite cells constitute a class of myogenic cells that differ distinctly from other embryonic myoblasts. Satellite cells arise from somites and first appear as a distinct myoblast type well before birth. Satellite cells from different muscles cannot be functionally distinguished from one another and are able to provide nuclei to all fibers without regard to phenotype. Thus, it is difficult to ascribe any significant function to establishing or stabilizing fiber type, even during regeneration. Within a muscle, satellite cells exhibit marked heterogeneity with respect to their proliferative behavior. The satellite cell population on a fiber can be partitioned into those that function as stem cells and those which are readily available for fusion. Recent studies have shown that the cells are not simply spindle shaped, but are very diverse in their morphology and have multiple branches emanating from the poles of the cells. This finding is consistent with other studies indicating that the cells have the capacity for extensive migration within, and perhaps between, muscles. Complexity of cell shape usually reflects increased cytoplasmic volume and organelles including a well developed Golgi, and is usually associated with growing postnatal muscle or muscles undergoing some form of induced adaptive change or repair. The appearance of activated satellite cells suggests some function of the cells in the adaptive process through elaboration and secretion of a product. Significant advances have been made in determining the potential secretion products that satellite cells make. The manner in which satellite cell proliferative and fusion behavior is controlled has also been studied. There seems to be little doubt that cellcell coupling is not how satellite cells and myofibers communicate. Rather satellite cell regulation is through a number of potential growth factors that arise from a number of sources. Critical to the understanding of this form

  1. Mechanics rules cell biology

    Directory of Open Access Journals (Sweden)

    Wang James HC

    2010-07-01

    Full Text Available Abstract Cells in the musculoskeletal system are subjected to various mechanical forces in vivo. Years of research have shown that these mechanical forces, including tension and compression, greatly influence various cellular functions such as gene expression, cell proliferation and differentiation, and secretion of matrix proteins. Cells also use mechanotransduction mechanisms to convert mechanical signals into a cascade of cellular and molecular events. This mini-review provides an overview of cell mechanobiology to highlight the notion that mechanics, mainly in the form of mechanical forces, dictates cell behaviors in terms of both cellular mechanobiological responses and mechanotransduction.

  2. Fuel cell catalyst degradation

    DEFF Research Database (Denmark)

    Arenz, Matthias; Zana, Alessandro

    2016-01-01

    Fuel cells are an important piece in our quest for a sustainable energy supply. Although there are several different types of fuel cells, the by far most popular is the proton exchange membrane fuel cell (PEMFC). Among its many favorable properties are a short start up time and a high power density...... increasing focus. Activity of the catalyst is important, but stability is essential. In the presented perspective paper, we review recent efforts to investigate fuel cell catalysts ex-situ in electrochemical half-cell measurements. Due to the amount of different studies, this review has no intention to give...

  3. Fuel Cell/Electrochemical Cell Voltage Monitor

    Science.gov (United States)

    Vasquez, Arturo

    2012-01-01

    A concept has been developed for a new fuel cell individual-cell-voltage monitor that can be directly connected to a multi-cell fuel cell stack for direct substack power provisioning. It can also provide voltage isolation for applications in high-voltage fuel cell stacks. The technology consists of basic modules, each with an 8- to 16-cell input electrical measurement connection port. For each basic module, a power input connection would be provided for direct connection to a sub-stack of fuel cells in series within the larger stack. This power connection would allow for module power to be available in the range of 9-15 volts DC. The relatively low voltage differences that the module would encounter from the input electrical measurement connection port, coupled with the fact that the module's operating power is supplied by the same substack voltage input (and so will be at similar voltage), provides for elimination of high-commonmode voltage issues within each module. Within each module, there would be options for analog-to-digital conversion and data transfer schemes. Each module would also include a data-output/communication port. Each of these ports would be required to be either non-electrical (e.g., optically isolated) or electrically isolated. This is necessary to account for the fact that the plurality of modules attached to the stack will normally be at a range of voltages approaching the full range of the fuel cell stack operating voltages. A communications/ data bus could interface with the several basic modules. Options have been identified for command inputs from the spacecraft vehicle controller, and for output-status/data feeds to the vehicle.

  4. Stress and stem cells.

    Science.gov (United States)

    Tower, John

    2012-01-01

    The unique properties and functions of stem cells make them particularly susceptible to stresses and also lead to their regulation by stress. Stem cell division must respond to the demand to replenish cells during normal tissue turnover as well as in response to damage. Oxidative stress, mechanical stress, growth factors, and cytokines signal stem cell division and differentiation. Many of the conserved pathways regulating stem cell self-renewal and differentiation are also stress-response pathways. The long life span and division potential of stem cells create a propensity for transformation (cancer) and specific stress responses such as apoptosis and senescence act as antitumor mechanisms. Quiescence regulated by CDK inhibitors and a hypoxic niche regulated by FOXO transcription factor function to reduce stress for several types of stem cells to facilitate long-term maintenance. Aging is a particularly relevant stress for stem cells, because repeated demands on stem cell function over the life span can have cumulative cell-autonomous effects including epigenetic dysregulation, mutations, and telomere erosion. In addition, aging of the organism impairs function of the stem cell niche and systemic signals, including chronic inflammation and oxidative stress. Copyright © 2012 Wiley Periodicals, Inc.

  5. Hematopoietic Stem Cells Therapies.

    Science.gov (United States)

    Chivu-Economescu, Mihaela; Rubach, Martin

    2017-01-01

    Stem cell-based therapies are recognized as a new way to treat various diseases and injuries, with a wide range of health benefits. The goal is to heal or replace diseased or destroyed organs or body parts with healthy new cells provided by stem cell transplantation. The current practical form of stem cell therapy is the hematopoietic stem cells transplant applied for the treatment of hematological disorders. There are over 2100 clinical studies in progress concerning hematopoietic stem cell therapies. All of them are using hematopoietic stem cells to treat various diseases like: cancers, leukemia, lymphoma, cardiac failure, neural disorders, auto-immune diseases, immunodeficiency, metabolic or genetic disorders. Several challenges are to be addressed prior to developing and applying large scale cell therapies: 1) to explain and control the mechanisms of differentiation and development toward a specific cell type needed to treat the disease, 2) to obtain a sufficient number of desired cell type for transplantation, 3) to overcome the immune rejection and 4) to show that transplanted cells fulfill their normal functions in vivo after transplants. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  6. Cell and Tissue Engineering

    CERN Document Server

    2012-01-01

    Cell and Tissue Engineering” introduces the principles and new approaches in cell and tissue engineering. It includes both the fundamentals and the current trends in cell and tissue engineering, in a way useful both to a novice and an expert in the field. The book is composed of 13 chapters all of which are written by the leading experts. It is organized to gradually assemble an insight in cell and tissue function starting form a molecular nano-level, extending to a cellular micro-level and finishing at the tissue macro-level. In specific, biological, physiological, biophysical, biochemical, medical, and engineering aspects are covered from the standpoint of the development of functional substitutes of biological tissues for potential clinical use. Topics in the area of cell engineering include cell membrane biophysics, structure and function of the cytoskeleton, cell-extracellular matrix interactions, and mechanotransduction. In the area of tissue engineering the focus is on the in vitro cultivation of ...

  7. Overview of Cell Synchronization.

    Science.gov (United States)

    Banfalvi, Gaspar

    2017-01-01

    The widespread interest in cell synchronization is maintained by the studies of control mechanism involved in cell cycle regulation. During the synchronization distinct subpopulations of cells are obtained representing different stages of the cell cycle. These subpopulations are then used to study regulatory mechanisms of the cycle at the level of macromolecular biosynthesis (DNA synthesis, gene expression, protein synthesis), protein phosphorylation, development of new drugs, etc. Although several synchronization methods have been described, it is of general interest that scientists get a compilation and an updated view of these synchronization techniques. This introductory chapter summarizes: (1) the basic concepts and principal criteria of cell cycle synchronizations, (2) the most frequently used synchronization methods, such as physical fractionation (flow cytometry, dielectrophoresis, cytofluorometric purification), chemical blockade, (3) synchronization of embryonic cells, (4) synchronization at low temperature, (5) comparison of cell synchrony techniques, (6) synchronization of unicellular organisms, and (7) the effect of synchronization on transfection.

  8. Microfluidic fuel cell systems

    Science.gov (United States)

    Ho, Bernard; Kjeang, Erik

    2011-06-01

    A microfluidic fuel cell is a microfabricated device that produces electrical power through electrochemical reactions involving a fuel and an oxidant. Microfluidic fuel cell systems exploit co-laminar flow on the microscale to separate the fuel and oxidant species, in contrast to conventional fuel cells employing an ion exchange membrane for this function. Since 2002 when the first microfluidic fuel cell was invented, many different fuels, oxidants, and architectures have been investigated conceptually and experimentally. In this mini-review article, recent advancements in the field of microfluidic fuel cell systems are documented, with particular emphasis on design, operation, and performance. The present microfluidic fuel cell systems are categorized by the fluidic phases of the fuel and oxidant streams, featuring gaseous/gaseous, liquid/gaseous, and liquid/liquid systems. The typical cell configurations and recent contributions in each category are analyzed. Key research challenges and opportunities are highlighted and recommendations for further work are provided.

  9. Biosensing with cell phones.

    Science.gov (United States)

    Preechaburana, Pakorn; Suska, Anke; Filippini, Daniel

    2014-07-01

    Continued progress in cell-phone devices has made them powerful mobile computers, equipped with sophisticated, permanent physical sensors embedded as the default configuration. By contrast, the incorporation of permanent biosensors in cell-phone units has been prevented by the multivocal nature of the stimuli and the reactions involved in biosensing and chemical sensing. Biosensing with cell phones entails the complementation of biosensing devices with the physical sensors and communication and processing capabilities of modern cell phones. Biosensing, chemical-sensing, environmental-sensing, and diagnostic capabilities would thus be supported and run on the residual capacity of existing cell-phone infrastructure. The technologies necessary to materialize such a scenario have emerged in different fields and applications. This article addresses the progress on cell-phone biosensing, the specific compromises, and the blend of technologies required to craft biosensing on cell phones. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Enteroendocrine cell types revisited

    DEFF Research Database (Denmark)

    Engelstoft, Maja S; Egerod, Kristoffer Lihme; Lund, Mari L

    2013-01-01

    The GI-tract is profoundly involved in the control of metabolism through peptide hormones secreted from enteroendocrine cells scattered throughout the gut mucosa. A large number of recently generated transgenic reporter mice have allowed for direct characterization of biochemical and cell...... biological properties of these previously highly elusive enteroendocrine cells. In particular the surprisingly broad co-expression of six functionally related hormones in the intestinal enteroendocrine cells indicates that it should be possible to control not only the hormone secretion but also the type...... and number of enteroendocrine cells. However, this will require a more deep understanding of the factors controlling differentiation, gene expression and specification of the enteroendocrine cells during their weekly renewal from progenitor cells in the crypts of the mucosa....

  11. Avian B cell development.

    Science.gov (United States)

    Masteller, E L; Pharr, G T; Funk, P E; Thompson, C B

    1997-01-01

    Development of B cells in chickens proceeds via a series of discrete developmental stages that includes the maturation of committed B cell progenitors in the specialized microenvironment of the bursa of Fabricius. The bursa has been shown to be required for the amplification of the B cell pool and selects for cells with productive immunoglobulin rearrangement events. Other events regulating chicken B cell development such as lymphocyte trafficking and apoptosis are just beginning to be elucidated. Within the bursa, the variable regions of immunoglobulin genes of B cell progenitors are diversified by a process of intrachromosomal gene conversion, where blocks of sequence information are transferred from pseudo-V regions to the recombined variable regions of the immunoglobulin genes. Recently gene conversion has been determined to play a role in the diversification of the immune repertoire in other species. In this review we focus on the current understanding and recent advances of B cell development in the chicken.

