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Sample records for astrocyte cell line

  1. Construction and identification of immortalized rat astrocyte cell line expressing enkephalin

    Institute of Scientific and Technical Information of China (English)

    XU Ying; TIAN Yu-ke; TIAN Xue-bi; AN Ke; YANG Hui

    2007-01-01

    Objective: To provide a sound cell source for further ex-vivo gene therapy for chronic pain, we attempt to develop an immortalized rat astrocyte cell line that expresses enkephalin regulated by doxycycline.Methods: Retrovirus infection method was employed to develop an immortalized rat astrocyte cell line that could express enkephalin regulated by doxycycline. The hPPE gene expression level of immoralized astroyte cells (IAC)/hPPE was detected by RT-PCR, indirect immunofiuorescence staining and radioimmunoassay.Results: IAC carrying Tet-on system transfected with preproenkephalin gene could secrete enkephalin that was regulated by doxycycline in a dose-dependent manner and hPPE gene activation could be repeated in on-off-on cycles through administration or removal of doxycycline.Conclusion: An immortalized rat astrocyte cell line that secrete enkephalin under the control of doxycycline is established successfully, which provides a research basis for transgenic cell transplantation for analgesia.

  2. Lauric Acid Stimulates Ketone Body Production in the KT-5 Astrocyte Cell Line.

    Science.gov (United States)

    Nonaka, Yudai; Takagi, Tetsuo; Inai, Makoto; Nishimura, Shuhei; Urashima, Shogo; Honda, Kazumitsu; Aoyama, Toshiaki; Terada, Shin

    2016-08-01

    Coconut oil has recently attracted considerable attention as a potential Alzheimer's disease therapy because it contains large amounts of medium-chain fatty acids (MCFAs) and its consumption is thought to stimulate hepatic ketogenesis, supplying an alternative energy source for brains with impaired glucose metabolism. In this study, we first reevaluated the responses of plasma ketone bodies to oral administration of coconut oil to rats. We found that the coconut oil-induced increase in plasma ketone body concentration was negligible and did not significantly differ from that observed after high-oleic sunflower oil administration. In contrast, the administration of coconut oil substantially increased the plasma free fatty acid concentration and lauric acid content, which is the major MCFA in coconut oil. Next, to elucidate whether lauric acid can activate ketogenesis in astrocytes with the capacity to generate ketone bodies from fatty acids, we treated the KT-5 astrocyte cell line with 50 and 100 μM lauric acid for 4 h. The lauric acid treatments increased the total ketone body concentration in the cell culture supernatant to a greater extent than oleic acid, suggesting that lauric acid can directly and potently activate ketogenesis in KT-5 astrocytes. These results suggest that coconut oil intake may improve brain health by directly activating ketogenesis in astrocytes and thereby by providing fuel to neighboring neurons. PMID:27430387

  3. Lauric Acid Stimulates Ketone Body Production in the KT-5 Astrocyte Cell Line.

    Science.gov (United States)

    Nonaka, Yudai; Takagi, Tetsuo; Inai, Makoto; Nishimura, Shuhei; Urashima, Shogo; Honda, Kazumitsu; Aoyama, Toshiaki; Terada, Shin

    2016-08-01

    Coconut oil has recently attracted considerable attention as a potential Alzheimer's disease therapy because it contains large amounts of medium-chain fatty acids (MCFAs) and its consumption is thought to stimulate hepatic ketogenesis, supplying an alternative energy source for brains with impaired glucose metabolism. In this study, we first reevaluated the responses of plasma ketone bodies to oral administration of coconut oil to rats. We found that the coconut oil-induced increase in plasma ketone body concentration was negligible and did not significantly differ from that observed after high-oleic sunflower oil administration. In contrast, the administration of coconut oil substantially increased the plasma free fatty acid concentration and lauric acid content, which is the major MCFA in coconut oil. Next, to elucidate whether lauric acid can activate ketogenesis in astrocytes with the capacity to generate ketone bodies from fatty acids, we treated the KT-5 astrocyte cell line with 50 and 100 μM lauric acid for 4 h. The lauric acid treatments increased the total ketone body concentration in the cell culture supernatant to a greater extent than oleic acid, suggesting that lauric acid can directly and potently activate ketogenesis in KT-5 astrocytes. These results suggest that coconut oil intake may improve brain health by directly activating ketogenesis in astrocytes and thereby by providing fuel to neighboring neurons.

  4. Influence of X-rays on early response gene expression in rat astrocytes and brain tumour cell lines

    International Nuclear Information System (INIS)

    The effects of ionizing radiation on c-fos, c-jun and jun-B mRNA levels were determined in cultures of rat perinatal type 1 astrocytes and two rat brain tumour cell lines, 175A and 9L. In astrocyte cultures X-ray doses as low as 1 Gy induced the expression of c-fos and jun-B but had essentially no effect on c-jun. The maximum increase in expression was found 1 h after irradiation, which then rapidly returned to control levels. These findings suggest that astrocytes may play a role in mediating the radiation response of the central nervous system via X-ray-induced changes in gene expression. In contrast, doses of up to 20 Gy had no effect on c-fos, c-jun and jun-B mRNA levels in the two brain tumour cell lines. In addition, whereas 12-0-tetradecanoylphorbol-13-acetate induced the expression of these genes in astrocytes, it had little or no effect on fos or jun expression in 9L or 175A cells. These results suggest that the signal transduction pathways mediating radiation-induced genes expression may be different in normal astrocytes and brain tumour cells. (author)

  5. Influence of drugs on gap junctions in glioma cell lines and primary astrocytes in vitro

    OpenAIRE

    Zahra eMoinfar; Hannes eDambach; Pedro Michael Faustmann

    2014-01-01

    Gap junctions (GJs) are hemichannels on cell membrane. Once they are intercellulary connected to the neighboring cells, they build a functional syncytium which allows rapid transfer of ions and molecules between cells. This characteristic makes GJs a potential modulator in proliferation, migration, and development of the cells. So far, several types of GJs are recognized on different brain cells as well as in glioma. Astrocytes, as one of the major cells that maintain neuronal homeostasis, ex...

  6. In vitro neuroprotective action of recombinant rat erythropoietin produced by astrocyte cell lines and comparative studies with erythropoietin produced by Chinese hamster ovary cells

    OpenAIRE

    Masuda, Seiji; Kada, Emi; Nagao, Masaya; Sasaki, Ryuzo

    1999-01-01

    In the central nervous system, astrocytes produce erythropoietin (Epo) and neurons express its receptor. To examine whether or not the brain Epo protects the in vitro cultured neurons from glutamate-induced cell death, we established rat astrocyte cell lines containing the plasmid for production of recombinant rat Epo. Epo partially purified from the culture medium showed a neuroprotective effect similar to that of rat Epo produced by Chinese hamster ovary (CHO) cells. Comparison was made in ...

  7. Influence of drugs on gap junctions in glioma cell lines and primary astrocytes in vitro

    Directory of Open Access Journals (Sweden)

    Zahra eMoinfar

    2014-05-01

    Full Text Available Gap junctions (GJs are hemichannels on cell membrane. Once they are intercellulary connected to the neighboring cells, they build a functional syncytium which allows rapid transfer of ions and molecules between cells. This characteristic makes GJs a potential modulator in proliferation, migration and development of the cells. So far, several types of GJs are recognized on different brain cells as well as in glioma. Astrocytes, as one of the major cells that maintain neuronal homeostasis, express different types of GJs that let them communicate with neurons, oligodendrocytes and endothelial cells of the blood brain barrier; however, the main GJ in astrocytes is connexin 43. There are different cerebral diseases in which astrocyte GJs might play a role. Several drugs have been reported to modulate gap junctional communication in the brain which can consequently have beneficial or detrimental effects on the course of treatment in certain diseases. However, the exact cellular mechanism behind those pharmaceutical efficacies on GJs is not well-understood. Accordingly, how specific drugs would affect GJs and what some consequent specific brain diseases would be are the interests of the authors of this chapter. We would focus on pharmaceutical effects on GJs on astrocytes in specific diseases where GJs could possibly play a role including: 1 migraine and a novel therapy for migraine with aura, 2 neuroautoimmune diseases and immunomodulatory drugs in the treatment of demyelinating diseases of the central nervous system such as multiple sclerosis, 3 glioma and antineoplastic and anti-inflammatory agents that are used in treating brain tumors and 4 epilepsy and anticonvulsants that are widely used for seizures therapy. All of the above-mentioned therapeutic categories can possibly affect GJs expression of astrocytes and the role is discussed in the upcoming chapter.

  8. astrocyte and astrocytoma cells

    DEFF Research Database (Denmark)

    Tfelt-Hansen, J.

    2008-01-01

    -transforming gene (PTTG), was found to be upregulated by the CaR in the H-500 cells, whereas calcium had no effect on PTTG expression in the U-87 astrocytoma cell line, but other proproliferative agents did upregulate PTTG in the U-87 cells. This makes PTTG a potential marker of malignancy and a therapeutic target...

  9. Impact of flattening-filter-free radiation on the clonogenic survival of astrocytic cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Steenken, Caroline; Fleckenstein, Jens; Kegel, Stefan; Jahnke, Lennart; Simeonova, Anna; Hartmann, Linda; Kuebler, Jens; Veldwijk, Marlon R.; Wenz, Frederik; Herskind, Carsten; Giordano, Frank Anton [Universitaetsmedizin Mannheim (UMM), Medical Faculty Mannheim, Heidelberg University, Department of Radiation Oncology, Mannheim (Germany)

    2015-07-15

    Flattening-filter-free (FFF) beams are increasingly used in radiotherapy as delivery times can be substantially reduced. However, the relative biologic effectiveness (RBE) of FFF may be increased relative to conventional flattened (FLAT) beams due to differences in energy spectra. Therefore, we investigated the effects of FFF and FLAT beams on the clonogenic survival of astrocytoma cells. Three cell lines (U251, U251-MGMT, and U87) were irradiated with 6-MV and 10-MV X-rays from a linear accelerator in FFF- or FLAT-beam modes at dose rates in the range of 0.5-24 Gy/min. The surviving fraction (SF) as function of dose (2-12 Gy) was determined by the colony formation assay and fitted by the linear-quadratic model. For both beams (FFF or FLAT), the cells were pelleted in conical 15-ml centrifuge tubes and irradiated at 2-cm depth in a 1 x 1-cm{sup 2} area on the central axis of a 30 x 30-cm{sup 2} field. Dosimetry was performed with a 0.3-cm{sup 3} rigid ionization chamber. RBE was determined for FFF versus FLAT irradiation. The RBE of FFF at 7.3-11.3 Gy was 1.027 ± 0.013 and 1.063 ± 0.018 relative to FLAT beams for 6- and 10-MV beams, respectively, and was only significantly higher than 1 for 10 MV. Significantly increased survival rates were seen for lower dose rates (0.5 Gy/min FLAT vs. 5 Gy/min FLAT) at higher doses (11.9 Gy), while no differences were seen at dose rates ≥ 1.4 Gy/min (1.4 Gy/min FFF vs. 14 Gy/min FFF and 2.4 Gy/min FFF vs. 24 Gy/min FFF). FFF beams showed only a slightly increased RBE relative to FLAT beams in this experimental set-up, which is unlikely to result in clinically relevant differences in outcome. (orig.) [German] Die Flattening-Filter-freie (FFF) Bestrahlungstechnik findet zunehmend Verwendung, da sich die Applikationsdauer der einzelnen Fraktionen deutlich verkuerzen laesst. Aufgrund der Unterschiede im Spektrum koennte die relative biologische Wirksamkeit (RBW) von FFF jedoch hoeher sein als bei konventioneller Technik (d.h. bei

  10. Astrocytes.

    Science.gov (United States)

    Kimelberg, Harold K.; Norenberg, Michael D.

    1989-01-01

    Describes the astrocytes' function as equal partners with neurons in both the normal and the abnormal brain. Discusses the developmental scaffolds, inert scar tissue, Huntington's disease, psychiatric disorders, and the identification of these brain cells. (RT)

  11. PrP{sup C} displays an essential protective role from oxidative stress in an astrocyte cell line derived from PrP{sup C} knockout mice

    Energy Technology Data Exchange (ETDEWEB)

    Bertuchi, Fernanda R. [Center for Natural Sciences and Humanities, Federal University of ABC - UFABC, Avenida dos Estados, 5001, Bloco B, 09210-170, Santo Andre, SP (Brazil); Bourgeon, Dominique M.G.; Landemberger, Michele C.; Martins, Vilma R. [International Center for Research and Education, A.C. Camargo Hospital, Rua Tagua 440, 01505-010 Sao Paulo, SP (Brazil); Cerchiaro, Giselle, E-mail: giselle.cerchiaro@ufabc.edu.br [Center for Natural Sciences and Humanities, Federal University of ABC - UFABC, Avenida dos Estados, 5001, Bloco B, 09210-170, Santo Andre, SP (Brazil)

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer PrP{sup C} in solution acts as a radical scavenger. Black-Right-Pointing-Pointer PrP{sup C} reduces hydrogen peroxide toxicity in astrocytes. Black-Right-Pointing-Pointer Increase in ROS disrupted the cell cycle in the PrP{sup C}-knockout astrocytes. Black-Right-Pointing-Pointer PrP{sup C} prevents the cell death independently of an SOD-like activity. -- Abstract: The PrP{sup C} protein, which is especially present in the cellular membrane of nervous system cells, has been extensively studied for its controversial antioxidant activity. In this study, we elucidated the free radical scavenger activity of purified murine PrP{sup C} in solution and its participation as a cell protector in astrocytes that were subjected to treatment with an oxidant. In vitro and using an EPR spin-trapping technique, we observed that PrP{sup C} decreased the oxidation of the DMPO trap in a Fenton reaction system (Cu{sup 2+}/ascorbate/H{sub 2}O{sub 2}), which was demonstrated by approximately 70% less DMPO/OH{sup {center_dot}}. In cultured PrP{sup C}-knockout astrocytes from mice, the absence of PrP{sup C} caused an increase in intracellular ROS (reactive oxygen species) generation during the first 3 h of H{sub 2}O{sub 2} treatment. This rapid increase in ROS disrupted the cell cycle in the PrP{sup C}-knockout astrocytes, which increased the population of cells in the sub-G1 phase when compared with cultured wild-type astrocytes. We conclude that PrP{sup C} in solution acts as a radical scavenger, and in astrocytes, it is essential for protection from oxidative stress caused by an external chemical agent, which is a likely condition in human neurodegenerative CNS disorders and pathological conditions such as ischemia.