  12. Human regulatory B cells control the TFH cell response.

    Science.gov (United States)

    Achour, Achouak; Simon, Quentin; Mohr, Audrey; Séité, Jean-François; Youinou, Pierre; Bendaoud, Boutahar; Ghedira, Ibtissem; Pers, Jacques-Olivier; Jamin, Christophe

    2017-07-01

    Follicular helper T (TFH) cells support terminal B-cell differentiation. Human regulatory B (Breg) cells modulate cellular responses, but their control of TFH cell-dependent humoral immune responses is unknown. We sought to assess the role of Breg cells on TFH cell development and function. Human T cells were polyclonally stimulated in the presence of IL-12 and IL-21 to generate TFH cells. They were cocultured with B cells to induce their terminal differentiation. Breg cells were included in these cultures, and their effects were evaluated by using flow cytometry and ELISA. B-cell lymphoma 6, IL-21, inducible costimulator, CXCR5, and programmed cell death protein 1 (PD-1) expressions increased on stimulated human T cells, characterizing TFH cell maturation. In cocultures they differentiated B cells into CD138(+) plasma and IgD(-)CD27(+) memory cells and triggered immunoglobulin secretions. Breg cells obtained by Toll-like receptor 9 and CD40 activation of B cells prevented TFH cell development. Added to TFH cell and B-cell cocultures, they inhibited B-cell differentiation, impeded immunoglobulin secretions, and expanded Foxp3(+)CXCR5(+)PD-1(+) follicular regulatory T cells. Breg cells modulated IL-21 receptor expressions on TFH cells and B cells, and their suppressive activities involved CD40, CD80, CD86, and intercellular adhesion molecule interactions and required production of IL-10 and TGF-β. Human Breg cells control TFH cell maturation, expand follicular regulatory T cells, and inhibit the TFH cell-mediated antibody secretion. These novel observations demonstrate a role for the Breg cell in germinal center reactions and suggest that deficient activities might impair the TFH cell-dependent control of humoral immunity and might lead to the development of aberrant autoimmune responses. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  13. Membrane Cells for Brine Electrolysis.

    Science.gov (United States)

    Tingle, M.

    1982-01-01

    Membrane cells were developed as alternatives to mercury and diaphragm cells for the electrolysis of brine. Compares the three types of cells, focusing on the advantages and disadvantages of membrane cells. (JN)

  14. Sickle Cell Crisis (For Teens)

    Science.gov (United States)

    ... Plan Hot Topics Flu Facts Arrhythmias Abuse Sickle Cell Crisis (Pain Crisis) KidsHealth > For Teens > Sickle Cell ... drepanocíticas (Crisis de dolor) What Is a Sickle Cell Crisis? Sickle cell disease changes the shape of ...

  15. Nevoid basal cell carcinoma syndrome

    Science.gov (United States)

    NBCC syndrome; Gorlin-Goltz syndrome; Basal cell nevus syndrome; BCNS; Basal cell cancer - nevoid basal cell carcinoma syndrome ... Nevoid basal cell carcinoma nevus syndrome is a rare genetic ... syndrome is known as PTCH ("patched"). The gene is passed down ...

  16. Functional interplay between cell cycle and cell phenotypes

    Science.gov (United States)

    Chen, Wei-Chiang; Wu, Pei-Hsun; Phillip, Jude M.; Khatau, Shyam B.; Choi, Jae Min; Dallas, Matthew R.; Konstantopoulos, Konstantinos; Sun, Sean X.; Lee, Jerry S.H.; Hodzic, Didier; Wirtz, Denis

    2013-01-01

    Cell cycle distribution of adherent cells is typically assessed using flow cytometry, which precludes the measurements of many cell properties and their cycle phase in the same environment. Here we develop and validate a microscopy system to quantitatively analyze the cell-cycle phase of thousands of adherent cells and their associated cell properties simultaneously. This assay demonstrates that population-averaged cell phenotypes can be written as a linear combination of cell-cycle fractions and phase-dependent phenotypes. By perturbing cell cycle through inhibition of cell-cycle regulators or changing nuclear morphology by depletion of structural proteins, our results reveal that cell cycle regulators and structural proteins can significantly interfere with each other’s prima facie functions. This study introduces a high-throughput method to simultaneously measure cell cycle and phenotypes at single-cell resolution, which reveals a complex functional interplay between cell cycle and cell phenotypes. PMID:23319145

  17. PEROVSKITE SOLAR CELLS (REVIEW ARTICLE)

    OpenAIRE

    Benli, Deniz

    2015-01-01

    A solar cell is a device that converts sunlight into electricity. There are different types of solar cells but this report mainly focuses on a type of new generation solar cell that has the name organo-metal halide perovskite, shortly perovskite solar cells. In this respect, the efficiency of power conversion is taken into account to replace the dominancy of traditional and second generation solar cell fields by perovskite solar cells. Perovskite solar cell is a type of solar cell including a...

  18. Nanofluidic fuel cell

    Science.gov (United States)

    Lee, Jin Wook; Kjeang, Erik

    2013-11-01

    Fuel cells are gaining momentum as a critical component in the renewable energy mix for stationary, transportation, and portable power applications. State-of-the-art fuel cell technology benefits greatly from nanotechnology applied to nanostructured membranes, catalysts, and electrodes. However, the potential of utilizing nanofluidics for fuel cells has not yet been explored, despite the significant opportunity of harnessing rapid nanoscale reactant transport in close proximity to the reactive sites. In the present article, a nanofluidic fuel cell that utilizes fluid flow through nanoporous media is conceptualized and demonstrated for the first time. This transformative concept captures the advantages of recently developed membraneless and catalyst-free fuel cell architectures paired with the enhanced interfacial contact area enabled by nanofluidics. When compared to previously reported microfluidic fuel cells, the prototype nanofluidic fuel cell demonstrates increased surface area, reduced activation overpotential, superior kinetic characteristics, and moderately enhanced fuel cell performance in the high cell voltage regime with up to 14% higher power density. However, the expected mass transport benefits in the high current density regime were constrained by high ohmic cell resistance, which could likely be resolved through future optimization studies.

  19. Mast Cell Function

    Science.gov (United States)

    da Silva, Elaine Zayas Marcelino; Jamur, Maria Célia

    2014-01-01

    Since first described by Paul Ehrlich in 1878, mast cells have been mostly viewed as effectors of allergy. It has been only in the past two decades that mast cells have gained recognition for their involvement in other physiological and pathological processes. Mast cells have a widespread distribution and are found predominantly at the interface between the host and the external environment. Mast cell maturation, phenotype and function are a direct consequence of the local microenvironment and have a marked influence on their ability to specifically recognize and respond to various stimuli through the release of an array of biologically active mediators. These features enable mast cells to act as both first responders in harmful situations as well as to respond to changes in their environment by communicating with a variety of other cells implicated in physiological and immunological responses. Therefore, the critical role of mast cells in both innate and adaptive immunity, including immune tolerance, has gained increased prominence. Conversely, mast cell dysfunction has pointed to these cells as the main offenders in several chronic allergic/inflammatory disorders, cancer and autoimmune diseases. This review summarizes the current knowledge of mast cell function in both normal and pathological conditions with regards to their regulation, phenotype and role. PMID:25062998

  20. CELL RESPIRATION STUDIES

    Science.gov (United States)

    Daland, Geneva A.; Isaacs, Raphael

    1927-01-01

    1. The oxygen consumption of blood of normal individuals, when the hemoglobin is saturated with oxygen, is practically zero within the limits of experimental error of the microspirometer used. 2. The oxygen consumed in a microspirometer by the blood of patients with chronic myelogenous leucemia with a high white blood cell count, and of one with leucocytosis from sepsis, was proportional to the number of adult polymorphonuclear neutrophils in the blood. 3. No correlation could be made between the rate of oxygen absorption and the total number of white blood cells in the blood, or the total number of immature cells, or the number of red blood cells, or the amount of oxyhemoglobin. 4. The blood of patients with chronic myelogenous leucemia continued to use oxygen in the microspirometer longer than that of normal individuals, and the hemoglobin, in the leucemic bloods, became desaturated even though exposed to air. 5. In blood in which the bulk. of the cells were immature and the mature cells few, the oxygen consumption was lower than in blood in which the mature cells predominated. The rate of oxygen consumption of the immature cells was relatively low as compared to the mature. 6. The slower rate of oxygen absorption by the immature leucocytes in chronic myelogenous leucemia as compared to the mature cells, places them, in accord with Warburg's reports, in the class of the malignant tissues in this respect rather than in the group of young or embryonic cells. PMID:19869329

  1. NKT cells in leishmaniasis.

    Science.gov (United States)

    Zamora-Chimal, Jaime; Hernández-Ruiz, Joselín; Becker, Ingeborg

    2017-04-01

    The role of NKT cells in the resistance or susceptibility towards Leishmania infections remains to be defined, since controversial data persist. The response of these cells seems to depend on many variables such as the infection site, the number of infecting parasites, the virulence of the strain and the Leishmania species. We here revise the activation pathways leading to NKT cell activation. NKT cells can be activated by the direct pathway, in which Leishmania glycolipids are presented by CD1d molecules on antigen presenting cells, such as dendritic cells (DC), leading to the secretion of diverse cytokines by NKT. NKT cells can also be activated by the indirect pathway, in which Leishmania glycolipids, such as LPG, stimulate TLR2 in DC, inducing their IL-12 production, which in turn activates NKT cells. The review further analyzes the role of NKT cells in disease development, both in humans as in mouse models. Finally we propose the activation of NKT cells for controlling Leishmania infections. Copyright © 2016 Elsevier GmbH. All rights reserved.

  2. T Cells in Fish.

    Science.gov (United States)

    Nakanishi, Teruyuki; Shibasaki, Yasuhiro; Matsuura, Yuta

    2015-09-25

    Cartilaginous and bony fish are the most primitive vertebrates with a thymus, and possess T cells equivalent to those in mammals. There are a number of studies in fish demonstrating that the thymus is the essential organ for development of T lymphocytes from early thymocyte progenitors to functionally competent T cells. A high number of T cells in the intestine and gills has been reported in several fish species. Involvement of CD4⁺ and CD8α⁺ T cells in allograft rejection and graft-versus-host reaction (GVHR) has been demonstrated using monoclonal antibodies. Conservation of CD4⁺ helper T cell functions among teleost fishes has been suggested in a number studies employing mixed leukocyte culture (MLC) and hapten/carrier effect. Alloantigen- and virus-specific cytotoxicity has also been demonstrated in ginbuna and rainbow trout. Furthermore, the important role of cell-mediated immunity rather than humoral immunity has been reported in the protection against intracellular bacterial infection. Recently, the direct antibacterial activity of CD8α⁺, CD4⁺ T-cells and sIgM⁺ cells in fish has been reported. In this review, we summarize the recent progress in T cell research focusing on the tissue distribution and function of fish T cells.

  3. Biology of Schwann cells.

    Science.gov (United States)

    Kidd, Grahame J; Ohno, Nobuhiko; Trapp, Bruce D

    2013-01-01

    The fundamental roles of Schwann cells during peripheral nerve formation and regeneration have been recognized for more than 100 years, but the cellular and molecular mechanisms that integrate Schwann cell and axonal functions continue to be elucidated. Derived from the embryonic neural crest, Schwann cells differentiate into myelinating cells or bundle multiple unmyelinated axons into Remak fibers. Axons dictate which differentiation path Schwann cells follow, and recent studies have established that axonal neuregulin1 signaling via ErbB2/B3 receptors on Schwann cells is essential for Schwann cell myelination. Extracellular matrix production and interactions mediated by specific integrin and dystroglycan complexes are also critical requisites for Schwann cell-axon interactions. Myelination entails expansion and specialization of the Schwann cell plasma membrane over millimeter distances. Many of the myelin-specific proteins have been identified, and transgenic manipulation of myelin genes have provided novel insights into myelin protein function, including maintenance of axonal integrity and survival. Cellular events that facilitate myelination, including microtubule-based protein and mRNA targeting, and actin based locomotion, have also begun to be understood. Arguably, the most remarkable facet of Schwann cell biology, however, is their vigorous response to axonal damage. Degradation of myelin, dedifferentiation, division, production of axonotrophic factors, and remyelination all underpin the substantial regenerative capacity of the Schwann cells and peripheral nerves. Many of these properties are not shared by CNS fibers, which are myelinated by oligodendrocytes. Dissecting the molecular mechanisms responsible for the complex biology of Schwann cells continues to have practical benefits in identifying novel therapeutic targets not only for Schwann cell-specific diseases but other disorders in which axons degenerate. Copyright © 2013 Elsevier B.V. All rights

  4. Myoepithelial cells in pathology

    Directory of Open Access Journals (Sweden)

    N Balachander

    2015-01-01

    Full Text Available Myoepithelial cells are a normal constituent of the salivary acini and ducts and are found between the epithelial cells and the basement membrane. Microscopically myoepithelial cells are thin and spindle-shaped and ultrastructurally they possess a number of Cytoplasmic processes that extend between and over the acinar and ductal-lining cells, and they show features of both smooth muscle and epithelium. They play a vital role during expulsion of saliva and regulates the electrolytic exchange. They also perform as tumor suppressors and are considered to play a very important role in differentiation of various salivary gland tumors and help in the diagnosis of tumors. Neoplastic myoepithelial cells in both benign and malignant tumors can take numerous forms including epithelioid, plasmacytoid, spindle and clear cell variant, and this variability largely accounts for difficulties in histopathological diagnosis.

  5. Cell-matrix adhesion.

    Science.gov (United States)

    Berrier, Allison L; Yamada, Kenneth M

    2007-12-01

    The complex interactions of cells with extracellular matrix (ECM) play crucial roles in mediating and regulating many processes, including cell adhesion, migration, and signaling during morphogenesis, tissue homeostasis, wound healing, and tumorigenesis. Many of these interactions involve transmembrane integrin receptors. Integrins cluster in specific cell-matrix adhesions to provide dynamic links between extracellular and intracellular environments by bi-directional signaling and by organizing the ECM and intracellular cytoskeletal and signaling molecules. This mini review discusses these interconnections, including the roles of matrix properties such as composition, three-dimensionality, and porosity, the bi-directional functions of cellular contractility and matrix rigidity, and cell signaling. The review concludes by speculating on the application of this knowledge of cell-matrix interactions in the formation of cell adhesions, assembly of matrix, migration, and tumorigenesis to potential future therapeutic approaches. 2007 Wiley-Liss, Inc.

  6. Cytoskeleton and Cell Motility

    CERN Document Server

    Risler, Thomas

    2011-01-01

    The present article is an invited contribution to the Encyclopedia of Complexity and System Science, Robert A. Meyers Ed., Springer New York (2009). It is a review of the biophysical mechanisms that underly cell motility. It mainly focuses on the eukaryotic cytoskeleton and cell-motility mechanisms. Bacterial motility as well as the composition of the prokaryotic cytoskeleton is only briefly mentioned. The article is organized as follows. In Section III, I first present an overview of the diversity of cellular motility mechanisms, which might at first glance be categorized into two different types of behaviors, namely "swimming" and "crawling". Intracellular transport, mitosis - or cell division - as well as other extensions of cell motility that rely on the same essential machinery are briefly sketched. In Section IV, I introduce the molecular machinery that underlies cell motility - the cytoskeleton - as well as its interactions with the external environment of the cell and its main regulatory pathways. Sec...

  7. Fish germ cells.

    Science.gov (United States)

    Xu, HongYan; Li, MingYou; Gui, JianFang; Hong, YunHan

    2010-04-01

    Fish, like many other animals, have two major cell lineages, namely the germline and soma. The germ-soma separation is one of the earliest events of embryonic development. Germ cells can be specifically labeled and isolated for culture and transplantation, providing tools for reproduction of endangered species in close relatives, such as surrogate production of trout in salmon. Haploid cell cultures, such as medaka haploid embryonic stem cells have recently been obtained, which are capable of mimicking sperm to produce fertile offspring, upon nuclear being directly transferred into normal eggs. Such fish originated from a mosaic oocyte that had a haploid meiotic nucleus and a transplanted haploid mitotic cell culture nucleus. The first semi-cloned fish is Holly. Here we review the current status and future directions of understanding and manipulating fish germ cells in basic research and reproductive technology.

  8. Mammary gland stem cells

    DEFF Research Database (Denmark)

    Fridriksdottir, Agla J R; Petersen, Ole W; Rønnov-Jessen, Lone

    2011-01-01

    Distinct subsets of cells, including cells with stem cell-like properties, have been proposed to exist in normal human breast epithelium and breast carcinomas. The cellular origins of epithelial cells contributing to gland development, tissue homeostasis and cancer are, however, still poorly...... and differences between mouse and human gland development with particular emphasis on the identity and localization of stem cells, and the influence of the surrounding microenvironment. It is concluded that while recent advances in the field have contributed immense insight into how the normal mammary gland...... develops and is maintained, significant discrepancies exist between the mouse and human gland which should be taken into consideration in current and future models of mammary stem cell biology....

  9. Gingival plasma cell granuloma

    Directory of Open Access Journals (Sweden)

    Amitkumar B Pandav

    2012-01-01

    Full Text Available Plasma cell granuloma, also known as inflammatory pseudotumor is a tumor-like lesion that manifests primarily in the lungs. But it may occur in various other anatomic locations like orbit, head and neck, liver and rarely in the oral cavity. We here report an exceedingly rare case of gingival plasma cell granuloma in a 58 year old woman who presented with upper gingival polypoidal growth. The histopathological examination revealed a mass composed of proliferation of benign spindle mesenchymal cells in a loose myxoid and fibrocollagenous stroma along with dense infiltrate of chronic inflammatory cells predominantly containing plasma cells. Immunohistochemistry for kappa and lambda light chains showed a polyclonal staining pattern confirming a diagnosis of plasma cell granuloma.

  10. NCAM regulates cell motility

    DEFF Research Database (Denmark)

    Prag, Søren; Lepekhin, Eugene A; Kolkova, Kateryna

    2002-01-01

    Cell migration is required during development of the nervous system. The regulatory mechanisms for this process, however, are poorly elucidated. We show here that expression of or exposure to the neural cell adhesion molecule (NCAM) strongly affected the motile behaviour of glioma cells...... independently of homophilic NCAM interactions. Expression of the transmembrane 140 kDa isoform of NCAM (NCAM-140) caused a significant reduction in cellular motility, probably through interference with factors regulating cellular attachment, as NCAM-140-expressing cells exhibited a decreased attachment...... to a fibronectin substratum compared with NCAM-negative cells. Ectopic expression of the cytoplasmic part of NCAM-140 also inhibited cell motility, presumably via the non-receptor tyrosine kinase p59(fyn) with which NCAM-140 interacts. Furthermore, we showed that the extracellular part of NCAM acted as a paracrine...

  11. The human cell atlas

    DEFF Research Database (Denmark)

    Regev, Aviv; Teichmann, Sarah A.; Lander, Eric S.

    2017-01-01

    The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international...... collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells...... in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early...

  12. Cell Therapy in Dermatology

    Science.gov (United States)

    Petrof, Gabriela; Abdul-Wahab, Alya; McGrath, John A.

    2014-01-01

    Harnessing the regenerative capacity of keratinocytes and fibroblasts from human skin has created new opportunities to develop cell-based therapies for patients. Cultured cells and bioengineered skin products are being used to treat patients with inherited and acquired skin disorders associated with defective skin, and further clinical trials of new products are in progress. The capacity of extracutaneous sources of cells such as bone marrow is also being investigated for its plasticity in regenerating skin, and new strategies, such as the derivation of inducible pluripotent stem cells, also hold great promise for future cell therapies in dermatology. This article reviews some of the preclinical and clinical studies and future directions relating to cell therapy in dermatology, particularly for inherited skin diseases associated with fragile skin and poor wound healing. PMID:24890834

  13. Basal cell carcinoma of the skin with areas of squamous cell carcinoma: a basosquamous cell carcinoma?

    OpenAIRE

    de Faria, J

    1985-01-01

    The diagnosis of basosquamous cell carcinoma is controversial. A review of cases of basal cell carcinoma showed 23 cases that had conspicuous areas of squamous cell carcinoma. This was distinguished from squamous differentiation and keratotic basal cell carcinoma by a comparative study of 40 cases of compact lobular and 40 cases of keratotic basal cell carcinoma. Areas of intermediate tumour differentiation between basal cell and squamous cell carcinoma were found. Basal cell carcinomas with ...

  14. Traction in smooth muscle cells varies with cell spreading

    Science.gov (United States)

    Tolic-Norrelykke, Iva Marija; Wang, Ning

    2005-01-01

    Changes in cell shape regulate cell growth, differentiation, and apoptosis. It has been suggested that the regulation of cell function by the cell shape is a result of the tension in the cytoskeleton and the distortion of the cell. Here we explore the association between cell-generated mechanical forces and the cell morphology. We hypothesized that the cell contractile force is associated with the degree of cell spreading, in particular with the cell length. We measured traction fields of single human airway smooth muscle cells plated on a polyacrylamide gel, in which fluorescent microbeads were embedded to serve as markers of gel deformation. The traction exerted by the cells at the cell-substrate interface was determined from the measured deformation of the gel. The traction was measured before and after treatment with the contractile agonist histamine, or the relaxing agonist isoproterenol. The relative increase in traction induced by histamine was negatively correlated with the baseline traction. On the contrary, the relative decrease in traction due to isoproterenol was independent of the baseline traction, but it was associated with cell shape: traction decreased more in elongated than in round cells. Maximum cell width, mean cell width, and projected area of the cell were the parameters most tightly coupled to both baseline and histamine-induced traction in this study. Wide and well-spread cells exerted larger traction than slim cells. These results suggest that cell contractility is controlled by cell spreading.

  15. Quantitative methods for analyzing cell-cell adhesion in development.

    Science.gov (United States)

    Kashef, Jubin; Franz, Clemens M

    2015-05-01

    During development cell-cell adhesion is not only crucial to maintain tissue morphogenesis and homeostasis, it also activates signalling pathways important for the regulation of different cellular processes including cell survival, gene expression, collective cell migration and differentiation. Importantly, gene mutations of adhesion receptors can cause developmental disorders and different diseases. Quantitative methods to measure cell adhesion are therefore necessary to understand how cells regulate cell-cell adhesion during development and how aberrations in cell-cell adhesion contribute to disease. Different in vitro adhesion assays have been developed in the past, but not all of them are suitable to study developmentally-related cell-cell adhesion processes, which usually requires working with low numbers of primary cells. In this review, we provide an overview of different in vitro techniques to study cell-cell adhesion during development, including a semi-quantitative cell flipping assay, and quantitative single-cell methods based on atomic force microscopy (AFM)-based single-cell force spectroscopy (SCFS) or dual micropipette aspiration (DPA). Furthermore, we review applications of Förster resonance energy transfer (FRET)-based molecular tension sensors to visualize intracellular mechanical forces acting on cell adhesion sites. Finally, we describe a recently introduced method to quantitate cell-generated forces directly in living tissues based on the deformation of oil microdroplets functionalized with adhesion receptor ligands. Together, these techniques provide a comprehensive toolbox to characterize different cell-cell adhesion phenomena during development. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Bioprinting with live cells

    OpenAIRE

    Özler, Saime Burçe; Ozler, Saime Burce; Küçükgül, Can; Kucukgul, Can; KOÇ, Bahattin; Koc, Bahattin

    2015-01-01

    Tissue engineering is an emerging multidisciplinary field to regenerate damaged or diseased tissues and organs. Traditional tissue engineering strategies involve seeding cells into porous scaffolds to regenerate tissues or organs. However, there are still some challenges such as difficulty in seeding different type of cells spatially into a scaffold, limited oxygen and nutrient delivery and removal of metabolic waste from scaffold and weak cell-adhesion to scaffold material need to be overcom...

  17. Immobilized Cell Research

    Science.gov (United States)

    1990-10-31

    substances (EPS or slime) by Pseudomonas biorcniediatcd. The membrane has the oxygen per- aeruginosa growing on surface of ultrafiltration mem- mcability...the same nutritional stresses, well- metabolism, mixed, free-living cells will react similarly. The exagger- Bringi and Shuler, Cornell University...mammalian cells. This technique could be as putida and a Hyphornicrobium species. Thes._ organ- used to measure the physiological status of cells in

  18. Direct hydrocarbon fuel cells

    Science.gov (United States)

    Barnett, Scott A.; Lai, Tammy; Liu, Jiang

    2010-05-04

    The direct electrochemical oxidation of hydrocarbons in solid oxide fuel cells, to generate greater power densities at lower temperatures without carbon deposition. The performance obtained is comparable to that of fuel cells used for hydrogen, and is achieved by using novel anode composites at low operating temperatures. Such solid oxide fuel cells, regardless of fuel source or operation, can be configured advantageously using the structural geometries of this invention.

  19. Lymphomas of large cells.

    Science.gov (United States)

    Staples, W G; Gétaz, E P

    1977-09-03

    Historial aspects of the classification of large-cell lymphomas are described. Immunological characterization of the lymphomas has been made possible by identification of T and B lymphocytes according to their cell membrane surface characteristics. The pathogenesis of lymphomas has been clarified by the germinal (follicular) centre cell concepts of Lennert and Lukes and Collins. The various classifications are presented and compared. Whether these subdivisions will have any relevance in the clinical context remains to be seen.

  20. Liver Cancer Stem Cells

    OpenAIRE

    Sameh Mikhail; Aiwu Ruth He

    2011-01-01

    Hepatocellular carcinoma is the most common primary malignancy of the liver in adults. It is also the fifth most common solid cancer worldwide and the third leading cause of cancer-related death. Recent research supports that liver cancer is a disease of adult stem cells. From the models of experimental hepatocarcinogenesis, there may be at least three distinct cell lineages with progenitor properties susceptible to neoplastic transformation. Identification of specific cell surface markers fo...