  12. PrPC displays an essential protective role from oxidative stress in an astrocyte cell line derived from PrPC knockout mice

    International Nuclear Information System (INIS)

    Highlights: ► PrPC in solution acts as a radical scavenger. ► PrPC reduces hydrogen peroxide toxicity in astrocytes. ► Increase in ROS disrupted the cell cycle in the PrPC-knockout astrocytes. ► PrPC prevents the cell death independently of an SOD-like activity. -- Abstract: The PrPC protein, which is especially present in the cellular membrane of nervous system cells, has been extensively studied for its controversial antioxidant activity. In this study, we elucidated the free radical scavenger activity of purified murine PrPC in solution and its participation as a cell protector in astrocytes that were subjected to treatment with an oxidant. In vitro and using an EPR spin-trapping technique, we observed that PrPC decreased the oxidation of the DMPO trap in a Fenton reaction system (Cu2+/ascorbate/H2O2), which was demonstrated by approximately 70% less DMPO/OH·. In cultured PrPC-knockout astrocytes from mice, the absence of PrPC caused an increase in intracellular ROS (reactive oxygen species) generation during the first 3 h of H2O2 treatment. This rapid increase in ROS disrupted the cell cycle in the PrPC-knockout astrocytes, which increased the population of cells in the sub-G1 phase when compared with cultured wild-type astrocytes. We conclude that PrPC in solution acts as a radical scavenger, and in astrocytes, it is essential for protection from oxidative stress caused by an external chemical agent, which is a likely condition in human neurodegenerative CNS disorders and pathological conditions such as ischemia.

  13. Peptide gH625 enters into neuron and astrocyte cell lines and crosses the blood–brain barrier in rats

    Directory of Open Access Journals (Sweden)

    Valiante S

    2015-03-01

    Full Text Available Salvatore Valiante,1,* Annarita Falanga,2,3,* Luisa Cigliano,1 Giuseppina Iachetta,1 Rosa Anna Busiello,1 Valeria La Marca,1 Massimiliano Galdiero,4 Assunta Lombardi,1 Stefania Galdiero1,2 1Department of Biology, 2Department of Pharmacy, 3DFM Scarl, University of Naples Federico II, 4Department of Experimental Medicine, II University of Naples, Naples, Italy *These authors contributed equally to this paper and are considered joint first authors Abstract: Peptide gH625, derived from glycoprotein H of herpes simplex virus type 1, can enter cells efficiently and deliver a cargo. Nanoparticles armed with gH625 are able to cross an in vitro model of the blood–brain barrier (BBB. In the present study, in vitro experiments were performed to investigate whether gH625 can enter and accumulate in neuron and astrocyte cell lines. The ability of gH625 to cross the BBB in vivo was also evaluated. gH625 was administered in vivo to rats and its presence in the liver and in the brain was detected. Within 3.5 hours of intravenous administration, gH625 can be found beyond the BBB in proximity to cell neurites. gH625 has no toxic effects in vivo, since it does not affect the maximal oxidative capacity of the brain or the mitochondrial respiration rate. Our data suggest that gH625, with its ability to cross the BBB, represents a novel nanocarrier system for drug delivery to the central nervous system. These results open up new possibilities for direct delivery of drugs into patients in the field of theranostics and might address the treatment of several human diseases. Keywords: drug delivery, neurons, astrocytes, blood–brain barrier, peptide

  14. Specialized contacts of astrocytes with astrocytes and with other cell types in the hypothalamus of the hamster.

    OpenAIRE

    Suarez Najera, I; Fernandez Ruiz, B; Garcia Segura, L M

    1980-01-01

    Adult hamsters were used for this electron microscopic study of the hypothalamic region. Specialized contacts between astrocytes and astrocytes, and between astrocytes and other cellular elements, are described and illustrated. The specialized inter-astrocytic junctions occur primarily in perivascular and subpial regions, but also in areas of high synaptic density. The junctions between astrocytic processes are of hemidesmosomal type. Astrocytes are connected to oligodendroglial cells by mean...

  15. Target cell-specific modulation of neuronal activity by astrocytes

    OpenAIRE

    Kozlov, A. S.; Angulo, M. C.; Audinat, E.; Charpak, S

    2006-01-01

    Interaction between astrocytes and neurons enriches the behavior of brain circuits. By releasing glutamate and ATP, astrocytes can directly excite neurons and modulate synaptic transmission. In the rat olfactory bulb, we demonstrate that the release of GABA by astrocytes causes long-lasting and synchronous inhibition of mitral and granule cells. In addition, astrocytes release glutamate, leading to a selective activation of granule-cell NMDA receptors. Thus, by releasing excitatory and inhibi...

  16. Astrocytes derived from trisomic human embryonic stem cells express markers of astrocytic cancer cells and premalignant stem-like progenitors

    Directory of Open Access Journals (Sweden)

    Iverson Linda E

    2010-04-01

    Full Text Available Abstract Background Trisomic variants of human embryonic stem cells (hESCs arise spontaneously in culture. Although trisomic hESCs share many properties with diploid hESCs, they also exhibit features of cancer stem cells. Since most hESC-based therapies will utilize differentiated derivatives, it is imperative to investigate the potential of trisomic hESCs to undergo malignant transformation during differentiation prior to their use in the clinical setting. Methods Diploid and trisomic hESCs were differentiated into astrocytic progenitors cells (APCs, RNA extracted and hybridized to human exon-specific microarrays. Global gene expression profiles of diploid and trisomic APCs were compared to that of an astrocytoma cell line and glioblastoma samples, analyzed by others, using the same microarray platform. Results Bioinformatic analysis of microarray data indicates that differentiated trisomic APCs exhibit global expression profiles with similarities to the malignant astrocytoma cell line. An analogous trend is observed in comparison to glioblastoma samples indicating that trisomic APCs express markers of astrocytic cancer cells. The analysis also allowed identification of transcripts predicted to be differentially expressed in brain tumor stem cells. These data indicate that in vitro differentiation of trisomic hESCs along astrocytic pathways give rise to cells exhibiting properties of premalignant astrocytic stem/progenitor cells. Conclusions Given their occult nature, opportunities to study premalignant stem/progenitor cells in human have been few. The ability to propagate and direct the differentiation of aneuploid hESCs provides a powerful in vitro system for investigating biological properties of human cells exhibiting features of premalignant stem cells. This in vitro culture system can be used to elucidate changes in gene expression occurring enroute to malignant transformation and to identify molecular markers of cancer stem

  17. From stem cell to astrocyte: Decoding the regulation of GFAP

    NARCIS (Netherlands)

    R. Kanski

    2014-01-01

    The research presented in this thesis focuses on glial fibrillary acidic protein (GFAP), the main intermediate filament (IF) in astrocytes and astrocyte subpopulations such as neural stem cells (NSCs). In neurodegenerative diseases or upon brain damage, astrocytes respond to an injury with an upregu

  18. Functional and phenotypic differences of pure populations of stem cell-derived astrocytes and neuronal precursor cells.

    Science.gov (United States)

    Kleiderman, Susanne; Sá, João V; Teixeira, Ana P; Brito, Catarina; Gutbier, Simon; Evje, Lars G; Hadera, Mussie G; Glaab, Enrico; Henry, Margit; Sachinidis, Agapios; Alves, Paula M; Sonnewald, Ursula; Leist, Marcel

    2016-05-01

    Availability of homogeneous astrocyte populations would facilitate research concerning cell plasticity (metabolic and transcriptional adaptations; innate immune responses) and cell cycle reactivation. Current protocols to prepare astrocyte cultures differ in their final content of immature precursor cells, preactivated cells or entirely different cell types. A new method taking care of all these issues would improve research on astrocyte functions. We found here that the exposure of a defined population of pluripotent stem cell-derived neural stem cells (NSC) to BMP4 results in pure, nonproliferating astrocyte cultures within 24-48 h. These murine astrocytes generated from embryonic stem cells (mAGES) expressed the positive markers GFAP, aquaporin 4 and GLT-1, supported neuronal function, and acquired innate immune functions such as the response to tumor necrosis factor and interleukin 1. The protocol was applicable to several normal or disease-prone pluripotent cell lines, and the corresponding mAGES all exited the cell cycle and lost most of their nestin expression, in contrast to astrocytes generated by serum-addition or obtained as primary cultures. Comparative gene expression analysis of mAGES and NSC allowed quantification of differences between the two cell types and a definition of an improved marker set to define astrocytes. Inclusion of several published data sets in this transcriptome comparison revealed the similarity of mAGES with cortical astrocytes in vivo. Metabolic analysis of homogeneous NSC and astrocyte populations revealed distinct neurochemical features: both cell types synthesized glutamine and citrate, but only mature astrocytes released these metabolites. Thus, the homogeneous cultures allowed an improved definition of NSC and astrocyte features. PMID:26689134

  19. Astrocytes Enhance Streptococcus suis-Glial Cell Interaction in Primary Astrocyte-Microglial Cell Co-Cultures.

    Science.gov (United States)

    Seele, Jana; Nau, Roland; Prajeeth, Chittappen K; Stangel, Martin; Valentin-Weigand, Peter; Seitz, Maren

    2016-06-13

    Streptococcus (S.) suis infections are the most common cause of meningitis in pigs. Moreover, S. suis is a zoonotic pathogen, which can lead to meningitis in humans, mainly in adults. We assume that glial cells may play a crucial role in host-pathogen interactions during S. suis infection of the central nervous system. Glial cells are considered to possess important functions during inflammation and injury of the brain in bacterial meningitis. In the present study, we established primary astrocyte-microglial cell co-cultures to investigate interactions of S. suis with glial cells. For this purpose, microglial cells and astrocytes were isolated from new-born mouse brains and characterized by flow cytometry, followed by the establishment of astrocyte and microglial cell mono-cultures as well as astrocyte-microglial cell co-cultures. In addition, we prepared microglial cell mono-cultures co-incubated with uninfected astrocyte mono-culture supernatants and astrocyte mono-cultures co-incubated with uninfected microglial cell mono-culture supernatants. After infection of the different cell cultures with S. suis, bacteria-cell association was mainly observed with microglial cells and most prominently with a non-encapsulated mutant of S. suis. A time-dependent induction of NO release was found only in the co-cultures and after co-incubation of microglial cells with uninfected supernatants of astrocyte mono-cultures mainly after infection with the capsular mutant. Only moderate cytotoxic effects were found in co-cultured glial cells after infection with S. suis. Taken together, astrocytes and astrocyte supernatants increased interaction of microglial cells with S. suis. Astrocyte-microglial cell co-cultures are suitable to study S. suis infections and bacteria-cell association as well as NO release by microglial cells was enhanced in the presence of astrocytes.

  20. Astrocytes Directly Influence Tumor Cell Invasion and Metastasis In Vivo

    OpenAIRE

    Wang, Ling; Cossette, Stephanie M.; Rarick, Kevin R.; Gershan, Jill; Michael B Dwinell; Harder, David R.; Ramchandran, Ramani

    2013-01-01

    Brain metastasis is a defining component of tumor pathophysiology, and the underlying mechanisms responsible for this phenomenon are not well understood. Current dogma is that tumor cells stimulate and activate astrocytes, and this mutual relationship is critical for tumor cell sustenance in the brain. Here, we provide evidence that primary rat neonatal and adult astrocytes secrete factors that proactively induced human lung and breast tumor cell invasion and metastasis capabilities. Among wh...

  1. Characterization of the BAC Id3-enhanced green fluorescent protein transgenic mouse line for in vivo imaging of astrocytes

    OpenAIRE

    Lamantia, Cassandra; Tremblay, Marie-Eve; Majewska, Ania

    2014-01-01

    Astrocytes are highly ramified glial cells with critical roles in brain physiology and pathology. Recently, breakthroughs in imaging technology have expanded our understanding of astrocyte function in vivo. The in vivo study of astrocytic dynamics, however, is limited by the tools available to label astrocytes and their processes. Here, we characterize the bacterial artificial chromosome transgenic Id3-EGFP knock-in mouse to establish its usefulness for in vivo imaging of astrocyte processes....

  2. Astrocytes directly influence tumor cell invasion and metastasis in vivo.

    Directory of Open Access Journals (Sweden)

    Ling Wang

    Full Text Available Brain metastasis is a defining component of tumor pathophysiology, and the underlying mechanisms responsible for this phenomenon are not well understood. Current dogma is that tumor cells stimulate and activate astrocytes, and this mutual relationship is critical for tumor cell sustenance in the brain. Here, we provide evidence that primary rat neonatal and adult astrocytes secrete factors that proactively induced human lung and breast tumor cell invasion and metastasis capabilities. Among which, tumor invasion factors namely matrix metalloprotease-2 (MMP-2 and MMP-9 were partly responsible for the astrocyte media-induced tumor cell invasion. Inhibiting MMPs reduced the ability of tumor cell to migrate and invade in vitro. Further, injection of astrocyte media-conditioned breast cancer cells in mice showed increased invasive activity to the brain and other distant sites. More importantly, blocking the preconditioned tumor cells with broad spectrum MMP inhibitor decreased the invasion and metastasis of the tumor cells, in particular to the brain in vivo. Collectively, our data implicate astrocyte-derived MMP-2 and MMP-9 as critical players that facilitate tumor cell migration and invasion leading to brain metastasis.

  3. Staurosporine induces different cell death forms in cultured rat astrocytes

    International Nuclear Information System (INIS)

    Astroglial cells are frequently involved in malignant transformation. Besides apoptosis, necroptosis, a different form of regulated cell death, seems to be related with glioblastoma genesis, proliferation, angiogenesis and invasion. In the present work we elucidated mechanisms of necroptosis in cultured astrocytes, and compared them with apoptosis, caused by staurosporine. Cultured rat cortical astrocytes were used for a cell death studies. Cell death was induced by different concentrations of staurosporine, and modified by inhibitors of apoptosis (z-vad-fmk) and necroptosis (nec-1). Different forms of a cell death were detected using flow cytometry. We showed that staurosporine, depending on concentration, induces both, apoptosis as well as necroptosis. Treatment with 10−7 M staurosporine increased apoptosis of astrocytes after the regeneration in a staurosporine free medium. When caspases were inhibited, apoptosis was attenuated, while necroptosis was slightly increased. Treatment with 10−6 M staurosporine induced necroptosis that occurred after the regeneration of astrocytes in a staurosporine free medium, as well as without regeneration period. Necroptosis was significantly attenuated by nec-1 which inhibits RIP1 kinase. On the other hand, the inhibition of caspases had no effect on necroptosis. Furthermore, staurosporine activated RIP1 kinase increased the production of reactive oxygen species, while an antioxidant BHA significantly attenuated necroptosis. Staurosporine can induce apoptosis and/or necroptosis in cultured astrocytes via different signalling pathways. Distinction between different forms of cell death is crucial in the studies of therapy-induced necroptosis

  4. An experimental study on astrocytes promoting production of neural stem cells derived from mouse embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    ZHOU Yu-feng; FANG Feng; FU Jin-rong; DONG Yong-sui; YE Du-yun; SHU Sai-nan; ZHEN Hong; LI Ge

    2005-01-01

    Background The production of neural stem cells (NSCs) derived from embryonic stem (ES) cells was usually very low according to previous studies, which was a major obstacle for meeting the needs of clinical application. This study aimed at investigating whether astrocytes could promote production of NSCs derived from ES cells in vitro.Methods Mouse ES cells line-D3 was used to differentiate into NSCs with astrocytes as inducing stromal cells by means of three-stage differentiation procedure. Another group without astrocytes served as control. The totipotency of ES cells was identified by observation of cells' morphology and formation of teratoma in severe combined immunodeficiency disease (SCID) mice. The quantity and purity of NSCs derived from ES cells were analyzed using clonogenic assay, immunohistochemical staining and flow cytometry assay. The plasticity of NSCs was detected by differentiating test. Octamer-binding transcription factor 4 (Oct-4) and nestin, the specific marker genes of ES cells and NSCs respectively, were detected continuously using reverse transcription-polymerase chain reaction (RT-PCR) method to monitor the process of cell differentiation. Results The ES cells of D3 line could maintain the ability of differentiating into cellular derivations of all three primary germ layers after continuous passage culture. At the end of two-stage of inducing process, 23.2±3.5 neurospheres per plate formed in astrocyte-induced group and only 0.8±0.3 per plate in the control group (clonogenic assay, P<0.01), and the ratio of nestin positive cells was (50.2±2.8)% in astrocyte-induced group and only (1.4±0.5)% in the control group (flow cytometry, P<0.01). With the induction undergoing, the expression of Oct-4 gradually decreased and then disappeared, while the expression of nestin was increased step by step, and the ratio of nestin positive cells was up to 91.4% by the three-stage differentiation. The nestin positive cells could be further induced into

  5. Are astrocytes executive cells within the central nervous system?

    Science.gov (United States)

    Sica, Roberto E; Caccuri, Roberto; Quarracino, Cecilia; Capani, Francisco

    2016-08-01

    Experimental evidence suggests that astrocytes play a crucial role in the physiology of the central nervous system (CNS) by modulating synaptic activity and plasticity. Based on what is currently known we postulate that astrocytes are fundamental, along with neurons, for the information processing that takes place within the CNS. On the other hand, experimental findings and human observations signal that some of the primary degenerative diseases of the CNS, like frontotemporal dementia, Parkinson's disease, Alzheimer's dementia, Huntington's dementia, primary cerebellar ataxias and amyotrophic lateral sclerosis, all of which affect the human species exclusively, may be due to astroglial dysfunction. This hypothesis is supported by observations that demonstrated that the killing of neurons by non-neural cells plays a major role in the pathogenesis of those diseases, at both their onset and their progression. Furthermore, recent findings suggest that astrocytes might be involved in the pathogenesis of some psychiatric disorders as well. PMID:27556379

  6. Neurorestorative Role of Stem Cells in Alzheimer's Disease: Astrocyte Involvement.

    Science.gov (United States)

    Choi, Sung S; Lee, Sang-Rae; Lee, Hong J

    2016-01-01

    Neurogenesis is maintained in both neonatal and adult brain, although it is dramatically reduced in aged neurogenic brain region such as the subgranular layer and subventricular zone of the dentate gyrus (DG). Astrocytes play important roles for survival and maintenance of neurons as well as maintenance of neurogenic niche in quiescent state. Aβ can induce astrocyte activation which give rise to produce reactive oxygen species (ROS) and cytotoxic cytokines and chemokines, and subsequently induce neuronal death. Unfortunately, the current therapeutic medicines have been limited to reduce the symptoms and delay the pathogenesis of Alzheimer's disease (AD), but not to cure it. Stem cells enhance neurogenesis and Aβ clearing as well as improved cognitive impairment. Neurotrophins and growth factors which are produced from both stem cells and astrocytes also have neuroprotective effects via neurogenesis. Secreted factors from both astrocytes and neural stem cells also are influenced in neurogenesis and neuron survival in neurodegenerative diseases. Transplanted stem cells overexpressing neurogenic factors may be an effective and therapeutic tool to enhance neurogenesis for AD. PMID:27018261

  7. Protoplasmic Astrocytes Enhance the Ability of Neural Stem Cells to Differentiate into Neurons In Vitro

    OpenAIRE

    Yuan Liu; Li Wang; Zaiyun Long; Lin Zeng; Yamin Wu

    2012-01-01

    Protoplasmic astrocytes have been reported to exhibit neuroprotective effects on neurons, but there has been no direct evidence for a functional relationship between protoplasmic astrocytes and neural stem cells (NSCs). In this study, we examined neuronal differentiation of NSCs induced by protoplasmic astrocytes in a co-culture model. Protoplasmic astrocytes were isolated from new-born and NSCs from the E13-15 cortex of rats respectively. The differentiated cells labeled with neuron-specific...

  8. Cell-cell contact viral transfer contributes to HIV infection and persistence in astrocytes

    OpenAIRE

    Luo, Xiaoyu; He, Johnny J.

    2014-01-01

    Astrocytes are the most abundant cells in the central nervous system and play important roles in HIV/neuroAIDS. Detection of HIV proviral DNA, RNA and early gene products but not late structural gene products in astrocytes in vivo and in vitro indicates that astrocytes are susceptible to HIV infection albeit in a restricted manner. We as well as others have shown that cell-free HIV is capable of entering CD4− astrocytes through human mannose receptor-mediated endocytosis. In this study, we to...

  9. In vitro differentiation of cultured human CD34+ cells into astrocytes

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    Katari Venkatesh

    2013-01-01

    Full Text Available Background: Astrocytes are abundantly present as glial cells in the brain and play an important role in the regenerative processes. The possible role of stem cell derived astrocytes in the spinal cord injuries is possible related to their influence at the synaptic junctions. Aim: The present study is focused on in vitro differentiation of cultured human CD34+ cells into astrocytes. Materials and Methods: Granulocyte-colony stimulating factor mobilized human CD34+ cells were isolated from peripheral blood using apheresis method from a donor. These cells were further purified by fluorescence-activated cell sorting and cultured in Dulbecco′s modified eagle′s medium. Thus, cultured cells were induced with astrocyte defined medium (ADM and in the differentiated astrocytes serine/threonine protein kinases (STPK and glutamine synthetase (GLUL activities were estimated. The expression of glial fibrillary acidic protein (GFAP and GLUL were confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR. Results: The cultured human CD34+ cells differentiated into astrocytes after 11 h of incubation in ADM. The RT-PCR experiment showed the expression of GLUL (1.5 kb and GFAP (2.9 kb in differentiated astrocytes. The high enzyme activities of GLUL and STPK in differentiated astrocytes compared with cultured human CD34+ cells confirmed astrocyte formation. Conclusion: In the present study, in vitro differentiation of stem cells with retinoic acid induction may result in the formation of astrocytes.

  10. "Cell therapy for stroke: use of local astrocytes"

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    Melek eChouchane

    2012-10-01

    Full Text Available Stroke refers to a variety of conditions caused by the occlusion or hemorrhage of blood vessels supplying the brain, which is one of the main causes of death and the leading cause of disability worldwide. In the last years, cell-based therapies have been proposed as a new approach to ameliorate post stroke deficits. However, the most appropriate type of cell to be used in such therapies, as well as their sources, remains a matter of intense research. A good candidate cell should, in principle, display high plasticity to generate diverse types of neurons and, at the same type, low risk to cause undesired outcomes, such as malignant transformation. Recently, a new approach grounded on the reprogramming of endogenous astrocytes towards neuronal fates emerged as an alternative to restore neurological functions in several central nervous system diseases. In this perspective, we review data about the potential of astrocytes to become functional neurons following expression of neurogenic genes and discuss the potential benefits and risks of reprogramming astrocytes in the glial scar to replace neurons lost after stroke.

  11. Comparison of the Gene Expression Profiles of Human Fetal Cortical Astrocytes with Pluripotent Stem Cell Derived Neural Stem Cells Identifies Human Astrocyte Markers and Signaling Pathways and Transcription Factors Active in Human Astrocytes

    OpenAIRE

    Nasir Malik; Xiantao Wang; Sonia Shah; Efthymiou, Anastasia G.; Bin Yan; Sabrina Heman-Ackah; Ming Zhan; Mahendra Rao

    2014-01-01

    Astrocytes are the most abundant cell type in the central nervous system (CNS) and have a multitude of functions that include maintenance of CNS homeostasis, trophic support of neurons, detoxification, and immune surveillance. It has only recently been appreciated that astrocyte dysfunction is a primary cause of many neurological disorders. Despite their importance in disease very little is known about global gene expression for human astrocytes. We have performed a microarray expression anal...

  12. Induction of adult human bone marrow mesenchymal stromal cells into functional astrocyte-like cells: potential for restorative treatment in Parkinson's disease.

    Science.gov (United States)

    Bahat-Stroomza, Merav; Barhum, Yael; Levy, Yossef S; Karpov, Olga; Bulvik, Shlomo; Melamed, Eldad; Offen, Daniel

    2009-09-01

    Parkinson's disease (PD) is a neurodegenerative disorder with its motor phenomena due mostly to loss of dopamine-producing neurons in the substantia nigra. Pharmacological treatments aimed to increase the deficient dopaminergic neurotransmission are effective in ameliorating the cardinal symptoms, but none of these therapies is curative. It has been suggested that treatment with neurotrophic factors (NTFs) might protect and prevent death of the surviving dopaminergic neurons and induce proliferation of their axonal nerve terminals with reinnervations of the deafferented striatum. However, long-term delivery of such proteins into the CNS is problematic. We therefore aimed to differentiate ex vivo human bone marrow-derived mesenchymal stromal cells into astrocyte-like cells, capable of generating NTFs for future transplantation into basal ganglia of PD patients. Indeed, mesenchymal stromal cells treated with our novel astrocyte differentiation medium, present astrocyte-like morphology and express the astrocyte markers S100beta, glutamine synthetase and glial fibrillary acidic protein. Moreover, these astrocyte-like cells produce and secrete significant amounts of glial cell line-derived neurotrophic factor (GDNF), nerve growth factor (NGF), and brain-derived neurotrophic factor as indicated by messenger RNA, real-time polymerase chain reaction, ELISA, and Western blot analyses. Such NTF-producing cells transplanted into the striatum of 6-hydroxydopamine-lesioned rats, a model of PD, produced a progressive reduction in the apomorphine-induced contralateral rotations as well as behavioral improvement in rotor-rod and the "sunflower seeds" eating motor tests. Histological assessments revealed that the engrafted cells survived and expressed astrocyte and human markers and acted to regenerate the damaged dopaminergic nerve terminal system. Findings indicate that our novel procedure to induce NTF-producing astrocyte-like cells derived from human bone marrow stromal cells

  13. GDNF facilitates differentiation of the adult dentate gyrus-derived neural precursor cells into astrocytes via STAT3

    International Nuclear Information System (INIS)