  1. Physics of adherent cells

    OpenAIRE

    Schwarz, Ulrich S.; Safran, Samuel S.

    2013-01-01

    One of the most unique physical features of cell adhesion to external surfaces is the active generation of mechanical force at the cell-material interface. This includes pulling forces generated by contractile polymer bundles and networks, and pushing forces generated by the polymerization of polymer networks. These forces are transmitted to the substrate mainly by focal adhesions, which are large, yet highly dynamic adhesion clusters. Tissue cells use these forces to sense the physical prope...

  2. Materials for fuel cells

    OpenAIRE

    Haile, Sossina M

    2003-01-01

    Because of their potential to reduce the environmental impact and geopolitical consequences of the use of fossil fuels, fuel cells have emerged as tantalizing alternatives to combustion engines. Like a combustion engine, a fuel cell uses some sort of chemical fuel as its energy source but, like a battery, the chemical energy is directly converted to electrical energy, without an often messy and relatively inefficient combustion step. In addition to high efficiency and low emissions, fuel cell...

  3. Nanocrystal Solar Cells

    Energy Technology Data Exchange (ETDEWEB)

    Gur, Ilan [Univ. of California, Berkeley, CA (United States)

    2006-01-01

    This dissertation presents the results of a research agenda aimed at improving integration and stability in nanocrystal-based solar cells through advances in active materials and device architectures. The introduction of 3-dimensional nanocrystals illustrates the potential for improving transport and percolation in hybrid solar cells and enables novel fabrication methods for optimizing integration in these systems. Fabricating cells by sequential deposition allows for solution-based assembly of hybrid composites with controlled and well-characterized dispersion and electrode contact. Hyperbranched nanocrystals emerge as a nearly ideal building block for hybrid cells, allowing the controlled morphologies targeted by templated approaches to be achieved in an easily fabricated solution-cast device. In addition to offering practical benefits to device processing, these approaches offer fundamental insight into the operation of hybrid solar cells, shedding light on key phenomena such as the roles of electrode-contact and percolation behavior in these cells. Finally, all-inorganic nanocrystal solar cells are presented as a wholly new cell concept, illustrating that donor-acceptor charge transfer and directed carrier diffusion can be utilized in a system with no organic components, and that nanocrystals may act as building blocks for efficient, stable, and low-cost thin-film solar cells.

  4. Transfection of nerve cells.

    Science.gov (United States)

    Salozhin, S V; Bol'shakov, A P

    2010-03-01

    Transfection is a method of transforming cells based on the introduction into living cells of plasmids encoding a particular protein or RNA. This review describes the main methods of transfection and considers their advantages and disadvantages. Most attention is paid to lentivirus transduction as one of the most efficient methods for transforming nerve cells. The development of current transfection systems based on lentivirus vectors is described and a brief review of studies performed using in vivo and in vitro lentivirus transfection of nerve cells is presented.

  5. Stem Cells in Neuroendocrinology

    National Research Council Canada - National Science Library

    Pfaff, Donald; Christen, Yves

    2016-01-01

    This volume starts with an elementary introduction covering stem cell methodologies used to produce specific types of neurons, possibilities for their therapeutic use, and warnings of technical problems...

  6. Prostate cancer stem cells.

    Science.gov (United States)

    Tu, Shi-Ming; Lin, Sue-Hwa

    2012-06-01

    Stem cells have long been implicated in prostate gland formation. The prostate undergoes regression after androgen deprivation and regeneration after testosterone replacement. Regenerative studies suggest that these cells are found in the proximal ducts and basal layer of the prostate. Many characteristics of prostate cancer indicate that it originates from stem cells. For example, the putative androgen receptor-negative (AR(-)) status of prostate stem cells renders them inherently insensitive to androgen blockade therapy. The androgen-regulated gene fusion TMPRSS2-ERG could be used to clarify both the cells of origin and the evolution of prostate cancer cells. In this review, we show that the hypothesis that distinct subtypes of cancer result from abnormalities within specific cell types-the stem cell theory of cancer-may instigate a major paradigm shift in cancer research and therapy. Ultimately, the stem cell theory of cancers will affect how we practice clinical oncology: our diagnosis, monitoring, and therapy of prostate and other cancers. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. Communicating artificial cells.

    Science.gov (United States)

    Lentini, Roberta; Yeh Martín, Noël; Mansy, Sheref S

    2016-10-01

    Intercellular chemical communication is commonly exploited for the engineering of living cells but has been largely ignored by efforts to build artificial cells. Since communication is a fundamental feature of life, the construction of artificial cells capable of chemical communication will likely lead to a deeper understanding of biology and allow for the development of life-like technologies. Herein we highlight recent progress towards the construction of artificial systems that are capable of chemically communicating with natural living cells. Artificial systems that exploit both biological and abiological material for function are discussed. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Applications of Cell Microencapsulation.

    Science.gov (United States)

    Opara, Emmanuel C

    2017-01-01

    The goal of this chapter is to provide an overview of the different purposes for which the cell microencapsulation technology can be used. These include immunoisolation of non-autologous cells used for cell therapy; immobilization of cells for localized (targeted) delivery of therapeutic products to ablate, repair, or regenerate tissue; simultaneous delivery of multiple therapeutic agents in cell therapy; spatial compartmentalization of cells in complex tissue engineering; expansion of cells in culture; and production of different probiotics and metabolites for industrial applications. For each of these applications, specific examples are provided to illustrate how the microencapsulation technology can be utilized to achieve the purpose. However, successful use of the cell microencapsulation technology for whatever purpose will ultimately depend upon careful consideration for the choice of the encapsulating polymers, the method of fabrication (cross-linking) of the microbeads, which affects the permselectivity, the biocompatibility and the mechanical strength of the microbeads as well as environmental parameters such as temperature, humidity, osmotic pressure, and storage solutions.The various applications discussed in this chapter are illustrated in the different chapters of this book and where appropriate relevant images of the microencapsulation products are provided. It is hoped that this outline of the different applications of cell microencapsulation would provide a good platform for tissue engineers, scientists, and clinicians to design novel tissue constructs and products for therapeutic and industrial applications.

  9. Biomaterials in cell microencapsulation.

    Science.gov (United States)

    Santos, Edorta; Zarate, Jon; Orive, Gorka; Hernández, Rosa M; Pedraz, José Luis

    2010-01-01

    The field of cell encapsulation is advancing rapidly. This cell-based technology permits the local and long-term delivery ofa desired therapeutic product reducing or even avoiding the need ofimmunosuppressant drugs. The choice of a suitable material preserving the viability and functionality of enclosed cells becomes fundamental if a therapeutic aim is intended. Alginate, which is by far the most frequently used biomaterial in the field of cell microencapsulation, has been demonstrated to be probably the best polymer for this purpose due to its biocompatibility, easy manipulation, gel forming capacity and in vivo performance.

  10. Cancer stem cell metabolism

    National Research Council Canada - National Science Library

    Peiris-Pagès, Maria; Martinez-Outschoorn, Ubaldo E; Pestell, Richard G; Sotgia, Federica; Lisanti, Michael P

    2016-01-01

    .... Cancer stem cells also seem to adapt their metabolism to microenvironmental changes by conveniently shifting energy production from one pathway to another, or by acquiring intermediate metabolic phenotypes...

  11. Viral Entry into Cells

    Science.gov (United States)

    D'Orsogna, Maria R.

    2010-09-01

    Successful viral infection of a healthy cell requires complex host-pathogen interactions. In this talk we focus on the dynamics specific to the HIV virus entering a eucaryotic cell. We model viral entry as a stochastic engagement of receptors and coreceptors on the cell surface. We also consider the transport of virus material to the cell nucleus by coupling microtubular motion to the concurrent biochemical transformations that render the viral material competent for nuclear entry. We discuss both mathematical and biological consequences of our model, such as the formulation of an effective integrodifferential boundary condition embodying a memory kernel and optimal timing in maximizing viral probabilities.

  12. Biopolymer networks in cells

    Science.gov (United States)

    Weitz, David

    2013-03-01

    This talk will discuss the role of biopolymer networks in cells. We probe their properties through measurements of fluctuating motions of particles within the cell. These motions have many similarities to thermal motion and, in fact, are often misinterpreted in the context of passive microrheology. Here, we demonstrate that the motion is, instead, driven by the presence of molecular motors within the cell, and we show how this motion can be interpreted quantitatively to determine the nature of the fluctuating forces in the cell due to the molecular motors. I acknowledge the essential input of Ming Guo and Fred MacKintosh and support from NSF and NIH.

  13. T Cells in Fish

    OpenAIRE

    Teruyuki Nakanishi; Yasuhiro Shibasaki; Yuta Matsuura

    2015-01-01

    Cartilaginous and bony fish are the most primitive vertebrates with a thymus, and possess T cells equivalent to those in mammals. There are a number of studies in fish demonstrating that the thymus is the essential organ for development of T lymphocytes from early thymocyte progenitors to functionally competent T cells. A high number of T cells in the intestine and gills has been reported in several fish species. Involvement of CD4+ and CD8α+ T cells in allograft rejection and graft-versus-ho...

  14. Littoral Cells 2005

    Data.gov (United States)

    California Department of Resources — Littoral cells along the California Coast. Originally digitized by Melanie Coyne from the Assessment and Atlas of Shoreline Erosion Along the California Coast...

  15. Microencapsulation Of Living Cells

    Science.gov (United States)

    Chang, Manchium; Kendall, James M.; Wang, Taylor G.

    1989-01-01

    In experimental technique, living cells and other biological materials encapsulated within submillimeter-diameter liquid-filled spheres. Sphere material biocompatible, tough, and compliant. Semipermeable, permitting relatively small molecules to move into and out of sphere core but preventing passage of large molecules. New technique promises to make such spherical capsules at high rates and in uniform, controllable sizes. Capsules injected into patient through ordinary hypodermic needle. Promising application for technique in treatment of diabetes. Also used to encapsulate pituitary cells and thyroid hormone adrenocortical cells for treatment of other hormonal disorders, to encapsulate other secreting cells for transplantation, and to package variety of pharmaceutical products and agricultural chemicals for controlled release.

  16. Separating biological cells

    Science.gov (United States)

    Brooks, D. E.

    1979-01-01

    Technique utilizing electric field to promote biological cell separation from suspending medium in zero gravity increases speed, reduces sedimentation, and improves efficiency of separation in normal gravity.

  17. Bacterial Cell Mechanics.

    Science.gov (United States)

    Auer, George K; Weibel, Douglas B

    2017-07-25

    Cellular mechanical properties play an integral role in bacterial survival and adaptation. Historically, the bacterial cell wall and, in particular, the layer of polymeric material called the peptidoglycan were the elements to which cell mechanics could be primarily attributed. Disrupting the biochemical machinery that assembles the peptidoglycan (e.g., using the β-lactam family of antibiotics) alters the structure of this material, leads to mechanical defects, and results in cell lysis. Decades after the discovery of peptidoglycan-synthesizing enzymes, the mechanisms that underlie their positioning and regulation are still not entirely understood. In addition, recent evidence suggests a diverse group of other biochemical elements influence bacterial cell mechanics, may be regulated by new cellular mechanisms, and may be triggered in different environmental contexts to enable cell adaptation and survival. This review summarizes the contributions that different biomolecular components of the cell wall (e.g., lipopolysaccharides, wall and lipoteichoic acids, lipid bilayers, peptidoglycan, and proteins) make to Gram-negative and Gram-positive bacterial cell mechanics. We discuss the contribution of individual proteins and macromolecular complexes in cell mechanics and the tools that make it possible to quantitatively decipher the biochemical machinery that contributes to bacterial cell mechanics. Advances in this area may provide insight into new biology and influence the development of antibacterial chemotherapies.

  18. Circulating Tumor Cells

    National Research Council Canada - National Science Library

    Vicki Plaks; Charlotte D. Koopman; Zena Werb

    2013-01-01

    .... During successful dissemination, tumor cells invade the surrounding tissue of the primary tumor, intravasate into blood and lymphatic vessels, translocate to distant tissues, extravasate, adapt...