    Highlights: •GDNF has no effect on ADP proliferation and apoptosis. •GDNF increases ADP differentiation into astrocyte. •A specific inhibitor of STAT3 decreases the astrogliogenic effect of GDNF. •STAT3 knockdown by lentiviral shRNA vector also decreases the astrogliogenic effect of GDNF. •GDNF increases the phosphorylation of STAT3. -- Abstract: While the pro-neurogenic actions of antidepressants in the adult hippocampal dentate gyrus (DG) are thought to be one of the mechanisms through which antidepressants exert their therapeutic actions, antidepressants do not increase proliferation of neural precursor cells derived from the adult DG. Because previous studies showed that antidepressants increase the expression and secretion of glial cell line-derived neurotrophic factor (GDNF) in C6 glioma cells derived from rat astrocytes and GDNF increases neurogenesis in adult DG in vivo, we investigated the effects of GDNF on the proliferation, differentiation and apoptosis of cultured neural precursor cells derived from the adult DG. Data showed that GDNF facilitated the differentiation of neural precursor cells into astrocytes but had no effect on their proliferation or apoptosis. Moreover, GDNF increased the phosphorylation of STAT3, and both a specific inhibitor of STAT3 and lentiviral shRNA for STAT3 decreased their differentiation into astrocytes. Taken together, our findings suggest that GDNF facilitates astrogliogenesis from neural precursor cells in adult DG through activating STAT3 and that this action might indirectly affect neurogenesis

  14. GDNF facilitates differentiation of the adult dentate gyrus-derived neural precursor cells into astrocytes via STAT3

    Energy Technology Data Exchange (ETDEWEB)

    Boku, Shuken, E-mail: shuboku@med.hokudai.ac.jp [Department of Psychiatry, Hokkaido University Graduate School of Medicine, Sapporo (Japan); Nakagawa, Shin [Department of Psychiatry, Hokkaido University Graduate School of Medicine, Sapporo (Japan); Takamura, Naoki [Pharmaceutical Laboratories, Dainippon Sumitomo Pharma Co. Ltd., Osaka (Japan); Kato, Akiko [Department of Psychiatry, Hokkaido University Graduate School of Medicine, Sapporo (Japan); Takebayashi, Minoru [Department of Psychiatry, National Hospital Organization Kure Medical Center, Kure (Japan); Hisaoka-Nakashima, Kazue [Department of Pharmacology, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima (Japan); Omiya, Yuki; Inoue, Takeshi; Kusumi, Ichiro [Department of Psychiatry, Hokkaido University Graduate School of Medicine, Sapporo (Japan)

    2013-05-17

    Highlights: •GDNF has no effect on ADP proliferation and apoptosis. •GDNF increases ADP differentiation into astrocyte. •A specific inhibitor of STAT3 decreases the astrogliogenic effect of GDNF. •STAT3 knockdown by lentiviral shRNA vector also decreases the astrogliogenic effect of GDNF. •GDNF increases the phosphorylation of STAT3. -- Abstract: While the pro-neurogenic actions of antidepressants in the adult hippocampal dentate gyrus (DG) are thought to be one of the mechanisms through which antidepressants exert their therapeutic actions, antidepressants do not increase proliferation of neural precursor cells derived from the adult DG. Because previous studies showed that antidepressants increase the expression and secretion of glial cell line-derived neurotrophic factor (GDNF) in C6 glioma cells derived from rat astrocytes and GDNF increases neurogenesis in adult DG in vivo, we investigated the effects of GDNF on the proliferation, differentiation and apoptosis of cultured neural precursor cells derived from the adult DG. Data showed that GDNF facilitated the differentiation of neural precursor cells into astrocytes but had no effect on their proliferation or apoptosis. Moreover, GDNF increased the phosphorylation of STAT3, and both a specific inhibitor of STAT3 and lentiviral shRNA for STAT3 decreased their differentiation into astrocytes. Taken together, our findings suggest that GDNF facilitates astrogliogenesis from neural precursor cells in adult DG through activating STAT3 and that this action might indirectly affect neurogenesis.

  15. Phenotypic transition of microglia into astrocyte-like cells associated with disease onset in a model of inherited ALS

    OpenAIRE

    Emiliano eTrias; Pablo eDíaz-Amarilla; Silvia eOlivera-Bravo; Eugenia eIsasi; Drechsel, Derek A.; Nathan eLopez; Charles Samuel Bradford; Kyle Edward Ireton; Beckman, Joseph S; Luis Hector Barbeito

    2013-01-01

    Microglia and reactive astrocytes accumulate in the spinal cord of rats expressing the Amyotrophic lateral sclerosis (ALS)-linked SOD1 G93A mutation. We previously reported that the rapid progression of paralysis in ALS rats is associated with the appearance of proliferative astrocyte-like cells that surround motor neurons. These cells, designated as Aberrant Astrocytes (AbA cells) because of their atypical astrocytic phenotype, exhibit high toxicity to motor neurons. However, the cellular or...

  16. Astrocytes and Müller cells changes during retinal degeneration in a transgenic rat model of retinitis pigmentosa.

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    Laura eFernández-Sánchez

    2015-12-01

    Full Text Available Purpose: Retinitis pigmentosa includes a group of progressive retinal degenerative diseases that affect the structure and function of photoreceptors. Secondarily to the loss of photoreceptors, there is a reduction in retinal vascularization, which seems to influence the cellular degenerative process. Retinal macroglial cells, astrocytes and Müller cells provide support for retinal neurons and are fundamental for maintaining normal retinal function. The aim of this study was to investigate the evolution of macroglial changes during retinal degeneration in P23H rats. Methods: Homozygous P23H line-3 rats aged from P18 to 18 months were used to study the evolution of the disease, and SD rats were used as controls. Immunolabeling with antibodies against GFAP, vimentin, and transducin were used to visualize macroglial cells and cone photoreceptors. Results: In P23H rats, increased GFAP labeling in Müller cells was observed as an early indicator of retinal gliosis. At 4 and 12 months of age, the apical processes of Müller cells in P23H rats clustered in firework-like structures, which were associated with ring-like shaped areas of cone degeneration in the outer nuclear layer. These structures were not observed at 16 months of age. The number of astrocytes was higher in P23H rats than in the SD matched controls at 4 and 12 months of age, supporting the idea of astrocyte proliferation. As the disease progressed, astrocytes exhibited a deteriorated morphology and marked hypertrophy. The increase in the complexity of the astrocytic processes correlated with greater connexin 43 expression and higher density of connexin 43 immunoreactive puncta within the ganglion cell layer of P23H versus SD rat retinas. Conclusions: In the P23H rat model of retinitis pigmentosa, the loss of photoreceptors triggers major changes in the number and morphology of glial cells affecting the inner retina.

  17. Induction of neural stem cell-like cells (NSCLCs) from mouse astrocytes by Bmi1

    International Nuclear Information System (INIS)

    Recently, Bmi1 was shown to control the proliferation and self-renewal of neural stem cells (NSCs). In this study, we demonstrated the induction of NSC-like cells (NSCLCs) from mouse astrocytes by Bmi1 under NSC culture conditions. These NSCLCs exhibited the morphology and growth properties of NSCs, and expressed NSC marker genes, including nestin, CD133, and Sox2. In vitro differentiation of NSCLCs resulted in differentiated cell populations containing astrocytes, neurons, and oligodendrocytes. Following treatment with histone deacetylase inhibitors (trichostatin A and valproic acid), the potential of NSCLCs for proliferation, dedifferentiation, and self-renewal was significantly inhibited. Our data indicate that multipotent NSCLCs can be generated directly from astrocytes by the addition of Bmi1

  18. Astrocyte-like glial cells physiologically regulate olfactory processing through the modification of ORN-PN synaptic strength in Drosophila.

    Science.gov (United States)

    Liu, He; Zhou, Bangyu; Yan, Wenjun; Lei, Zhengchang; Zhao, Xiaoliang; Zhang, Ke; Guo, Aike

    2014-09-01

    Astrocyte-like glial cells are abundant in the central nervous system of adult Drosophila and exhibit morphology similar to astrocytes of mammals. Previous evidence has shown that astrocyte-like glial cells are strongly associated with synapses in the antennal lobe (AL), the first relay of the olfactory system, where olfactory receptor neurons (ORNs) transmit information into projection neurons (PNs). However, the function of astrocyte-like glia in the AL remains obscure. In this study, using in vivo calcium imaging, we found that astrocyte-like glial cells exhibited spontaneous microdomain calcium elevations. Using simultaneous manipulation of glial activity and monitoring of neuronal function, we found that the astrocyte-like glial activation, but not ensheathing glial activation, could inhibit odor-evoked responses of PNs. Ensheathing glial cells are another subtype of glia, and are of functional importance in the AL. Electrophysiological experiments indicated that astrocyte-like glial activation decreased the amplitude and slope of excitatory postsynaptic potentials evoked through electrical stimulation of the antennal nerve. These results suggest that astrocyte-like glial cells may regulate olfactory processing through negative regulation of ORN-PN synaptic strength. Beyond the antennal lobe we observed astrocyte-like glial spontaneous calcium activities in the ventromedial protocerebrum, indicating that astrocyte-like glial spontaneous calcium elevations might be general in the adult fly brain. Overall, our study demonstrates a new function for astrocyte-like glial cells in the physiological modulation of olfactory information transmission, possibly through regulating ORN-PN synapse strength.

  19. Transplantation of stem cell-derived astrocytes for thetreatment of amyotrophic lateral sclerosis and spinal cordinjury

    Institute of Scientific and Technical Information of China (English)

    Charles Nicaise; Dinko Mitrecic; Aditi Falnikar; Angelo C Lepore

    2015-01-01

    Neglected for years, astrocytes are now recognized tofulfill and support many, if not all, homeostatic functionsof the healthy central nervous system (CNS). Duringneurodegenerative diseases such as amyotrophiclateral sclerosis (ALS) and spinal cord injury (SCI),astrocytes in the vicinity of degenerating areasundergo both morphological and functional changesthat might compromise their intrinsic properties.Evidence from human and animal studies show thatdeficient astrocyte functions or loss-of-astrocytes largelycontribute to increased susceptibility to cell death forneurons, oligodendrocytes and axons during ALS andSCI disease progression. Despite exciting advances inexperimental CNS repair, most of current approachesthat are translated into clinical trials focus on thereplacement or support of spinal neurons throughstem cell transplantation, while none focus on thespecific replacement of astroglial populations. Knowingthe important functions carried out by astrocytesin the CNS, astrocyte replacement-based therapiesmight be a promising approach to alleviate overallastrocyte dysfunction, deliver neurotrophic support todegenerating spinal tissue and stimulate endogenousCNS repair abilities. Enclosed in this review, we gatheredexperimental evidence that argue in favor of astrocytetransplantation during ALS and SCI. Based on theirintrinsic properties and according to the cell typetransplanted, astrocyte precursors or stem cell-derivedastrocytes promote axonal growth, support mechanismsand cells involved in myelination, are able to modulatethe host immune response, deliver neurotrophic factorsand provide protective molecules against oxidative orexcitotoxic insults, amongst many possible benefits.Embryonic or adult stem cells can even be geneticallyengineered in order to deliver missing gene productsand therefore maximize the chance of neuroprotectionand functional recovery. However, before broad clinicaltranslation, further preclinical data on safety

  20. Heterogeneity of astrocytes: from development to injury - single cell gene expression.

    Directory of Open Access Journals (Sweden)

    Vendula Rusnakova

    Full Text Available Astrocytes perform control and regulatory functions in the central nervous system; heterogeneity among them is still a matter of debate due to limited knowledge of their gene expression profiles and functional diversity. To unravel astrocyte heterogeneity during postnatal development and after focal cerebral ischemia, we employed single-cell gene expression profiling in acutely isolated cortical GFAP/EGFP-positive cells. Using a microfluidic qPCR platform, we profiled 47 genes encoding glial markers and ion channels/transporters/receptors participating in maintaining K(+ and glutamate homeostasis per cell. Self-organizing maps and principal component analyses revealed three subpopulations within 10-50 days of postnatal development (P10-P50. The first subpopulation, mainly immature glia from P10, was characterized by high transcriptional activity of all studied genes, including polydendrocytic markers. The second subpopulation (mostly from P20 was characterized by low gene transcript levels, while the third subpopulation encompassed mature astrocytes (mainly from P30, P50. Within 14 days after ischemia (D3, D7, D14, additional astrocytic subpopulations were identified: resting glia (mostly from P50 and D3, transcriptionally active early reactive glia (mainly from D7 and permanent reactive glia (solely from D14. Following focal cerebral ischemia, reactive astrocytes underwent pronounced changes in the expression of aquaporins, nonspecific cationic and potassium channels, glutamate receptors and reactive astrocyte markers.

  1. Globular adiponectin induces a pro-inflammatory response in human astrocytic cells

    Energy Technology Data Exchange (ETDEWEB)

    Wan, Zhongxiao; Mah, Dorrian; Simtchouk, Svetlana [School of Health and Exercise Sciences, University of British Columbia Okanagan, Kelowna, BC (Canada); Klegeris, Andis [Department of Biology, University of British Columbia Okanagan, Kelowna, BC (Canada); Little, Jonathan P., E-mail: jonathan.little@ubc.ca [School of Health and Exercise Sciences, University of British Columbia Okanagan, Kelowna, BC (Canada)

    2014-03-28

    Highlights: • Adiponectin receptors are expressed in human astrocytes. • Globular adiponectin induces secretion of IL-6 and MCP-1 from cultured astrocytes. • Adiponectin may play a pro-inflammatory role in astrocytes. - Abstract: Neuroinflammation, mediated in part by activated brain astrocytes, plays a critical role in the development of neurodegenerative disorders, including Alzheimer’s disease (AD). Adiponectin is the most abundant adipokine secreted from adipose tissue and has been reported to exert both anti- and pro-inflammatory effects in peripheral tissues; however, the effects of adiponectin on astrocytes remain unknown. Shifts in peripheral concentrations of adipokines, including adiponectin, could contribute to the observed link between midlife adiposity and increased AD risk. The aim of the present study was to characterize the effects of globular adiponectin (gAd) on pro-inflammatory cytokine mRNA expression and secretion in human U373 MG astrocytic cells and to explore the potential involvement of nuclear factor (NF)-κB, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) and phosphatidylinositide 3-kinases (PI3 K) signaling pathways in these processes. We demonstrated expression of adiponectin receptor 1 (adipoR1) and adipoR2 in U373 MG cells and primary human astrocytes. gAd induced secretion of interleukin (IL)-6 and monocyte chemoattractant protein (MCP)-1, and gene expression of IL-6, MCP-1, IL-1β and IL-8 in U373 MG cells. Using specific inhibitors, we found that NF-κB, p38MAPK and ERK1/2 pathways are involved in gAd-induced induction of cytokines with ERK1/2 contributing the most. These findings provide evidence that gAd may induce a pro-inflammatory phenotype in human astrocytes.