  19. The dyad palindromic glutathione transferase P enhancer binds multiple factors including AP1.

    Science.gov (United States)

    Diccianni, M B; Imagawa, M; Muramatsu, M

    1992-10-11

    Glutathione Transferase P (GST-P) gene expression is dominantly regulated by an upstream enhancer (GPEI) consisting of a dyad of palindromically oriented imperfect TPA (12-O-tetradecanoyl-phorbol-13-acetate)-responsive elements (TRE). GPEI is active in AP1-lacking F9 cells as well in AP1-containing HeLa cells. Despite GPEI's similarity to a TRE, c-jun co-transfection has only a minimal effect on transactivation. Antisense c-jun and c-fos co-transfection experiments further demonstrate the lack of a role for AP1 in GPEI mediated trans-activation in F9 cells, although endogenously present AP1 can influence GPEI in HeLa cells. Co-transfection of delta fosB with c-jun, which forms an inactive c-Jun/delta FosB heterodimer that binds TRE sequences, inhibits GPEI-mediated transcription in AP1-lacking F9 cells as well as AP1-containing HeLa cells. These data suggest novel factor(s) other than AP1 are influencing GPEI. Binding studies reveal multiple nucleoproteins bind to GPEI. These factors are likely responsible for the high level of GPEI-mediated transcription observed in the absence of AP1 and during hepatocarcinogenesis.

  20. Regulation of beta cell replication

    DEFF Research Database (Denmark)

    Lee, Ying C; Nielsen, Jens Høiriis

    2008-01-01

    Beta cell mass, at any given time, is governed by cell differentiation, neogenesis, increased or decreased cell size (cell hypertrophy or atrophy), cell death (apoptosis), and beta cell proliferation. Nutrients, hormones and growth factors coupled with their signalling intermediates have been...... suggested to play a role in beta cell mass regulation. In addition, genetic mouse model studies have indicated that cyclins and cyclin-dependent kinases that determine cell cycle progression are involved in beta cell replication, and more recently, menin in association with cyclin-dependent kinase...... inhibitors has been demonstrated to be important in beta cell growth. In this review, we consider and highlight some aspects of cell cycle regulation in relation to beta cell replication. The role of cell cycle regulation in beta cell replication is mostly from studies in rodent models, but whether...

  1. c-Myc-Dependent Cell Competition in Human Cancer Cells.

    Science.gov (United States)

    Patel, Manish S; Shah, Heta S; Shrivastava, Neeta

    2017-07-01

    Cell Competition is an interaction between cells for existence in heterogeneous cell populations of multicellular organisms. This phenomenon is involved in initiation and progression of cancer where heterogeneous cell populations compete directly or indirectly for the survival of the fittest based on differential gene expression. In Drosophila, cells having lower dMyc expression are eliminated by cell competition through apoptosis when present in the milieu of cells having higher dMyc expression. Thus, we designed a study to develop c-Myc (human homolog) dependent in vitro cell competition model of human cancer cells. Cells with higher c-Myc were transfected with c-myc shRNA to prepare cells with lower c-Myc and then co-cultured with the same type of cells having a higher c-Myc in equal ratio. Cells with lower c-Myc showed a significant decrease in numbers when compared with higher c-Myc cells, suggesting "loser" and "winner" status of cells, respectively. During microscopy, engulfment of loser cells by winner cells was observed with higher expression of JNK in loser cells. Furthermore, elimination of loser cells was prevented significantly, when co-cultured cells were treated with the JNK (apoptosis) inhibitor. Above results indicate elimination of loser cells in the presence of winner cells by c-Myc-dependent mechanisms of cell competition in human cancer cells. This could be an important mechanism in human tumors where normal cells are eliminated by c-Myc-overexpressed tumor cells. J. Cell. Biochem. 118: 1782-1791, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  2. Fuel cells and fuel cell catalysts

    Science.gov (United States)

    Masel, Richard I.; Rice, Cynthia A.; Waszczuk, Piotr; Wieckowski, Andrzej

    2006-11-07

    A direct organic fuel cell includes a formic acid fuel solution having between about 10% and about 95% formic acid. The formic acid is oxidized at an anode. The anode may include a Pt/Pd catalyst that promotes the direct oxidation of the formic acid via a direct reaction path that does not include formation of a CO intermediate.

  3. Small cell glioblastoma or small cell carcinoma

    DEFF Research Database (Denmark)

    Hilbrandt, Christine; Sathyadas, Sathya; Dahlrot, Rikke H

    2013-01-01

    was admitted to the hospital with left-sided loss of motor function. A MRI revealed a 6 cm tumor in the right temporoparietal area. The histology was consistent with both glioblastoma multiforme (GBM) and small cell lung carcinoma (SCLC) but IHC was suggestive of a SCLC metastasis. PET-CT revealed...

  4. Single-cell sequencing in stem cell biology.

    Science.gov (United States)

    Wen, Lu; Tang, Fuchou

    2016-04-15

    Cell-to-cell variation and heterogeneity are fundamental and intrinsic characteristics of stem cell populations, but these differences are masked when bulk cells are used for omic analysis. Single-cell sequencing technologies serve as powerful tools to dissect cellular heterogeneity comprehensively and to identify distinct phenotypic cell types, even within a 'homogeneous' stem cell population. These technologies, including single-cell genome, epigenome, and transcriptome sequencing technologies, have been developing rapidly in recent years. The application of these methods to different types of stem cells, including pluripotent stem cells and tissue-specific stem cells, has led to exciting new findings in the stem cell field. In this review, we discuss the recent progress as well as future perspectives in the methodologies and applications of single-cell omic sequencing technologies.

  5. Tetraspanins in Mast Cells

    Directory of Open Access Journals (Sweden)

    Martin eKöberle

    2012-05-01

    Full Text Available Mast cells are key mediators of the immune system, most prominently known for their role in eliciting harmful allergic reactions. Mast cell mediator release (e. g. by degranulation is triggered by Fc{epsilon}RI recognition of antigen – IgE complexes. Until today no therapeutic targeting of this and other mast cell activation pathways is established. Among possible new candidates there are tetraspanins that have been described on mast cells already several years ago.Tetraspanins are transmembrane proteins acting as scaffolds, mediating local clustering of their interaction partners and thus amplify their activities. More recently, tetraspanins were also found to exert intrinsic receptor functions. Tetraspanins have been found to be crucial components of fundamental biological processes like cell motility and adhesion. In immune cells, they not only boost the effectiveness of antigen presentation by clustering MHC molecules, they are also key players in all kinds of degranulation events and immune receptor clustering. This review focuses on the contribution of tetraspanins clustered with Fc{epsilon}RI or residing in granule membranes to classical mast cells functions but also undertakes an outlook on the possible contribution of tetraspanins to newly described mast cell functions and discusses possible drugging strategies.

  6. Criticality in cell differentiation

    Indian Academy of Sciences (India)

    Recentexperimental studies provide considerable support to the idea of criticality in cell differentiation and in other biologicalprocesses like the development of the fruit fly embryo. In this review, an elementary introduction is given to the concept ofcriticality in cell differentiation. The correspondence between the signatures of ...

  7. Dental pulp stem cells

    DEFF Research Database (Denmark)

    Ashri, N. Y.; Ajlan, S. A.; Aldahmash, Abdullah M.

    2015-01-01

    Inflammatory periodontal disease is a major cause of loss of tooth-supporting structures. Novel approaches for regeneration of periodontal apparatus is an area of intensive research. Periodontal tissue engineering implies the use of appropriate regenerative cells, delivered through a suitable sca...... an updated review on dental pulp stem cells and their applications in periodontal regeneration, in combination with different scaffolds and growth factors....

  8. Human mesenchymal stem cells

    DEFF Research Database (Denmark)

    Abdallah, Basem; Kassem, Moustapha

    2008-01-01

    introduced into clinical medicine in variety of applications and through different ways of administration. Here, we discuss approaches for isolation, characterization and directing differentiation of human mesenchymal stem cells (hMSC). An update of the current clinical use of the cells is also provided....

  9. Biosensors for Cell Analysis.

    Science.gov (United States)

    Zhou, Qing; Son, Kyungjin; Liu, Ying; Revzin, Alexander

    2015-01-01

    Biosensors first appeared several decades ago to address the need for monitoring physiological parameters such as oxygen or glucose in biological fluids such as blood. More recently, a new wave of biosensors has emerged in order to provide more nuanced and granular information about the composition and function of living cells. Such biosensors exist at the confluence of technology and medicine and often strive to connect cell phenotype or function to physiological or pathophysiological processes. Our review aims to describe some of the key technological aspects of biosensors being developed for cell analysis. The technological aspects covered in our review include biorecognition elements used for biosensor construction, methods for integrating cells with biosensors, approaches to single-cell analysis, and the use of nanostructured biosensors for cell analysis. Our hope is that the spectrum of possibilities for cell analysis described in this review may pique the interest of biomedical scientists and engineers and may spur new collaborations in the area of using biosensors for cell analysis.

  10. T-cell costimulation

    DEFF Research Database (Denmark)

    Owens, T

    1996-01-01

    The CD40L molecule expressed by CD4+ regulatory T lymphocytes is known to deliver signals that activate B cells and macrophages. It now appears that CD40L regulates T cells themselves, during both their development and their participation in adaptive immune responses....

  11. Sickle cell anemia

    African Journals Online (AJOL)

    salah

    The cells sickle at the oxygen tension normally found in the venous blood. When the level of healthy red cells be- comes too low, this can lead directly or indirectly to a variety of c`omplications which include hemolytic crisis and anaemia, jaundice, colelithiasis, aplas- tic crisis, autosplenectomy, sequestra- tion crisis, dactylitis ...

  12. Chromophobe Renal Cell Carcinoma.

    Directory of Open Access Journals (Sweden)

    Jyotsna Vijaykumar Wader

    2013-04-01

    Full Text Available Renal cell carcinoma is the most common neoplasm of the kidney comprised of different histological variants. Chromophobe renal cell carcinoma (ChRCC is a rare subtype of renal cell carcinoma (RCC mainly diagnosed in the sixth decade of life. It is important to identify this entity because it has significantly better prognosis than the clear cell (conventional and papillary renal cell carcinomas. The chromophobe renal cell carcinoma should be differentiated from oncocytoma and clear cell carcinoma. We report a case of a 70 year-old male who presented with a six month history of hematuria, left sided flank pain and a palpable non-tender lump in the left lumbar region. On radiology, the possibility of a left renal neoplasm was raised. A left radical nephrectomy was done and histopathological diagnosis of Type 2 (mixed chromophobe renal cell carcinoma was given. We present this case owing to its relative rarity of incidence, difficulties encountered and differential diagnoses to be considered during diagnosis as the prognosis and management protocols differ with different variants.

  13. Human innate lymphoid cells

    NARCIS (Netherlands)

    Hazenberg, Mette D.; Spits, Hergen

    2014-01-01

    Innate lymphoid cells (ILCs) are lymphoid cells that do not express rearranged receptors and have important effector and regulatory functions in innate immunity and tissue remodeling. ILCs are categorized into 3 groups based on their distinct patterns of cytokine production and the requirement of

  14. Human innate lymphoid cells

    NARCIS (Netherlands)

    Mjösberg, Jenny; Spits, Hergen

    2016-01-01

    Innate lymphoid cells (ILCs) are increasingly acknowledged as important mediators of immune homeostasis and pathology. ILCs act as early orchestrators of immunity, responding to epithelium-derived signals by expressing an array of cytokines and cell-surface receptors, which shape subsequent immune

  15. Juxtaglomerular Cell Phenotypic Plasticity

    NARCIS (Netherlands)

    A.G. Martini (Alexandre); A.H.J. Danser (Jan)

    2017-01-01

    textabstractRenin is the first and rate-limiting step of the renin-angiotensin system. The exclusive source of renin in the circulation are the juxtaglomerular cells of the kidney, which line the afferent arterioles at the entrance of the glomeruli. Normally, renin production by these cells suffices

  16. Solar cell. Taiyo denchi

    Energy Technology Data Exchange (ETDEWEB)

    Mitsui, K.; Shitsutani, T. (Mitsubishi electric Corp., Tokyo (Japan))

    1991-09-10

    This invention provides a highly efficient soalr cell which requires no accurate conformity of collector electrodes, especially a highly efficient tandem solar cell. This invention comprises a collector electrode placed in an effective light receiving zone on the surface of the 2nd electroconductive semiconductor layer formed on the 1st electroconductive semiconductor substrate, the 1st electrode placed in the periphery of light receiving zone and comprising a common electrode connected to the above-mentioned collector electrode, and the 2nd electrode formed on the back side of above-mentioned semiconductor substrate in zones except the zone facing the effective light-receiving zone. In case of using as a tandem solar cell, the above-mentioned solar cell is used as the 1st solar cell, and, as the 2nd solar cell which is incidented by solar light which passed through it, a solar cell having no electrode is used on the surface which faces the 1st solar cell. 4 figs.

  17. The Constitution by Cell

    Science.gov (United States)

    Greenhut, Stephanie; Jones, Megan

    2010-01-01

    On their visit to the National Archives Experience in Washington, D.C., students in Jenni Ashley and Gay Brock's U.S. history classes at the Potomac School in McLean, Virginia, participated in a pilot program called "The Constitution by Cell." Armed with their cell phones, a basic understanding of the Constitution, and a willingness to…

  18. Cell Phones for Education

    Science.gov (United States)

    Roberson, James H.; Hagevik, Rita A.

    2008-01-01

    Cell phones are fast becoming an integral part of students' everyday lives. They are regarded as important companions and tools for personal expression. School-age children are integrating the cell phone as such, and thus placing a high value on them. Educators endeavor to instill in students a high value for education, but often meet with…

  19. Fuel cell sesquicentennial

    Science.gov (United States)

    Cohn, E. M.

    1979-01-01

    The development of fuel cell technology is summarized, and the potential for utility-type fuel cell installations is assessed on the occasion of the 150th anniversary of the construction of the first fuel cell by Sir William Grove. The only functional fuel-cell systems developed to date, the hydrogen-oxygen cells used by NASA, are indicated, and hydrazine and alcohol (methanol) cells are considered. Areas requiring development before the implementation of fuel cells as general purpose utility-type electric generators include catalysts for naturally occurring hydrocarbons or processes for low-cost methanol or hydrazine production, efficient means of scrubbing and enriching air, self-regulating systems, and 15- to 20-fold power density increases. It is argued that although ideas for eliminating certain of the above-mentioned problems have been proposed, fuel-cell systems can never be expected to equal the efficiency, reliability and low cost of conventional power plants, and thus developmental support should be discontinued.

  20. Granular Cell Tumor

    African Journals Online (AJOL)

    ultrastructure and immunochemical staining. 4 strongly suggest Schwann cell derivation . hyperplasia at the edges of the tumor. Necrosis within the tumor was absent, no mitosis was. Granular cell tumors are seldom diagnosed identified in the section and the edges of the accurately clinically. The lesion in this case was.

  1. Cells and Hypotonic Solutions.

    Science.gov (United States)

    Bery, Julia

    1985-01-01

    Describes a demonstration designed to help students better understand the response of plant and animal cells to hypotonic solutions. The demonstration uses a balloon inside a flexible, thin-walled cardboard box. Air going in corresponds to water entering by osmosis, and, like real cells, if stretched enough, the balloon will burst. (DH)

  2. Basal cell cancer (image)

    Science.gov (United States)

    ... biopsy is needed to prove the diagnosis of basal cell carcinoma. Treatment varies depending on the size, depth, and location of the cancer. Early treatment by a dermatologist may result in a cure ... is required to watch for new sites of basal cell cancer.

  3. Cell phone explosion.

    Science.gov (United States)

    Atreya, Alok; Kanchan, Tanuj; Nepal, Samata; Pandey, Bhuwan Raj

    2016-03-01

    Cell phone explosions and resultant burn injuries are rarely reported in the scientific literature. We report a case of cell phone explosion that occurred when a young male was listening to music while the mobile was plugged in for charging. © The Author(s) 2015.

  4. Cancer stem cells revisited

    NARCIS (Netherlands)

    Batlle, Eduard; Clevers, Hans

    2017-01-01

    The cancer stem cell (CSC) concept was proposed four decades ago, and states that tumor growth, analogous to the renewal of healthy tissues, is fueled by small numbers of dedicated stem cells. It has gradually become clear that many tumors harbor CSCs in dedicated niches, and yet their

  5. Programmed cell death

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1995-12-31

    The purpose of this conference to provide a multidisciplinary forum for exchange of state-of-the-art information on the role programmed cell death plays in normal development and homeostasis of many organisms. This volume contains abstracts of papers in the following areas: invertebrate development; immunology/neurology; bcl-2 family; biochemistry; programmed cell death in viruses; oncogenesis; vertebrate development; and diseases.

  6. MICROBIAL FUEL CELL

    DEFF Research Database (Denmark)

    2008-01-01

    A novel microbial fuel cell construction for the generation of electrical energy. The microbial fuel cell comprises: (i) an anode electrode, (ii) a cathode chamber, said cathode chamber comprising an in let through which an influent enters the cathode chamber, an outlet through which an effluent...

  7. Liver Cell Culture Devices

    NARCIS (Netherlands)

    Andria, B.; Bracco, A.; Cirino, G.; Chamuleau, R. A. F. M.

    2010-01-01

    In the last 15 years many different liver cell culture devices, consisting of functional liver cells and artificial materials, have been developed. They have been devised for numerous different applications, such as temporary organ replacement (a bridge to liver transplantation or native liver

  8. Mesangial cell biology

    Energy Technology Data Exchange (ETDEWEB)

    Abboud, Hanna E., E-mail: Abboud@uthscsa.edu

    2012-05-15

    Mesangial cells originate from the metanephric mesenchyme and maintain structural integrity of the glomerular microvascular bed and mesangial matrix homeostasis. In response to metabolic, immunologic or hemodynamic injury, these cells undergo apoptosis or acquire an activated phenotype and undergo hypertrophy, proliferation with excessive production of matrix proteins, growth factors, chemokines and cytokines. These soluble factors exert autocrine and paracrine effects on the cells or on other glomerular cells, respectively. MCs are primary targets of immune-mediated glomerular diseases such as IGA nephropathy or metabolic diseases such as diabetes. MCs may also respond to injury that primarily involves podocytes and endothelial cells or to structural and genetic abnormalities of the glomerular basement membrane. Signal transduction and oxidant stress pathways are activated in MCs and likely represent integrated input from multiple mediators. Such responses are convenient targets for therapeutic intervention. Studies in cultured MCs should be supplemented with in vivo studies as well as examination of freshly isolated cells from normal and diseases glomeruli. In addition to ex vivo morphologic studies in kidney cortex, cells should be studied in their natural environment, isolated glomeruli or even tissue slices. Identification of a specific marker of MCs should help genetic manipulation as well as selective therapeutic targeting of these cells. Identification of biological responses of MCs that are not mediated by the renin–angiotensin system should help development of novel and effective therapeutic strategies to treat diseases characterized by MC pathology.

  9. Sickle Cell Trait

    Science.gov (United States)

    ... Websites About Us Information For… Media Policy Makers Sickle Cell Trait Language: English (US) Español (Spanish) Recommend on Facebook Tweet Share Compartir Get Screened for Sickle Cell Trait Did you know there’s more than one way ...

  10. Electrochemical cell stack assembly

    Science.gov (United States)

    Jacobson, Craig P.; Visco, Steven J.; De Jonghe, Lutgard C.

    2010-06-22

    Multiple stacks of tubular electrochemical cells having a dense electrolyte disposed between an anode and a cathode preferably deposited as thin films arranged in parallel on stamped conductive interconnect sheets or ferrules. The stack allows one or more electrochemical cell to malfunction without disabling the entire stack. Stack efficiency is enhanced through simplified gas manifolding, gas recycling, reduced operating temperature and improved heat distribution.

  11. Molecular weight-dependent genetic information transfer with disulfide-linked polyethylenimine-based nonviral vectors.

    Science.gov (United States)

    Parhiz, Hamideh; Hashemi, Maryam; Hatefi, Arash; Shier, Wayne Thomas; Farzad, Sara Amel; Ramezani, Mohammad

    2013-07-01

    One strategy for improving gene vector properties of polyethylenimine is to facilitate individual transfection mechanism steps. This study investigates (i) improving transfection efficiency by attaching peptide nuclear localization signals (nuclear localization signals: SV40 large T antigen nuclear localization signal or C-terminus of histone H1) to polyethylenimine (10 kDa) and (ii) using disulfide linkages, which are expected to be stable during polyplex formation, but cleaved inside cells giving improved gene release. Nuclear localization signal-containing polyplexes exhibited low cytotoxicity, whereas transfection efficiency with high molecular weight plasmid DNA increased up to 3.6 times that of underivatized polyethylenimine in Neuro2A cells at higher molar ratio of polyethylenimine-nitrogen to DNA-phosphate (N/P) ratios. However, with luciferase-specific low molecular weight small interfering RNA in Neuro2A/EGFPLuc cells, nuclear localization signal-containing polyplexes with disulfide linkages caused substantial cytotoxicity at N/P ratios >15 and no consistent significant reduction in luciferase expression. Possible explanations for molecular weight-dependent differences in genetic information transfer by polyplexes containing disulfide-linked nuclear localization signals are discussed.

  12. Characterization of a New Trioxilin and a Sulfoquinovosyl Diacylglycerol with Anti-Inflammatory Properties from the Dinoflagellate Oxyrrhis marina

    Directory of Open Access Journals (Sweden)

    Eun Young Yoon

    2017-02-01

    Full Text Available Two new compounds—a trioxilin and a sulfoquinovosyl diacylglycerol (SQDG—were isolated from the methanolic extract of the heterotrophic dinoflagellate Oxyrrhis marina cultivated by feeding on dried yeasts. The trioxilin was identified as (4Z,8E,13Z,16Z,19Z -7(S,10(S,11(S-trihydroxydocosapentaenoic acid (1, and the SQDG was identified as (2S-1-O-hexadecanosy-2-O-docosahexaenoyl-3-O-(6-sulfo-α-d-quinovopyranosyl-glycerol (2 by a combination of nuclear magnetic resonance (NMR spectra, mass analyses, and chemical reactions. The two compounds were associated with docosahexaenoic acid, which is a major component of O. marina. The two isolated compounds showed significant nitric oxide inhibitory activity on lipopolysaccharide-induced RAW264.7 cells. Compound 2 showed no cytotoxicity against hepatocarcinoma (HepG2, neuroblastoma (Neuro-2a, and colon cancer (HCT-116 cells, while weak cytotoxicity was observed for compound 1 against Neuro-2a cells.

  13. Biological Activities of Oleanolic Acid Derivatives from Calendula officinalis Seeds.

    Science.gov (United States)

    Zaki, Ahmed; Ashour, Ahmed; Mira, Amira; Kishikawa, Asuka; Nakagawa, Toshinori; Zhu, Qinchang; Shimizu, Kuniyoshi

    2016-05-01

    Phytochemical examination of butanol fraction of Calendula officinalis seeds led to the isolation of two compounds identified as 28-O-β-D-glucopyranosyl-oleanolic acid 3-O-β-D-glucopyranosyl (1→3)-β-D-glucopyranosiduronic acid (CS1) and oleanolic acid 3-O-β-D-glucopyranosyl (1→3)-β-D-glucopyranosiduronic acid (CS2). Biological evaluation was carried out for these two compounds such as melanin biosynthesis inhibitory, hyaluronic acid production activities, anti obesity using lipase inhibition and adipocyte differentiation as well as evaluation of the protective effect against hydrogen peroxide induced neurotoxicity in neuro-2A cells. The results showed that, compound CS2 has a melanin biosynthesis stimulatory activity; however, compound CS1 has a potent stimulatory effect for the production of hyaluronic acid on normal human dermal fibroblast from adult (NHDF-Ad). Both compounds did not show any inhibitory effect on both lipase and adipocyte differentiation. Compound CS2 could protect neuro-2A cells and increased cell viability against H2 O2 . These activities (melanin biosynthesis stimulatory and protective effect against H2 O2 of CS2 and hyaluronic acid productive activities of these triterpene derivatives) have been reported for the first time. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  14. Retinal stem cells and potential cell transplantation treatments

    Directory of Open Access Journals (Sweden)

    Tai-Chi Lin

    2014-11-01

    Full Text Available The retina, histologically composed of ten delicate layers, is responsible for light perception and relaying electrochemical signals to the secondary neurons and visual cortex. Retinal disease is one of the leading clinical causes of severe vision loss, including age-related macular degeneration, Stargardt's disease, and retinitis pigmentosa. As a result of the discovery of various somatic stem cells, advances in exploring the identities of embryonic stem cells, and the development of induced pluripotent stem cells, cell transplantation treatment for retinal diseases is currently attracting much attention. The sources of stem cells for retinal regeneration include endogenous retinal stem cells (e.g., neuronal stem cells, Müller cells, and retinal stem cells from the ciliary marginal zone and exogenous stem cells (e.g., bone mesenchymal stem cells, adipose-derived stem cells, embryonic stem cells, and induced pluripotent stem cells. The success of cell transplantation treatment depends mainly on the cell source, the timing of cell harvesting, the protocol of cell induction/transplantation, and the microenvironment of the recipient's retina. This review summarizes the different sources of stem cells for regeneration treatment in retinal diseases and surveys the more recent achievements in animal studies and clinical trials. Future directions and challenges in stem cell transplantation are also discussed.