  2. Calcium-mediated transductive systems and functionally active gap junctions in astrocyte-like GL15 cells

    Directory of Open Access Journals (Sweden)

    Steimberg Nathalie

    2001-05-01

    Full Text Available Abstract Background It has been proposed that GL15, a human cell line derived from glioblastoma multiforme, is a possible astroglial-like cell model, based on the presence of cytoplasmic glial fibrillary acidic protein. Results The aim of this work was to delineate the functional characteristics of GL15 cells using various experimental approaches, including the study of morphology, mechanism of induction of intracellular Ca2+ increase by different physiological agonists, and the presence and permeability of the gap-junction system during cell differentiation. Immunostaining experiments showed the presence and localization of specific glial markers, such as glial fibrillary acidic protein and S100B, and the lack of the neuronal marker S100A. Notably, all the Ca2+ pathways present in astrocytes were detected in GL15 cells. In particular, oscillations in intracellular Ca2+ levels were recorded either spontaneously, or in the presence of ATP or glutamate (but not KCl. Immunolabelling assays and confocal microscopy, substantiated by Western blot analyses, revealed the presence of connexin43, a subunit of astrocyte gap-junction channels. The protein is organised in characteristic spots on the plasma membrane at cell-cell contact regions, and its presence and distribution depends on the differentiative status of the cell. Finally, a microinjection/dye-transfer assay, employed to determine gap-junction functionality, clearly demonstrated that the cells were functionally coupled, albeit to varying degrees, in differentiated and undifferentiated phenotypes. Conclusions In conclusion, results from this study support the use of the GL15 cell line as a suitable in vitro astrocyte model, which provides a valuable guide for studying glial physiological features at various differentiation phases.

  3. Effects of lipopolysaccharide on oligodendrocyte progenitor cells are mediated by astrocytes and microglia.

    Science.gov (United States)

    Pang, Y; Cai, Z; Rhodes, P G

    2000-11-15

    Oligodendrocytes are the primary cells injured in periventricular leukomalacia (PVL), a predominant form of brain white matter lesion in preterm infants. To explore the possible linkage between white matter injury and maternal infection, purified rat O-2A progenitor (Oligodendrocyte-type 2 astrocyte progenitor) cell cultures were used as a model in studying the effects of lipopolysaccharide (LPS), an endotoxin, on survival and differentiation of oligodendrocytes and the involvement of other glial cells in the effects of LPS. O-2A progenitor cells were cultured from optic nerves of 7-day-old rat pups in a chemically defined medium (CDM). Astrocyte and microglia cell cultures were prepared from the cortex of 1-day-old rat brains in the CDM. Direct treatment of LPS (1 microg/ml) to O-2A cells had no effect on viability or differentiation of these cells. When O-2A progenitor cells were cultured in the conditioned medium obtained from either astrocyte or microglial cell cultures for 48 hr, survival rate and differentiation of O-2A cells into mature oligodendrocytes were greatly enhanced as measured by the MTT assay and immunocytochemistry. The conditioned medium obtained from astrocytes or microglia treated with LPS for 48 hr, however, failed to show such a promotional effect on viability and differentiation of O-2A cells. When 5 microg/ml LPS was used to stimulate astrocytes or microglia, the conditioned medium from these glial cell cultures caused O-2A cell injury. The overall results indicate that astrocytes and microglia may promote viability and differentiation of O-2A progenitor cells under physiological conditions, but they may also mediate cytotoxic effects of LPS on oligodendrocytes under an infectious disease biochemical environment.

  4. Non-cell autonomous influence of the astrocyte system xc− on hypoglycaemic neuronal cell death

    Directory of Open Access Journals (Sweden)

    Sandra J Hewett

    2012-02-01

    Full Text Available Despite longstanding evidence that hypoglycaemic neuronal injury is mediated by glutamate excitotoxicity, the cellular and molecular mechanisms involved remain incompletely defined. Here, we demonstrate that the excitotoxic neuronal death that follows GD (glucose deprivation is initiated by glutamate extruded from astrocytes via system xc− – an amino acid transporter that imports l-cystine and exports l-glutamate. Specifically, we find that depriving mixed cortical cell cultures of glucose for up to 8 h injures neurons, but not astrocytes. Neuronal death is prevented by ionotropic glutamate receptor antagonism and is partially sensitive to tetanus toxin. Removal of amino acids during the deprivation period prevents – whereas addition of l-cystine restores – GD-induced neuronal death, implicating the cystine/glutamate antiporter, system xc−. Indeed, drugs known to inhibit system xc− ameliorate GD-induced neuronal death. Further, a dramatic reduction in neuronal death is observed in chimaeric cultures consisting of neurons derived from WT (wild-type mice plated on top of astrocytes derived from sut mice, which harbour a naturally occurring null mutation in the gene (Slc7a11 that encodes the substrate-specific light chain of system xc− (xCT. Finally, enhancement of astrocytic system xc− expression and function via IL-1β (interleukin-1β exposure potentiates hypoglycaemic neuronal death, the process of which is prevented by removal of l-cystine and/or addition of system xc− inhibitors. Thus, under the conditions of GD, our studies demonstrate that astrocytes, via system xc−, have a direct, non-cell autonomous effect on cortical neuron survival.

  5. Non-cell autonomous influence of the astrocyte system xc- on hypoglycaemic neuronal cell death.

    Science.gov (United States)

    Jackman, Nicole A; Melchior, Shannon E; Hewett, James A; Hewett, Sandra J

    2012-02-08

    Despite longstanding evidence that hypoglycaemic neuronal injury is mediated by glutamate excitotoxicity, the cellular and molecular mechanisms involved remain incompletely defined. Here, we demonstrate that the excitotoxic neuronal death that follows GD (glucose deprivation) is initiated by glutamate extruded from astrocytes via system xc---an amino acid transporter that imports L-cystine and exports L-glutamate. Specifically, we find that depriving mixed cortical cell cultures of glucose for up to 8 h injures neurons, but not astrocytes. Neuronal death is prevented by ionotropic glutamate receptor antagonism and is partially sensitive to tetanus toxin. Removal of amino acids during the deprivation period prevents--whereas addition of L-cystine restores--GD-induced neuronal death, implicating the cystine/glutamate antiporter, system xc-. Indeed, drugs known to inhibit system xc- ameliorate GD-induced neuronal death. Further, a dramatic reduction in neuronal death is observed in chimaeric cultures consisting of neurons derived from WT (wild-type) mice plated on top of astrocytes derived from sut mice, which harbour a naturally occurring null mutation in the gene (Slc7a11) that encodes the substrate-specific light chain of system xc- (xCT). Finally, enhancement of astrocytic system xc- expression and function via IL-1β (interleukin-1β) exposure potentiates hypoglycaemic neuronal death, the process of which is prevented by removal of l-cystine and/or addition of system xc- inhibitors. Thus, under the conditions of GD, our studies demonstrate that astrocytes, via system xc-, have a direct, non-cell autonomous effect on cortical neuron survival.

  6. Monitoring Astrocytic Proteome Dynamics by Cell Type-Specific Protein Labeling.

    Directory of Open Access Journals (Sweden)

    Anke Müller

    Full Text Available The ability of the nervous system to undergo long-term plasticity is based on changes in cellular and synaptic proteomes. While many studies have explored dynamic alterations in neuronal proteomes during plasticity, there has been less attention paid to the astrocytic counterpart. Indeed, progress in identifying cell type-specific proteomes is limited owing to technical difficulties. Here, we present a cell type-specific metabolic tagging technique for a mammalian coculture model based on the bioorthogonal amino acid azidonorleucine and the mutated Mus musculus methionyl-tRNA synthetaseL274G enabling azidonorleucine introduction into de novo synthesized proteins. Azidonorleucine incorporation resulted in cell type-specific protein labeling and retained neuronal or astrocytic cell viability. Furthermore, we were able to label astrocytic de novo synthesized proteins and identified both Connexin-43 and 60S ribosomal protein L10a upregulated upon treatment with Brain-derived neurotrophic factor in astrocytes of a neuron-glia coculture. Taken together, we demonstrate the successful dissociation of astrocytic from neuronal proteomes by cell type-specific metabolic labeling offering new possibilities for the analyses of cell type-specific proteome dynamics.

  7. Phorbol 12-myristate 13-acetate induces protein kinase ceta-specific proliferative response in astrocytic tumor cells.

    Science.gov (United States)

    Hussaini, I M; Karns, L R; Vinton, G; Carpenter, J E; Redpath, G T; Sando, J J; VandenBerg, S R

    2000-07-21

    Protein kinase C (PKC) activation has been implicated in cellular proliferation in neoplastic astrocytes. The roles for specific PKC isozymes in regulating this glial response, however, are not well understood. The aim of this study was to characterize the expression of PKC isozymes and the role of PKC-eta expression in regulating cellular proliferation in two well characterized astrocytic tumor cell lines (U-1242 MG and U-251 MG) with different properties of growth in cell culture. Both cell lines expressed an array of conventional (alpha, betaI, betaII, and gamma) and novel (theta and epsilon) PKC isozymes that can be activated by phorbol myristate acetate (PMA). Another novel PKC isozyme, PKC-eta, was only expressed by U-251 MG cells. In contrast, PKC-delta was readily detected in U-1242 MG cells but was present only at low levels in U-251 MG cells. PMA (100 nm) treatment for 24 h increased cell proliferation by over 2-fold in the U-251 MG cells, whereas it decreased the mitogenic response in the U-1242 MG cells by over 90%. When PKC-eta was stably transfected into U-1242 MG cells, PMA increased cell proliferation by 2.2-fold, similar to the response of U-251 MG cells. The cell proliferation induced by PMA in both the U-251 MG and U-1242-PKC-eta cells was blocked by the PKC inhibitor bisindolylmaleimide (0.5 micrometer) and the MEK inhibitor, PD 98059 (50 micrometer). Transient transfection of wild type U-251 with PKC-eta antisense oligonucleotide (1 micrometer) also blocked the PMA-induced increase in [(3)H]thymidine incorporation. The data demonstrate that two glioblastoma lines, with functionally distinct proliferative responses to PMA, express different novel PKC isozymes and that the differential expression of PKC-eta plays a determining role in the different proliferative capacity. PMID:10806212

  8. Insulin promotes glycogen storage and cell proliferation in primary human astrocytes.

    Directory of Open Access Journals (Sweden)

    Martin Heni

    Full Text Available INTRODUCTION: In the human brain, there are at least as many astrocytes as neurons. Astrocytes are known to modulate neuronal function in several ways. Thus, they may also contribute to cerebral insulin actions. Therefore, we examined whether primary human astrocytes are insulin-responsive and whether their metabolic functions are affected by the hormone. METHODS: Commercially available Normal Human Astrocytes were grown in the recommended medium. Major players in the insulin signaling pathway were detected by real-time RT-PCR and Western blotting. Phosphorylation events were detected by phospho-specific antibodies. Glucose uptake and glycogen synthesis were assessed using radio-labeled glucose. Glycogen content was assessed by histochemistry. Lactate levels were measured enzymatically. Cell proliferation was assessed by WST-1 assay. RESULTS: We detected expression of key proteins for insulin signaling, such as insulin receptor β-subunit, insulin receptor substrat-1, Akt/protein kinase B and glycogen synthase kinase 3, in human astrocytes. Akt was phosphorylated and PI-3 kinase activity increased following insulin stimulation in a dose-dependent manner. Neither increased glucose uptake nor lactate secretion after insulin stimulation could be evidenced in this cell type. However, we found increased insulin-dependent glucose incorporation into glycogen. Furthermore, cell numbers increased dose-dependently upon insulin treatment. DISCUSSION: This study demonstrated that human astrocytes are insulin-responsive at the molecular level. We identified glycogen synthesis and cell proliferation as biological responses of insulin signaling in these brain cells. Hence, this cell type may contribute to the effects of insulin in the human brain.

  9. Death and survival of neuronal and astrocytic cells in ischemic brain injury: a role of autophagy

    Institute of Scientific and Technical Information of China (English)

    Min XU; Hui-ling ZHANG

    2011-01-01

    Autophagy is a highly regulated cellular mechanism that leads to degradation of long-lived proteins and dysfunctional organelles. The process has been implicated in a variety of physiological and pathological conditions relevant to neurological diseases. Recent studies show the existence of autophagy in cerebral ischemia, but no consensus has yet been reached regarding the functions of autophagy in this condition. This article highlights the activation of autophagy during cerebral ischemia and/or reperfusion, especially in neurons and astrocytes, as well as the role of autophagy in neuronal or astrocytic cell death and survival. We propose that physiological levels of autophagy, presumably caused by mild to modest hypoxia or ischemia, appear to be protective. However, high levels of autophagy caused by severe hypoxia or ischemia and/or reperfusion may cause self-digestion and eventual neuronal and astrocytic cell death. We also discuss that oxidative and endoplasmic reticulum (ER) stresses in cerebral hypoxia or ischemia and/or reperfusion are potent stimuli of autophagy in neurons and astrocytes. In addition, we review the evidence suggesting a considerable overlap between autophagy on one hand, and apoptosis, necrosis and necroptosis on the other hand, in determining the outcomes and final morphology of damaged neurons and astrocytes.