  15. Cell of Origin and Cancer Stem Cell Phenotype in Medulloblastomas

    Science.gov (United States)

    2015-07-01

    dominant role over some oncogene function.In addition, we recently reported that cancer stem cells (CSCs)- stem cell like cells in tumors that have stem ... cell properties and tumor initiating ability- retain epigenetic memories of their cells of origin (Chow et al., 2014). We showed that CSCs derived from

  16. Mesenchymal stem cells: cell biology and potential use in therapy

    DEFF Research Database (Denmark)

    Kassem, Moustapha; Kristiansen, Malthe; Abdallah, Basem M

    2004-01-01

    Mesenchymal stem cells are clonogenic, non-haematopoietic stem cells present in the bone marrow and are able to differentiate into multiple mesoderm-type cell lineages e.g. osteoblasts, chondrocytes, endothelial-cells and also non-mesoderm-type lineages e.g. neuronal-like cells. Several methods a...

  17. The cell biology of T-dependent B cell activation

    DEFF Research Database (Denmark)

    Owens, T; Zeine, R

    1989-01-01

    The requirement that CD4+ helper T cells recognize antigen in association with class II Major Histocompatibility Complex (MHC) encoded molecules constrains T cells to activation through intercellular interaction. The cell biology of the interactions between CD4+ T cells and antigen-presenting cells...

  18. Digital Microfluidic Cell Culture.

    Science.gov (United States)

    Ng, Alphonsus H C; Li, Bingyu Betty; Chamberlain, M Dean; Wheeler, Aaron R

    2015-01-01

    Digital microfluidics (DMF) is a droplet-based liquid-handling technology that has recently become popular for cell culture and analysis. In DMF, picoliter- to microliter-sized droplets are manipulated on a planar surface using electric fields, thus enabling software-reconfigurable operations on individual droplets, such as move, merge, split, and dispense from reservoirs. Using this technique, multistep cell-based processes can be carried out using simple and compact instrumentation, making DMF an attractive platform for eventual integration into routine biology workflows. In this review, we summarize the state-of-the-art in DMF cell culture, and describe design considerations, types of DMF cell culture, and cell-based applications of DMF.

  19. Cell Radiation Experiment System

    Science.gov (United States)

    Morrison, Dennis R.

    2010-01-01

    The cell radiation experiment system (CRES) is a perfused-cell culture apparatus, within which cells from humans or other animals can (1) be maintained in homeostasis while (2) being exposed to ionizing radiation during controlled intervals and (3) being monitored to determine the effects of radiation and the repair of radiation damage. The CRES can be used, for example, to determine effects of drug, radiation, and combined drug and radiation treatments on both normal and tumor cells. The CRES can also be used to analyze the effects of radiosensitive or radioprotectant drugs on cells subjected to radiation. The knowledge gained by use of the CRES is expected to contribute to the development of better cancer treatments and of better protection for astronauts, medical-equipment operators, and nuclear-power-plant workers, and others exposed frequently to ionizing radiation.

  20. Physical probing of cells

    Science.gov (United States)

    Rehfeldt, Florian; Schmidt, Christoph F.

    2017-11-01

    In the last two decades, it has become evident that the mechanical properties of the microenvironment of biological cells are as important as traditional biochemical cues for the control of cellular behavior and fate. The field of cell and matrix mechanics is quickly growing and so is the development of the experimental approaches used to study active and passive mechanical properties of cells and their surroundings. Within this topical review we will provide a brief overview, on the one hand, over how cellular mechanics can be probed physically, how different geometries allow access to different cellular properties, and, on the other hand, how forces are generated in cells and transmitted to the extracellular environment. We will describe the following experimental techniques: atomic force microscopy, traction force microscopy, magnetic tweezers, optical stretcher and optical tweezers pointing out both their advantages and limitations. Finally, we give an outlook on the future of the physical probing of cells.

  1. Cell-Size Control.

    Science.gov (United States)

    Amodeo, Amanda A; Skotheim, Jan M

    2016-04-01

    Cells of a given type maintain a characteristic cell size to function efficiently in their ecological or organismal context. They achieve this through the regulation of growth rates or by actively sensing size and coupling this signal to cell division. We focus this review on potential size-sensing mechanisms, including geometric, external cue, and titration mechanisms. Mechanisms that titrate proteins against DNA are of particular interest because they are consistent with the robust correlation of DNA content and cell size. We review the literature, which suggests that titration mechanisms may underlie cell-size sensing in Xenopus embryos, budding yeast, and Escherichia coli, whereas alternative mechanisms may function in fission yeast. Copyright © 2016 Cold Spring Harbor Laboratory Press; all rights reserved.

  2. Single-Cell Metabolomics.

    Science.gov (United States)

    Emara, Samy; Amer, Sara; Ali, Ahmed; Abouleila, Yasmine; Oga, April; Masujima, Tsutomu

    2017-01-01

    The dynamics of a cell is always changing. Cells move, divide, communicate, adapt, and are always reacting to their surroundings non-synchronously. Currently, single-cell metabolomics has become the leading field in understanding the phenotypical variations between them, but sample volumes, low analyte concentrations, and validating gentle sample techniques have proven great barriers toward achieving accurate and complete metabolomics profiling. Certainly, advanced technologies such as nanodevices and microfluidic arrays are making great progress, and analytical techniques, such as matrix-assisted laser desorption ionization (MALDI), are gaining popularity with high-throughput methodology. Nevertheless, live single-cell mass spectrometry (LCSMS) values the sample quality and precision, turning once theoretical speculation into present-day applications in a variety of fields, including those of medicine, pharmaceutical, and agricultural industries. While there is still room for much improvement, it is clear that the metabolomics field is progressing toward analysis and discoveries at the single-cell level.

  3. Nanostructured inorganic solar cells

    Energy Technology Data Exchange (ETDEWEB)

    Musselman, Kevin P. [Cambridge Univ. (United Kingdom). Dept. of Physics; Schmidt-Mende, Lukas [Ludwig-Maximilians Univ. Muenchen (DE). Dept. of Physics and Center for NanoScience (CeNS)

    2011-07-01

    Recent progress in the development of nanostructured inorganic solar cells is reviewed. Nanostructuring of inorganic solar cells offers the possibility of reducing the cost of photovoltaics by allowing smaller amounts of lower-grade photovoltaic semiconductors to be used. Various fabrication methods used to nanostructure traditional photovoltaic semiconductors are detailed and the performance of resulting devices is discussed. The synthesis of solar cells by solution-based methods using less traditional, abundant materials is identified as a promising route to widescale photovoltaic electricity generation, and nanostructured solar cell geometries are highlighted as essential in this approach. Templating and self-assembling methods used to produce appropriate low-cost nanostructures from solutions are detailed, and the performance of preliminary ultra-low-cost cells made with these structures is reviewed. (orig.)

  4. Photovoltaic solar cell

    Science.gov (United States)

    Nielson, Gregory N.; Gupta, Vipin P.; Okandan, Murat; Watts, Michael R.

    2015-09-08

    A photovoltaic solar concentrator is disclosed with one or more transverse-junction solar cells (also termed point contact solar cells) and a lens located above each solar cell to concentrate sunlight onto the solar cell to generate electricity. Piezoelectric actuators tilt or translate each lens to track the sun using a feedback-control circuit which senses the electricity generated by one or more of the solar cells. The piezoelectric actuators can be coupled through a displacement-multiplier linkage to provide an increased range of movement of each lens. Each lens in the solar concentrator can be supported on a frame (also termed a tilt plate) having three legs, with the movement of the legs being controlled by the piezoelectric actuators.

  5. Solar cell radiation handbook

    Science.gov (United States)

    Tada, H. Y.; Carter, J. R., Jr.; Anspaugh, B. E.; Downing, R. G.

    1982-01-01

    The handbook to predict the degradation of solar cell electrical performance in any given space radiation environment is presented. Solar cell theory, cell manufacturing and how they are modeled mathematically are described. The interaction of energetic charged particles radiation with solar cells is discussed and the concept of 1 MeV equivalent electron fluence is introduced. The space radiation environment is described and methods of calculating equivalent fluences for the space environment are developed. A computer program was written to perform the equivalent fluence calculations and a FORTRAN listing of the program is included. Data detailing the degradation of solar cell electrical parameters as a function of 1 MeV electron fluence are presented.

  6. Stem cell factor.

    Science.gov (United States)

    McNiece, I K; Briddell, R A

    1995-07-01

    Stem cell factor (SCF) is the ligand for the tyrosine kinase receptor c-kit, which is expressed on both primitive and mature hematopoietic progenitor cells. In vitro, SCF synergizes with other growth factors, such as granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage-colony-stimulating factor, and interleukin-3 to stimulate the proliferation and differentiation of cells of the lymphoid, myeloid, erythroid, and megakaryocytic lineages. In vivo, SCF also synergizes with other growth factors and has been shown to enhance the mobilization of peripheral blood progenitor cells in combination with G-CSF. In phase I/II clinical studies administration of the combination of SCF and G-CSF resulted in a two- to threefold increase in cells that express the CD34 antigen compared with G-CSF alone. Other potential clinical uses include ex vivo expansion protocols and in vitro culture for gene therapy.

  7. Solar cell. Taiyo denchi

    Energy Technology Data Exchange (ETDEWEB)

    Matsumoto, Hideo; Sato, Katsumi; Hokuyo, Shigeru.

    1989-08-09

    In the conventional soalr cell, adhesives flow out to outside of the interconnector before it cures when the glass covers are fitted, causing the stress relief part going out of function; this results in the damage of the cell, the expansion of the distance between the cells at assembling, which means a trend for larger size of the cell. This is especially a demerit when mounted onto the artificial satellite. This invention aims to prevent the break of the elements and the interconnectors by making the assembled unit smaller. In other words, it contains a solar cell element having electrodes on a the light-receiving surface, a transparent cover adhered onto the electrode and the light receiving surface, and an interconnector at the bottom of this cover; numerous throughholes at the parts from the element-contact part to the externally exposed edge. This prevented the flow out of the adhesive. 3 figs.

  8. Toward sustainable fuel cells

    DEFF Research Database (Denmark)

    Stephens, Ifan; Rossmeisl, Jan; Chorkendorff, Ib

    2016-01-01

    A quarter of humanity's current energy consumption is used for transportation (1). Low-temperature hydrogen fuel cells offer much promise for replacing this colossal use of fossil fuels with renewables; these fuel cells produce negligible emissions and have a mileage and filling time equal...... to a regular gasoline car. However, current fuel cells require 0.25 g of platinum (Pt) per kilowatt of power (2) as catalysts to drive the electrode reactions. If the entire global annual production of Pt were devoted to fuel cell vehicles, fewer than 10 million vehicles could be produced each year, a mere 10......% of the annual automotive vehicle production. Lowering the Pt loading in a fuel cell to a sustainable level requires the reactivity of Pt to be tuned so that it accelerates oxygen reduction more effectively (3). Two reports in this issue address this challenge (4, 5)....

  9. HTPEM Fuel Cell Impedance

    DEFF Research Database (Denmark)

    Vang, Jakob Rabjerg

    As part of the process to create a fossil free Denmark by 2050, there is a need for the development of new energy technologies with higher efficiencies than the current technologies. Fuel cells, that can generate electricity at higher efficiencies than conventional combustion engines, can...... potentially play an important role in the energy system of the future. One of the fuel cell technologies, that receives much attention from the Danish scientific community is high temperature proton exchange membrane (HTPEM) fuel cells based on polybenzimidazole (PBI) with phosphoric acid as proton conductor....... This type of fuel cell operates at higher temperature than comparable fuel cell types and they distinguish themselves by high CO tolerance. Platinum based catalysts have their efficiency reduced by CO and the effect is more pronounced at low temperature. This Ph.D. Thesis investigates this type of fuel...

  10. Cell fusions in mammals

    DEFF Research Database (Denmark)

    Larsson, Lars-Inge; Bjerregaard, Bolette; Talts, Jan Fredrik

    2008-01-01

    Cell fusions are important to fertilization, placentation, development of skeletal muscle and bone, calcium homeostasis and the immune defense system. Additionally, cell fusions participate in tissue repair and may be important to cancer development and progression. A large number of factors appear...... to regulate cell fusions, including receptors and ligands, membrane domain organizing proteins, proteases, signaling molecules and fusogenic proteins forming alpha-helical bundles that bring membranes close together. The syncytin family of proteins represent true fusogens and the founding member, syncytin-1......, has been documented to be involved in fusions between placental trophoblasts, between cancer cells and between cancer cells and host ells. We review the literature with emphasis on the syncytin family and propose that syncytins may represent universal fusogens in primates and rodents, which work...