  10. Carnosine decreased neuronal cell death through targeting glutamate system and astrocyte mitochondrial bioenergetics in cultured neuron/astrocyte exposed to OGD/recovery.

    Science.gov (United States)

    Ouyang, Li; Tian, Yueyang; Bao, Yun; Xu, Huijuan; Cheng, Jiaoyan; Wang, Bingyu; Shen, Yao; Chen, Zhong; Lyu, Jianxin

    2016-06-01

    Previously, we showed that carnosine upregulated the expression level of glutamate transporter 1 (GLT-1), which has been recognized as an important participant in the astrocyte-neuron lactate shuttle (ANLS), with ischemic model in vitro and in vivo. This study was designed to investigate the protective effect of carnosine on neuron/astrocyte co-cultures exposed to OGD/recovery, and to explore whether the ANLS or any other mechanism contributes to carnosine-induced neuroprotection on neuron/astrocyte. Co-cultures were treated with carnosine and exposed to OGD/recovery. Cell death and the extracellular levels of glutamate and GABA were measured. The mitochondrial respiration and glycolysis were detected by Seahorse Bioscience XF96 Extracellular Flux Analyzer. Results showed that carnosine decreased neuronal cell death, increased extracellular GABA level, and abolished the increase in extracellular glutamate and reversed the mitochondrial energy metabolism disorder induced by OGD/recovery. Carnosine also upregulated the mRNA level of neuronal glutamate transporter EAAC1 at 2h after OGD. Dihydrokainate, a specific inhibitor of GLT-1, decreased glycolysis but it did not affect mitochondrial respiration of the cells, and it could not reverse the increase in mitochondrial OXPHOS induced by carnosine in the co-cultures. The levels of mRNAs for monocarboxylate transporter1, 4 (MCT1, 4), which were expressed in astrocytes, and MCT2, the main neuronal MCT, were significantly increased at the early stage of recovery. Carnosine only partly reversed the increased expression of astrocytic MCT1 and MCT4. These results suggest that regulating astrocytic energy metabolism and extracellular glutamate and GABA levels but not the ANLS are involved in the carnosine-induced neuroprotection. PMID:27040711

  11. MK-801 treatment affects glycolysis in oligodendrocytes more than in astrocytes and neuronal cells: insights for schizophrenia

    Directory of Open Access Journals (Sweden)

    Paul C Guest

    2015-05-01

    Full Text Available As a multifactorial disease, the underlying causes of schizophrenia require analysis by multiplex methods such as proteomics to allow identification of whole protein networks. Previous post-mortem proteomic studies on brain tissues from schizophrenia patients have demonstrated changes in activation of glycolytic and energy metabolism pathways. However, it is not known whether these changes occur in neurons or in glial cells. To address this question, we treated neuronal, astrocyte and oligodendrocyte cell lines with the NMDA receptor antagonist MK-801 and measured the levels of six glycolytic enzymes by Western blot analysis. MK-801 acts on the glutamatergic system and has been proposed as a pharmacological means of modeling schizophrenia. Treatment with MK-801 resulted in significant changes in the levels of glycolytic enzymes in all cell types. Most of the differences were found in oligodendrocytes, which had altered levels of hexokinase 1 (HK1, enolase 2 (ENO2, phosphoglycerate kinase (PGK and phosphoglycerate mutase 1 (PGAM1 after acute MK-801 treatment (8 hours, and HK1, ENO2, PGK and triosphosphate isomerase (TPI following long term treatment (72 hours. Addition of the antipsychotic clozapine to the cultures resulted in counter-regulatory effects to the MK-801 treatment by normalizing the levels of ENO2 and PGK in both the acute and long term cultures. In astrocytes, MK-801 affected only aldolase C (ALDOC under both acute conditions and HK1 and ALDOC following long term treatment, and TPI was the only enzyme affected under long term conditions in the neuronal cells. In conclusion, MK-801 affects glycolysis in oligodendrocytes to a larger extent than neuronal cells and this may be modulated by antipsychotic treatment. Although cell culture studies do not necessarily reflect the in vivo pathophysiology and drug effects within the brain, these results suggest that neurons, astrocytes and oligodendrocytes are affected differently in

  12. Methamphetamine alters the normal progression by inducing cell cycle arrest in astrocytes.

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    Austin R Jackson

    Full Text Available Methamphetamine (MA is a potent psychostimulant with a high addictive capacity, which induces many deleterious effects on the brain. Chronic MA abuse leads to cognitive dysfunction and motor impairment. MA affects many cells in the brain, but the effects on astrocytes of repeated MA exposure is not well understood. In this report, we used Gene chip array to analyze the changes in the gene expression profile of primary human astrocytes treated with MA for 3 days. Range of genes were found to be differentially regulated, with a large number of genes significantly downregulated, including NEK2, TTK, TOP2A, and CCNE2. Gene ontology and pathway analysis showed a highly significant clustering of genes involved in cell cycle progression and DNA replication. Further pathway analysis showed that the genes downregulated by multiple MA treatment were critical for G2/M phase progression and G1/S transition. Cell cycle analysis of SVG astrocytes showed a significant reduction in the percentage of cell in the G2/M phase with a concomitant increase in G1 percentage. This was consistent with the gene array and validation data, which showed that repeated MA treatment downregulated the genes associated with cell cycle regulation. This is a novel finding, which explains the effect of MA treatment on astrocytes and has clear implication in neuroinflammation among the drug abusers.

  13. Knockdown of astrocyte elevated gene-1 inhibits proliferation and enhancing chemo-sensitivity to cisplatin or doxorubicin in neuroblastoma cells

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    Xie Li

    2009-02-01

    Full Text Available Abstract Background Astrocyte elevated gene-1 (AEG-1 was originally characterized as a HIV-1-inducible gene in primary human fetal astrocyte. Recent studies highlight a potential role of AEG-1 in promoting tumor progression and metastasis. The aim of this study was to investigate if AEG-1 serves as a potential therapeutic target of human neuroblastoma. Methods We employed RNA interference to reduce AEG-1 expression in human neuroblastoma cell lines and analyzed their phenotypic changes. Results We found that the knockdown of AEG-1 expression in human neuroblastoma cells significantly inhibited cell proliferation and apoptosis. The specific downregulation induced cell arrest in the G0/G1 phase of cell cycle. In the present study, we also observed a significant enhancement of chemo-sensitivity to cisplatin and doxorubicin by knockdown of AEG-1. Conclusion Our study suggests that overexpressed AEG-1 enhance the tumorogenic properties of neuroblastoma cells. The inhibition of AEG-1 expression could be a new adjuvant therapy for neuroblastoma.

  14. Cerebellar stem cells do not produce neurons and astrocytes in adult mouse

    Energy Technology Data Exchange (ETDEWEB)

    Su, Xin; Guan, Wuqiang; Yu, Yong-Chun; Fu, Yinghui, E-mail: fuyh@fudan.edu.cn

    2014-07-18

    Highlights: • No new neurons and astrocytes are generated in adult mouse cerebellum. • Very few mash1{sup +} or nestin{sup +} stem cells exist, and most of them are quiescent. • Cell proliferation rate is diversified among cerebellar regions and decreases over time. - Abstract: Although previous studies implied that cerebellar stem cells exist in some adult mammals, little is known about whether these stem cells can produce new neurons and astrocytes. In this study by bromodeoxyuridine (BrdU) intraperitoneal (i.p.) injection, we found that there are abundant BrdU{sup +} cells in adult mouse cerebellum, and their quantity and density decreases significantly over time. We also found cell proliferation rate is diversified in different cerebellar regions. Among these BrdU{sup +} cells, very few are mash1{sup +} or nestin{sup +} stem cells, and the vast majority of cerebellar stem cells are quiescent. Data obtained by in vivo retrovirus injection indicate that stem cells do not produce neurons and astrocytes in adult mouse cerebellum. Instead, some cells labeled by retrovirus are Iba1{sup +} microglia. These results indicate that very few stem cells exist in adult mouse cerebellum, and none of these stem cells contribute to neurogenesis and astrogenesis under physiological condition.

  15. Cerebellar stem cells do not produce neurons and astrocytes in adult mouse

    International Nuclear Information System (INIS)

    Highlights: • No new neurons and astrocytes are generated in adult mouse cerebellum. • Very few mash1+ or nestin+ stem cells exist, and most of them are quiescent. • Cell proliferation rate is diversified among cerebellar regions and decreases over time. - Abstract: Although previous studies implied that cerebellar stem cells exist in some adult mammals, little is known about whether these stem cells can produce new neurons and astrocytes. In this study by bromodeoxyuridine (BrdU) intraperitoneal (i.p.) injection, we found that there are abundant BrdU+ cells in adult mouse cerebellum, and their quantity and density decreases significantly over time. We also found cell proliferation rate is diversified in different cerebellar regions. Among these BrdU+ cells, very few are mash1+ or nestin+ stem cells, and the vast majority of cerebellar stem cells are quiescent. Data obtained by in vivo retrovirus injection indicate that stem cells do not produce neurons and astrocytes in adult mouse cerebellum. Instead, some cells labeled by retrovirus are Iba1+ microglia. These results indicate that very few stem cells exist in adult mouse cerebellum, and none of these stem cells contribute to neurogenesis and astrogenesis under physiological condition

  16. Therapeutic potential of mesenchymal stromal cells and MSC conditioned medium in Amyotrophic Lateral Sclerosis (ALS--in vitro evidence from primary motor neuron cultures, NSC-34 cells, astrocytes and microglia.

    Directory of Open Access Journals (Sweden)

    Hui Sun

    Full Text Available Administration of mesenchymal stromal cells (MSC improves functional outcome in the SOD1G93A mouse model of the degenerative motor neuron disorder amyotrophic lateral sclerosis (ALS as well as in models of other neurological disorders. We have now investigated the effect of the interaction between MSC and motor neurons (derived from both non-transgenic and mutant SOD1G93A transgenic mice, NSC-34 cells and glial cells (astrocytes, microglia (derived again from both non-transgenic and mutant SOD1G93A ALS transgenic mice in vitro. In primary motor neurons, NSC-34 cells and astrocytes, MSC conditioned medium (MSC CM attenuated staurosporine (STS - induced apoptosis in a concentration-dependent manner. Studying MSC CM-induced expression of neurotrophic factors in astrocytes and NSC-34 cells, we found that glial cell line-derived neurotrophic factor (GDNF and ciliary neurotrophic factor (CNTF gene expression in astrocytes were significantly enhanced by MSC CM, with differential responses of non-transgenic and mutant astrocytes. Expression of Vascular Endothelial Growth Factor (VEGF in NSC-34 cells was significantly upregulated upon MSC CM-treatment. MSC CM significantly reduced the expression of the cytokines TNFα and IL-6 and iNOS both in transgenic and non-transgenic astrocytes. Gene expression of the neuroprotective chemokine Fractalkine (CX3CL1 was also upregulated in mutant SOD1G93A transgenic astrocytes by MSC CM treatment. Correspondingly, MSC CM increased the respective receptor, CX3CR1, in mutant SOD1G93A transgenic microglia. Our data demonstrate that MSC modulate motor neuronal and glial response to apoptosis and inflammation. MSC therefore represent an interesting candidate for further preclinical and clinical evaluation in ALS.

  17. Oxidative Stress Induction of DJ-1 Protein in Reactive Astrocytes Scavenges Free Radicals and Reduces Cell Injury

    Directory of Open Access Journals (Sweden)

    Takashi Yanagida

    2009-01-01

    Full Text Available Astrocytes, one of the predominant types of glial cells, function as both supportive and metabolic cells for the brain. Under cerebral ischemia/reperfusion-induced oxidative conditions, astrocytes accumulate and activate in the ischemic region. DJ-1 has recently been shown to be a sensor of oxidative stress in living cells. However, the function of astrocytic DJ-1 is still unknown. In the present study, to clarify the effect of astrocytic DJ-1 protein under massive oxidative insult, we used a focal ischemic rat model that had been subjected to middle cerebral artery occlusion (MCAO and reperfusion. We then investigated changes in the distribution of DJ-1 in astrocytes, DJ-1 release from cultured astrocytes, and the effects of recombinant DJ-1 protein on hydrogen peroxide (H2O2-induced death in normal and DJ-1-knockdown SH-SY5Y cells and on in vitro scavenging of hydroxyl radicals (•OH by electron spin resonance spectrometry. At 24 h after 2-h MCAO and reperfusion, an infarct lesion was markedly observed using magnetic resonance imaging and 2,3,5-triphenyltetrazolium chloride staining. In addition, reactive astrocytes enhanced DJ-1 expression in the penumbral zone of the ischemic core and that DJ-1 protein was extracellularly released from astrocytes by H2O2 in in vitro primary cultures. Although DJ-1-knockdown SH-SY5Y cells were markedly vulnerable to oxidative stress, treatment with glutathione S-transferase-tagged recombinant human DJ-1 protein (GST-DJ-1 significantly inhibited H2O2-induced cell death. In addition, GST-DJ-1 protein directly scavenged •OH. These results suggest that oxidative stress induces the release of astrocytic DJ-1 protein, which may contribute to astrocyte-mediated neuroprotection.

  18. Blast shockwaves propagate Ca(2+) activity via purinergic astrocyte networks in human central nervous system cells.

    Science.gov (United States)

    Ravin, Rea; Blank, Paul S; Busse, Brad; Ravin, Nitay; Vira, Shaleen; Bezrukov, Ludmila; Waters, Hang; Guerrero-Cazares, Hugo; Quinones-Hinojosa, Alfredo; Lee, Philip R; Fields, R Douglas; Bezrukov, Sergey M; Zimmerberg, Joshua

    2016-01-01

    In a recent study of the pathophysiology of mild, blast-induced traumatic brain injury (bTBI) the exposure of dissociated, central nervous system (CNS) cells to simulated blast resulted in propagating waves of elevated intracellular Ca(2+). Here we show, in dissociated human CNS cultures, that these calcium waves primarily propagate through astrocyte-dependent, purinergic signaling pathways that are blocked by P2 antagonists. Human, compared to rat, astrocytes had an increased calcium response and prolonged calcium wave propagation kinetics, suggesting that in our model system rat CNS cells are less responsive to simulated blast. Furthermore, in response to simulated blast, human CNS cells have increased expressions of a reactive astrocyte marker, glial fibrillary acidic protein (GFAP) and a protease, matrix metallopeptidase 9 (MMP-9). The conjoint increased expression of GFAP and MMP-9 and a purinergic ATP (P2) receptor antagonist reduction in calcium response identifies both potential mechanisms for sustained changes in brain function following primary bTBI and therapeutic strategies targeting abnormal astrocyte activity. PMID:27162174

  19. Blast shockwaves propagate Ca2+ activity via purinergic astrocyte networks in human central nervous system cells

    Science.gov (United States)

    Ravin, Rea; Blank, Paul S.; Busse, Brad; Ravin, Nitay; Vira, Shaleen; Bezrukov, Ludmila; Waters, Hang; Guerrero-Cazares, Hugo; Quinones-Hinojosa, Alfredo; Lee, Philip R.; Fields, R. Douglas; Bezrukov, Sergey M.; Zimmerberg, Joshua

    2016-01-01

    In a recent study of the pathophysiology of mild, blast-induced traumatic brain injury (bTBI) the exposure of dissociated, central nervous system (CNS) cells to simulated blast resulted in propagating waves of elevated intracellular Ca2+. Here we show, in dissociated human CNS cultures, that these calcium waves primarily propagate through astrocyte-dependent, purinergic signaling pathways that are blocked by P2 antagonists. Human, compared to rat, astrocytes had an increased calcium response and prolonged calcium wave propagation kinetics, suggesting that in our model system rat CNS cells are less responsive to simulated blast. Furthermore, in response to simulated blast, human CNS cells have increased expressions of a reactive astrocyte marker, glial fibrillary acidic protein (GFAP) and a protease, matrix metallopeptidase 9 (MMP-9). The conjoint increased expression of GFAP and MMP-9 and a purinergic ATP (P2) receptor antagonist reduction in calcium response identifies both potential mechanisms for sustained changes in brain function following primary bTBI and therapeutic strategies targeting abnormal astrocyte activity. PMID:27162174

  20. Astrocytes Upregulate Survival Genes in Tumor Cells and Induce Protection from Chemotherapy

    Directory of Open Access Journals (Sweden)

    Sun-Jin Kim

    2011-03-01

    Full Text Available In the United States, more than 40% of cancer patients develop brain metastasis. The median survival for untreated patients is 1 to 2 months, which may be extended to 6 months with conventional radiotherapy and chemotherapy. The growth and survival of metastasis depend on the interaction of tumor cells with host factors in the organ microenvironment. Brain metastases are surrounded and infiltrated by activated astrocytes and are highly resistant to chemotherapy. We report here that coculture of human breast cancer cells or lung cancer cells with murine astrocytes (but not murine fibroblasts led to the up-regulation of survival genes, including GSTA5, BCL2L1, and TWIST1, in the tumor cells. The degree of up-regulation directly correlated with increased resistance to all tested chemotherapeutic agents. We further show that the up-regulation of the survival genes and consequent resistance are dependent on the direct contact between the astrocytes and tumor cells through gap junctions and are therefore transient. Knocking down these genes with specific small interfering RNA rendered the tumor cells sensitive to chemotherapeutic agents. These data clearly demonstrate that host cells in the microenvironment influence the biologic behavior of tumor cells and reinforce the contention that the organ microenvironment must be taken into consideration during the design of therapy.

  1. L-type voltage-operated calcium channels contribute to astrocyte activation In vitro.

    Science.gov (United States)

    Cheli, Veronica T; Santiago González, Diara A; Smith, Jessica; Spreuer, Vilma; Murphy, Geoffrey G; Paez, Pablo M

    2016-08-01

    We have found a significant upregulation of L-type voltage-operated Ca(++) channels (VOCCs) in reactive astrocytes. To test if VOCCs are centrally involved in triggering astrocyte reactivity, we used in vitro models of astrocyte activation in combination with pharmacological inhibitors, siRNAs and the Cre/lox system to reduce the activity of L-type VOCCs in primary cortical astrocytes. The endotoxin lipopolysaccharide (LPS) as well as high extracellular K(+) , glutamate, and ATP promote astrogliosis in vitro. L-type VOCC inhibitors drastically reduce the number of reactive cells, astrocyte hypertrophy, and cell proliferation after these treatments. Astrocytes transfected with siRNAs for the Cav1.2 subunit that conducts L-type Ca(++) currents as well as Cav1.2 knockout astrocytes showed reduce Ca(++) influx by ∼80% after plasma membrane depolarization. Importantly, Cav1.2 knock-down/out prevents astrocyte activation and proliferation induced by LPS. Similar results were found using the scratch wound assay. After injuring the astrocyte monolayer, cells extend processes toward the cell-free scratch region and subsequently migrate and populate the scratch. We found a significant increase in the activity of L-type VOCCs in reactive astrocytes located in the growing line in comparison to quiescent astrocytes situated away from the scratch. Moreover, the migration of astrocytes from the scratching line as well as the number of proliferating astrocytes was reduced in Cav1.2 knock-down/out cultures. In summary, our results suggest that Cav1.2 L-type VOCCs play a fundamental role in the induction and/or proliferation of reactive astrocytes, and indicate that the inhibition of these Ca(++) channels may be an effective way to prevent astrocyte activation. GLIA 2016. GLIA 2016;64:1396-1415. PMID:27247164

  2. Human iPS cell-derived astrocyte transplants preserve respiratory function after spinal cord injury.

    Science.gov (United States)

    Li, Ke; Javed, Elham; Scura, Daniel; Hala, Tamara J; Seetharam, Suneil; Falnikar, Aditi; Richard, Jean-Philippe; Chorath, Ashley; Maragakis, Nicholas J; Wright, Megan C; Lepore, Angelo C

    2015-09-01

    Transplantation-based replacement of lost and/or dysfunctional astrocytes is a promising therapy for spinal cord injury (SCI) that has not been extensively explored, despite the integral roles played by astrocytes in the central nervous system (CNS). Induced pluripotent stem (iPS) cells are a clinically-relevant source of pluripotent cells that both avoid ethical issues of embryonic stem cells and allow for homogeneous derivation of mature cell types in large quantities, potentially in an autologous fashion. Despite their promise, the iPS cell field is in its infancy with respect to evaluating in vivo graft integration and therapeutic efficacy in SCI models. Astrocytes express the major glutamate transporter, GLT1, which is responsible for the vast majority of glutamate uptake in spinal cord. Following SCI, compromised GLT1 expression/function can increase susceptibility to excitotoxicity. We therefore evaluated intraspinal transplantation of human iPS cell-derived astrocytes (hIPSAs) following cervical contusion SCI as a novel strategy for reconstituting GLT1 expression and for protecting diaphragmatic respiratory neural circuitry. Transplant-derived cells showed robust long-term survival post-injection and efficiently differentiated into astrocytes in injured spinal cord of both immunesuppressed mice and rats. However, the majority of transplant-derived astrocytes did not express high levels of GLT1, particularly at early times post-injection. To enhance their ability to modulate extracellular glutamate levels, we engineered hIPSAs with lentivirus to constitutively express GLT1. Overexpression significantly increased GLT1 protein and functional GLT1-mediated glutamate uptake levels in hIPSAs both in vitro and in vivo post-transplantation. Compared to human fibroblast control and unmodified hIPSA transplantation, GLT1-overexpressing hIPSAs reduced (1) lesion size within the injured cervical spinal cord, (2) morphological denervation by respiratory phrenic motor

  3. The speed of swelling kinetics modulates cell volume regulation and calcium signaling in astrocytes: A different point of view on the role of aquaporins.

    Science.gov (United States)

    Mola, Maria Grazia; Sparaneo, Angelo; Gargano, Concetta Domenica; Spray, David C; Svelto, Maria; Frigeri, Antonio; Scemes, Eliana; Nicchia, Grazia Paola

    2016-01-01

    Regulatory volume decrease (RVD) is a process by which cells restore their original volume in response to swelling. In this study, we have focused on the role played by two different Aquaporins (AQPs), Aquaporin-4 (AQP4), and Aquaporin-1 (AQP1), in triggering RVD and in mediating calcium signaling in astrocytes under hypotonic stimulus. Using biophysical techniques to measure water flux through the plasma membrane of wild-type (WT) and AQP4 knockout (KO) astrocytes and of an astrocyte cell line (DI TNC1) transfected with AQP4 or AQP1, we here show that AQP-mediated fast swelling kinetics play a key role in triggering and accelerating RVD. Using calcium imaging, we show that AQP-mediated fast swelling kinetics also significantly increases the amplitude of calcium transients inhibited by Gadolinium and Ruthenium Red, two inhibitors of the transient receptor potential vanilloid 4 (TRPV4) channels, and prevented by removing extracellular calcium. Finally, inhibition of TRPV4 or removal of extracellular calcium does not affect RVD. All together our study provides evidence that (1) AQP influenced swelling kinetics is the main trigger for RVD and in mediating calcium signaling after hypotonic stimulus together with TRPV4, and (2) calcium influx from the extracellular space and/or TRPV4 are not essential for RVD to occur in astrocytes.

  4. The speed of swelling kinetics modulates cell volume regulation and calcium signaling in astrocytes: A different point of view on the role of aquaporins.

    Science.gov (United States)

    Mola, Maria Grazia; Sparaneo, Angelo; Gargano, Concetta Domenica; Spray, David C; Svelto, Maria; Frigeri, Antonio; Scemes, Eliana; Nicchia, Grazia Paola

    2016-01-01

    Regulatory volume decrease (RVD) is a process by which cells restore their original volume in response to swelling. In this study, we have focused on the role played by two different Aquaporins (AQPs), Aquaporin-4 (AQP4), and Aquaporin-1 (AQP1), in triggering RVD and in mediating calcium signaling in astrocytes under hypotonic stimulus. Using biophysical techniques to measure water flux through the plasma membrane of wild-type (WT) and AQP4 knockout (KO) astrocytes and of an astrocyte cell line (DI TNC1) transfected with AQP4 or AQP1, we here show that AQP-mediated fast swelling kinetics play a key role in triggering and accelerating RVD. Using calcium imaging, we show that AQP-mediated fast swelling kinetics also significantly increases the amplitude of calcium transients inhibited by Gadolinium and Ruthenium Red, two inhibitors of the transient receptor potential vanilloid 4 (TRPV4) channels, and prevented by removing extracellular calcium. Finally, inhibition of TRPV4 or removal of extracellular calcium does not affect RVD. All together our study provides evidence that (1) AQP influenced swelling kinetics is the main trigger for RVD and in mediating calcium signaling after hypotonic stimulus together with TRPV4, and (2) calcium influx from the extracellular space and/or TRPV4 are not essential for RVD to occur in astrocytes. PMID:26413835

  5. Extracellular Microvesicles from Astrocytes Contain Functional Glutamate Transporters: Regulation by Protein Kinase C and Cell Activation

    Directory of Open Access Journals (Sweden)

    Romain-Daniel eGosselin

    2013-12-01

    Full Text Available Glutamate transport through astrocytic excitatory amino-acid transporters (EAAT-1 and EAAT-2 is paramount for neural homeostasis. EAAT-1 has been reported in secreted extracellular microvesicles (eMV, such as exosomes and because the Protein Kinase C (PKC family controls the sub-cellular distribution of EAATs, we have explored whether PKCs drive EAATs into eMV. Using rat primary astrocytes, confocal immunofluorescence and ultracentrifugation on sucrose gradient we here report that PKC activation by phorbol myristate acetate (PMA reorganizes EAAT-1 distribution and reduces functional [3H]-aspartate reuptake. Western-blots show that EAAT-1 is present in eMV from astrocyte conditioned medium, together with NaK ATPase and glutamine synthetase all being further increased after PMA treatment. However, nanoparticle tracking analysis reveals that PKC activation did not change particle concentration. Functional analysis indicates that eMV have the capacity to reuptake [3H]-aspartate. In vivo, we demonstrate that spinal astrocytic reaction induced by peripheral nerve lesion (spared nerve injury, SNI is associated with a phosphorylation of PKC δ together with a shift of EAAT distribution ipsilaterally. Ex vivo, spinal explants from SNI rats release eMV with an increased content of NaK ATPase, EAAT-1 and EAAT-2. These data indicate PKC and cell activation as important regulators of EAAT-1 incorporation in eMV, and raise the possibility that microvesicular EAAT-1 may exert extracellular functions. Beyond a putative role in neuropathic pain, this phenomenon may be important for understanding neural homeostasis and a wide range of neurological diseases associated with astrocytic reaction as well as non-neurological diseases linked to eMV release.