  11. Cell Control Engineering

    DEFF Research Database (Denmark)

    Lynggaard, Hans Jørgen Birk; Alting, Leo

    1996-01-01

    The engineering process of creating cell control systems is described, and a Cell Control Engineering (CCE) concept is defined. The purpose is to assist people, representing different disciplines in the organisation, to implement cell controllers by addressing the complexity of having many systems...... in physically and logically different and changing manufacturing environments. The defined CCE concept combines state-of-the-art of commercially available enabling technologies for automation system software development, generic cell control models and guidelines for the complete engineering process....... It facilitates the understanding of the task and structure of cell controllers and uses this knowledge directly in the implementation of the system. By applying generic models CCE facilitates reuse of software components and maintenance of applications. In many enterprises, software makes up an increasing part...

  12. Gastric Epithelial Stem Cells

    Science.gov (United States)

    MILLS, JASON C.; SHIVDASANI, RAMESH A.

    2013-01-01

    Advances in our understanding of stem cells in the gastrointestinal tract include the identification of molecular markers of stem and early progenitor cells in the small intestine. Although gastric epithelial stem cells have been localized, little is known about their molecular biology. Recent reports describe the use of inducible Cre recombinase activity to indelibly label candidate stem cells and their progeny in the distal stomach, (ie, the antrum and pylorus). No such lineage labeling of epithelial stem cells has been reported in the gastric body (corpus). Among stem cells in the alimentary canal, those of the adult corpus are unique in that they lie close to the lumen and increase proliferation following loss of a single mature progeny lineage, the acid-secreting parietal cell. They are also unique in that they neither depend on Wnt signaling nor express the surface marker Lgr5. Because pathogenesis of gastric adenocarcinoma has been associated with abnormal patterns of gastric differentiation and with chronic tissue injury, there has been much research on the response of stomach epithelial stem cells to inflammation. Chronic inflammation, as induced by infection with Helicobacter pylori, affects differentiation and promotes metaplasias. Several studies have identified cellular and molecular mechanisms in spasmolytic polypeptide–expressing (pseudopyloric) metaplasia. Researchers have also begun to identify signaling pathways and events that take place during embryonic development that eventually establish the adult stem cells to maintain the specific features and functions of the stomach mucosa. We review the cytologic, molecular, functional, and developmental properties of gastric epithelial stem cells. PMID:21144849

  13. A Quantitative Approach to Evaluate the Impact of Fluorescent Labeling on Membrane-Bound HIV-Gag Assembly by Titration of Unlabeled Proteins.

    Directory of Open Access Journals (Sweden)

    Julia Gunzenhäuser

    Full Text Available The assembly process of the human immunodeficiency virus 1 (HIV-1 is driven by the viral polyprotein Gag. Fluorescence imaging of Gag protein fusions is widely performed and has revealed important information on viral assembly. Gag fusion proteins are commonly co-transfected with an unlabeled form of Gag to prevent labeling artifacts such as morphological defects and decreased infectivity. Although viral assembly is widely studied on individual cells, the efficiency of the co-transfection rescue has never been tested at the single cell level. Here, we first develop a methodology to quantify levels of unlabeled to labeled Gag in single cells using a fluorescent reporter protein for unlabeled Gag and fluorescence correlation spectroscopy. Using super-resolution imaging based on photoactivated localization microscopy (PALM combined with molecular counting we then study the nanoscale morphology of Gag clusters as a function of unlabeled to labeled Gag ratios in single cells. We show that for a given co-transfection ratio, individual cells express a wide range of protein ratios, necessitating a quantitative read-out for the expression of unlabeled Gag. Further, we show that monomerically labeled Gag assembles into membrane-bound clusters that are morphologically indistinguishable from mixtures of unlabeled and labeled Gag.

  14. CCL22-specific T Cells

    DEFF Research Database (Denmark)

    Martinenaite, Evelina; Munir Ahmad, Shamaila; Hansen, Morten

    2016-01-01

    Tumor cells and tumor-infiltrating macrophages produce the chemokine CCL22, which attracts regulatory T cells (Tregs) into the tumor microenvironment, decreasing anticancer immunity. Here, we investigated the possibility of targeting CCL22-expressing cells by activating specific T cells. We...... analyzed the CCL22 protein signal sequence, identifying a human leukocyte antigen A2- (HLA-A2-) restricted peptide epitope, which we then used to stimulate peripheral blood mononuclear cells (PMBCs) to expand populations of CCL22-specific T cells in vitro. T cells recognizing an epitope derived from...... the signal-peptide of CCL22 will recognize CCL22-expressing cells even though CCL22 is secreted out of the cell. CCL22-specific T cells recognized and killed CCL22-expressing cancer cells. Furthermore, CCL22-specific T cells lysed acute monocytic leukemia cells in a CCL22 expression-dependent manner. Using...

  15. Hilar mossy cell circuitry controlling dentate granule cell excitability

    Directory of Open Access Journals (Sweden)

    Seiichiro eJinde

    2013-02-01

    Full Text Available Glutamatergic hilar mossy cells of the dentate gyrus can either excite or inhibit distant granule cells, depending on whether their direct excitatory projections to granule cells or their projections to local inhibitory interneurons dominate. However, it remains controversial whether the net effect of mossy cell loss is granule cell excitation or inhibition. Clarifying this controversy has particular relevance to temporal lobe epilepsy, which is marked by dentate granule cell hyperexcitability and extensive loss of dentate hilar mossy cells. Two diametrically opposed hypotheses have been advanced to explain this granule cell hyperexcitability – the dormant basket cell and the irritable mossy cell hypotheses. The dormant basket cell hypothesis proposes that mossy cells normally exert a net inhibitory effect on granule cells and therefore their loss causes dentate granule cell hyperexcitability. The irritable mossy cell hypothesis takes the opposite view that mossy cells normally excite granule cells and that the surviving mossy cells in epilepsy increase their activity, causing granule cell excitation. The inability to eliminate mossy cells selectively has made it difficult to test these two opposing hypotheses. To this end, we developed a transgenic toxin-mediated, mossy cell-ablation mouse line. Using these mutants, we demonstrated that the extensive elimination of hilar mossy cells causes granule cell hyperexcitability, although the mossy cell loss observed appeared insufficient to cause clinical epilepsy. In this review, we focus on this topic and also suggest that different interneuron populations may mediate mossy cell-induced translamellar lateral inhibition and intralamellar recurrent inhibition. These unique local circuits in the dentate hilar region may be centrally involved in the functional organization of the dentate gyrus.

  16. Are cranial germ cell tumours really tumours of germ cells?

    Science.gov (United States)

    Scotting, P J

    2006-12-01

    Germ cell tumours of the brain and those that occur in the gonads are believed to share a common origin from germ cell progenitors. This 'germ cell theory' rests upon similar histopathology between these tumours in different locations and the belief that endogenous somatic cells of the brain could not give rise to the range of cell types seen in germ cell tumours. An alternative 'embryonic cell theory' has been proposed for some classes of cranial germ cell tumours, but this still relies on the misplacement of cells in the brain (in this case the earliest embryonic stem cells) during early embryonic development. Recent evidence has demonstrated that neural stem cells of the brain can also give rise to many of the cell types seen in germ cell tumours. These data suggest that endogenous progenitor cells of the brain are a plausible alternative origin for these tumours. This idea is of central importance for studies aiming to elucidate the mechanisms of tumour development. The application of modern molecular analyses to reveal how tumour cells have altered with respect to their cell of origin relies on the certain identification of the cell from which the particular tumour arose. If the identity of this cell is mistaken, then studies to elucidate the mechanisms by which the progenitor cell has been subverted from its normal behaviour will not yield useful information. In addition, it will prove impossible to generate an appropriate animal model in which to study the underlying causes of those tumours. This article makes the case that current assumptions of the origins of cranial germ cell tumours are unreliable. It reviews the evidence in favour of the 'germ cell theory' and argues in favour of a 'brain cell theory' in which endogenous neural progenitor cells of the brain are the likely origin for these tumours. Thus, the case is made that cranial germ cell tumours, like other brain tumours, arise by the transformation of progenitor cells normally resident in the

  17. Oscillating Cell Culture Bioreactor

    Science.gov (United States)

    Freed, Lisa E.; Cheng, Mingyu; Moretti, Matteo G.

    2010-01-01

    To better exploit the principles of gas transport and mass transport during the processes of cell seeding of 3D scaffolds and in vitro culture of 3D tissue engineered constructs, the oscillatory cell culture bioreactor provides a flow of cell suspensions and culture media directly through a porous 3D scaffold (during cell seeding) and a 3D construct (during subsequent cultivation) within a highly gas-permeable closed-loop tube. This design is simple, modular, and flexible, and its component parts are easy to assemble and operate, and are inexpensive. Chamber volume can be very low, but can be easily scaled up. This innovation is well suited to work with different biological specimens, particularly with cells having high oxygen requirements and/or shear sensitivity, and different scaffold structures and dimensions. The closed-loop changer is highly gas permeable to allow efficient gas exchange during the cell seeding/culturing process. A porous scaffold, which may be seeded with cells, is fixed by means of a scaffold holder to the chamber wall with scaffold/construct orientation with respect to the chamber determined by the geometry of the scaffold holder. A fluid, with/without biological specimens, is added to the chamber such that all, or most, of the air is displaced (i.e., with or without an enclosed air bubble). Motion is applied to the chamber within a controlled environment (e.g., oscillatory motion within a humidified 37 C incubator). Movement of the chamber induces relative motion of the scaffold/construct with respect to the fluid. In case the fluid is a cell suspension, cells will come into contact with the scaffold and eventually adhere to it. Alternatively, cells can be seeded on scaffolds by gel entrapment prior to bioreactor cultivation. Subsequently, the oscillatory cell culture bioreactor will provide efficient gas exchange (i.e., of oxygen and carbon dioxide, as required for viability of metabolically active cells) and controlled levels of fluid

  18. Quantitative imaging of epithelial cell scattering identifies specific inhibitors of cell motility and cell-cell dissociation

    NARCIS (Netherlands)

    Loerke, D.; le Duc, Q.; Blonk, I.; Kerstens, A.; Spanjaard, E.; Machacek, M.; Danuser, G.; de Rooij, J.

    2012-01-01

    The scattering of cultured epithelial cells in response to hepatocyte growth factor (HGF) is a model system that recapitulates key features of metastatic cell behavior in vitro, including disruption of cell-cell adhesions and induction of cell migration. We have developed image analysis tools that

  19. Single-cell model of prokaryotic cell cycle.

    Science.gov (United States)

    Abner, Kristo; Aaviksaar, Tõnis; Adamberg, Kaarel; Vilu, Raivo

    2014-01-21

    One of the recognized prokaryotic cell cycle theories is Cooper-Helmstetter (CH) theory which relates start of DNA replication to particular (initiation) cell mass, cell growth and division. Different aspects of this theory have been extensively studied in the past. In the present study CH theory was applied at single cell level. Universal equations were derived for different cell parameters (cell mass and volume, surface area, DNA amount and content) depending on constructivist cell cycle parameters (unit mass, replication and division times, cell age, cell cycle duration) based on selected growth laws of cell mass (linear, exponential). The equations derived can be integrated into single-cell models for the analysis and design of bacterial cells. © 2013 Published by Elsevier Ltd.

  20. Regulatory T cells induced by B cells: a novel subpopulation of regulatory T cells.

    Science.gov (United States)

    Chien, Chien-Hui; Chiang, Bor-Luen

    2017-11-18

    Regulatory T cells play a crucial role in the homeostasis of the immune response. In addition to CD4+Foxp3+ regulatory T cells, several subsets of Foxp3- regulatory T cells, such as T helper 3 (Th3) cells and type 1 regulatory T (Tr1) cells, have been described in mice and human. Accumulating evidence shows that naïve B cells contribute to tolerance and are able to promote regulatory T cell differentiation. Naïve B cells can convert CD4+CD25- T cells into CD25+Foxp3- regulatory T cells, named Treg-of-B cells by our group. Treg-of-B cells express LAG3, ICOS, GITR, OX40, PD1, and CTLA4 and secrete IL-10. Intriguingly, B-T cell-cell contact but not IL-10 is essential for Treg-of-B cells induction. Moreover, Treg-of-B cells possess both IL-10-dependent and IL-10-independent inhibitory functions. Treg-of-B cells exert suppressive activities in antigen-specific and non-antigen-specific manners in vitro and in vivo. Here, we review the phenotype and function of Foxp3+ regulatory T cells, Th3 cells, Tr1 cells, and Treg-of-B cells.