  6. Endothelial Cells and Astrocytes: A Concerto en Duo in Ischemic Pathophysiology

    Directory of Open Access Journals (Sweden)

    Vincent Berezowski

    2012-01-01

    Full Text Available The neurovascular/gliovascular unit has recently gained increased attention in cerebral ischemic research, especially regarding the cellular and molecular changes that occur in astrocytes and endothelial cells. In this paper we summarize the recent knowledge of these changes in association with edema formation, interactions with the basal lamina, and blood-brain barrier dysfunctions. We also review the involvement of astrocytes and endothelial cells with recombinant tissue plasminogen activator, which is the only FDA-approved thrombolytic drug after stroke. However, it has a narrow therapeutic time window and serious clinical side effects. Lastly, we provide alternative therapeutic targets for future ischemia drug developments such as peroxisome proliferator- activated receptors and inhibitors of the c-Jun N-terminal kinase pathway. Targeting the neurovascular unit to protect the blood-brain barrier instead of a classical neuron-centric approach in the development of neuroprotective drugs may result in improved clinical outcomes after stroke.

  7. RNA Localization in Astrocytes

    DEFF Research Database (Denmark)

    Thomsen, Rune

    2012-01-01

    of 18S ribosomal RNA and the Rab13, Pkp4, Ankrd25, and Inpp1 mRNAs in astrocyte protrusions. The Boyden chamber isolated RNA from both primary astrocytes and C8S cells was analyzed by next generation sequencing (NGS), which revealed that >250 polyadenylated (polyA) RNA species accumulated in the cell...

  8. Activated astrocytes enhance the dopaminergic differentiation of stem cells and promote brain repair through bFGF.

    Science.gov (United States)

    Yang, Fan; Liu, Yunhui; Tu, Jie; Wan, Jun; Zhang, Jie; Wu, Bifeng; Chen, Shanping; Zhou, Jiawei; Mu, Yangling; Wang, Liping

    2014-12-17

    Astrocytes provide neuroprotective effects against degeneration of dopaminergic (DA) neurons and play a fundamental role in DA differentiation of neural stem cells. Here we show that light illumination of astrocytes expressing engineered channelrhodopsin variant (ChETA) can remarkably enhance the release of basic fibroblast growth factor (bFGF) and significantly promote the DA differentiation of human embryonic stem cells (hESCs) in vitro. Light activation of transplanted astrocytes in the substantia nigra (SN) also upregulates bFGF levels in vivo and promotes the regenerative effects of co-transplanted stem cells. Importantly, upregulation of bFGF levels, by specific light activation of endogenous astrocytes in the SN, enhances the DA differentiation of transplanted stem cells and promotes brain repair in a mouse model of Parkinson's disease (PD). Our study indicates that astrocyte-derived bFGF is required for regulation of DA differentiation of the stem cells and may provide a strategy targeting astrocytes for treatment of PD.

  9. Generation of reactive astrocytes from NG2 cells is regulated by sonic hedgehog.

    Science.gov (United States)

    Honsa, Pavel; Valny, Martin; Kriska, Jan; Matuskova, Hana; Harantova, Lenka; Kirdajova, Denisa; Valihrach, Lukas; Androvic, Peter; Kubista, Mikael; Anderova, Miroslava

    2016-09-01

    NG2 cells, a fourth glial cell type in the adult mammalian central nervous system, produce oligodendrocytes in the healthy nervous tissue, and display wide differentiation potential under pathological conditions, where they could give rise to reactive astrocytes. The factors that control the differentiation of NG2 cells after focal cerebral ischemia (FCI) are largely unknown. Here, we used transgenic Cspg4-cre/Esr1/ROSA26Sortm14(CAG-tdTomato) mice, in which tamoxifen administration triggers the expression of red fluorescent protein (tomato) specifically in NG2 cells and cells derived therefrom. Differentiation potential (in vitro and in vivo) of tomato-positive NG2 cells from control or postischemic brains was determined using the immunohistochemistry, single cell RT-qPCR and patch-clamp method. The ischemic injury was induced by middle cerebral artery occlusion, a model of FCI. Using genetic fate-mapping method, we identified sonic hedgehog (Shh) as an important factor that influences differentiation of NG2 cells into astrocytes in vitro. We also manipulated Shh signaling in the adult mouse brain after FCI. Shh signaling activation significantly increased the number of astrocytes derived from NG2 cells in the glial scar around the ischemic lesion, while Shh signaling inhibition caused the opposite effect. Since Shh signaling modifications did not change the proliferation rate of NG2 cells, we can conclude that Shh has a direct influence on the differentiation of NG2 cells and therefore, on the formation and composition of a glial scar, which consequently affects the degree of the brain damage. GLIA 2016;64:1518-1531. PMID:27340757

  10. The development of blood-retinal barrier during the interaction of astrocytes with vascular wall cells

    Institute of Scientific and Technical Information of China (English)

    Huanling Yao; Tianshi Wang; Jiexin Deng; Ding Liu; Xiaofei Li; Jinbo Deng

    2014-01-01

    Astrocytes are intimately involved in the formation and development of retinal vessels. Astrocyte dysfunction is a major cause of blood-retinal barrier injury and other retinal vascular diseases. In this study, the development of the retinal vascular system and the formation of the blood-ret-inal barrier in mice were investigated using immunolfuorescence staining, gelatin-ink perfusion, and transmission electron microscopy. The results showed that the retinal vascular system of mice develops from the optic disc after birth, and radiates out gradually to cover the entire retina, taking the papilla optica as the center. First, the superifcial vasculature is formed on the inner retinal layer;then, the vasculature extends into the inner and outer edges of the retinal inner nuclear layer, forming the deep vasculature that is parallel to the superifcial vasculature. The blood-retinal barrier is mainly composed of endothelium, basal lamina and the end-feet of astrocytes, which become mature during mouse development. Initially, the naive endothelial cells were immature with few organelles and many microvilli. The basal lamina was uniform in thickness, and the glial end-feet surrounded the outer basal lamina incompletely. In the end, the blood-retinal barrier matures with smooth endothelia connected through tight junctions, rela-tively thin and even basal lamina, and relatively thin glial cell end-feet. These ifndings indicate that the development of the vasculature in the retina follows the rules of“center to periphery”and“superifcial layer to deep layers”. Its development and maturation are spatially and tempo-rally consistent with the functional performance of retinal neurons and photosensitivity. The blood-retinal barrier gradually becomes mature via the process of interactions between astro-cytes and blood vessel cells.

  11. Brucella abortus Induces the Secretion of Proinflammatory Mediators from Glial Cells Leading to Astrocyte Apoptosis

    Science.gov (United States)

    García Samartino, Clara; Delpino, M. Victoria; Pott Godoy, Clara; Di Genaro, María Silvia; Pasquevich, Karina A.; Zwerdling, Astrid; Barrionuevo, Paula; Mathieu, Patricia; Cassataro, Juliana; Pitossi, Fernando; Giambartolomei, Guillermo H.

    2010-01-01

    Central nervous system (CNS) invasion by bacteria of the genus Brucella results in an inflammatory disorder called neurobrucellosis. In this study we present in vivo and in vitro evidence that B. abortus and its lipoproteins activate the innate immunity of the CNS, eliciting an inflammatory response that leads to astrogliosis, a characteristic feature of neurobrucellosis. Intracranial injection of heat-killed B. abortus (HKBA) or outer membrane protein 19 (Omp19), a B. abortus lipoprotein model, induced astrogliosis in mouse striatum. Moreover, infection of astrocytes and microglia with B. abortus induced the secretion of interleukin (IL)−6, IL-1β, tumor necrosis factor (TNF)-α, macrophage chemoattractant protein−1, and KC (CXCL1). HKBA also induced these inflammatory mediators, suggesting the involvement of a structural component of the bacterium. Accordingly, Omp19 induced the same cytokine and chemokine secretion pattern. B. abortus infection induced astrocyte, but not microglia, apoptosis. Indeed, HKBA and Omp19 elicited not only astrocyte apoptosis but also proliferation, two features observed during astrogliosis. Apoptosis induced by HKBA and L-Omp19 was completely suppressed in cells of TNF receptor p55−/− mice or when the general caspase inhibitor Z-VAD-FMK was added to cultures. Hence, TNF-α signaling via TNF receptor (TNFR) 1 through the coupling of caspases determines apoptosis. Our results provide proof of the principle that Brucella lipoproteins could be key virulence factors in neurobrucellosis and that astrogliosis might contribute to neurobrucellosis pathogenesis. PMID:20093491

  12. Targeting astrocytes in major depression

    OpenAIRE

    Verkhratsky, Alexej; Peng, Liang; Gu, Li; Li, Baoman

    2015-01-01

    Astrocytes represent a highly heterogeneous population of neural cells primarily responsible for the homeostasis of the central nervous system. Astrocytes express multiple receptors for neurotransmitters, including the serotonin 5-HT2B receptors and interact with neurones at the synapse. Astroglia contribute to neurological diseases through homeostatic response, neuroprotection and reactivity. In major depression, astrocytes show signs of degeneration and are decreased in numbe...

  13. Curcumin alleviates oxidative stress and mitochondrial dysfunction in astrocytes.

    Science.gov (United States)

    Daverey, Amita; Agrawal, Sandeep K

    2016-10-01

    Oxidative stress plays a critical role in various neurodegenerative diseases, thus alleviating oxidative stress is a potential strategy for therapeutic intervention and/or prevention of neurodegenerative diseases. In the present study, alleviation of oxidative stress through curcumin is investigated in A172 (human glioblastoma cell line) and HA-sp (human astrocytes cell line derived from the spinal cord) astrocytes. H2O2 was used to induce oxidative stress in astrocytes (A172 and HA-sp). Data show that H2O2 induces activation of astrocytes in dose- and time-dependent manner as evident by increased expression of GFAP in A172 and HA-sp cells after 24 and 12h respectively. An upregulation of Prdx6 was also observed in A172 and HA-sp cells after 24h of H2O2 treatment as compared to untreated control. Our data also showed that curcumin inhibits oxidative stress-induced cytoskeleton disarrangement, and impedes the activation of astrocytes by inhibiting upregulation of GFAP, vimentin and Prdx6. In addition, we observed an inhibition of oxidative stress-induced inflammation, apoptosis and mitochondria fragmentation after curcumin treatment. Therefore, our results suggest that curcumin not only protects astrocytes from H2O2-induced oxidative stress but also reverses the mitochondrial damage and dysfunction induced by oxidative stress. This study also provides evidence for protective role of curcumin on astrocytes by showing its effects on attenuating reactive astrogliosis and inhibiting apoptosis. PMID:27423629

  14. Immune Privilege as an Intrinsic CNS Property: Astrocytes Protect the CNS against T-Cell-Mediated Neuroinflammation

    Directory of Open Access Journals (Sweden)

    Ulrike Gimsa

    2013-01-01

    Full Text Available Astrocytes have many functions in the central nervous system (CNS. They support differentiation and homeostasis of neurons and influence synaptic activity. They are responsible for formation of the blood-brain barrier (BBB and make up the glia limitans. Here, we review their contribution to neuroimmune interactions and in particular to those induced by the invasion of activated T cells. We discuss the mechanisms by which astrocytes regulate pro- and anti-inflammatory aspects of T-cell responses within the CNS. Depending on the microenvironment, they may become potent antigen-presenting cells for T cells and they may contribute to inflammatory processes. They are also able to abrogate or reprogram T-cell responses by inducing apoptosis or secreting inhibitory mediators. We consider apparently contradictory functions of astrocytes in health and disease, particularly in their interaction with lymphocytes, which may either aggravate or suppress neuroinflammation.

  15. Heterogeneity of Astrocytic Form and Function

    OpenAIRE

    Oberheim, Nancy Ann; Goldman, Steven A.; NEDERGAARD, Maiken

    2012-01-01

    Astrocytes participate in all essential CNS functions, including blood flow regulation, energy metabolism, ion and water homeostasis, immune defence, neurotransmission, and adult neurogenesis. It is thus not surprising that astrocytic morphology and function differ between regions, and that different subclasses of astrocytes exist within the same brain region. Recent lines of work also show that the complexity of protoplasmic astrocytes increases during evolution. Human astrocytes are structu...

  16. Astrocyte-Derived CCL2 is Associated with M1 Activation and Recruitment of Cultured Microglial Cells

    Directory of Open Access Journals (Sweden)

    Mingfeng He

    2016-02-01

    Full Text Available Background/Aims: Microglia are an essential player in central nervous system inflammation. Recent studies have demonstrated that the astrocytic chemokine, CCL2, is associated with microglial activation in vivo. However, CCL2-induced microglial activation has not yet been studied in vitro. The purpose of the current study was to understand the role of astrocyte-derived CCL2 in microglial activation and to elucidate the underlying mechanism(s. Methods: Primary astrocytes were pre-treated with CCL2 siRNA and stimulated with TNF-α. The culture medium (CM was collected and added to cultures of microglia, which were incubated with and without CCR2 inhibitor. Microglial cells were analyzed by quantitative RT-PCR to determine whether they polarized to the M1 or M2 state. Microglial migratory ability was assessed by transwell migration assay. Results: TNF-α stimulated the release of CCL2 from astrocytes, even if the culture media containing TNF-α was replaced with fresh media after 3 h. CM from TNF-α-stimulated astrocytes successfully induced microglial activation, which was ascertained by increased activation of M1 and enhanced migration ability. In contrast, CM from astrocytes pretreated with CCL2 siRNA showed no effect on microglial activation, compared to controls. Additionally, microglia pre-treated with RS102895, a CCR2 inhibitor, were resistant to activation by CM from TNF-α-stimulated astrocytes. Conclusion: This study demonstrates that the CCL2/CCR2 pathway of astrocyte-induced microglial activation is associated with M1 polarization and enhanced migration ability, indicating that this pathway could be a useful target to ameliorate inflammation in the central nervous system.

  17. Group B Streptococcus interactions with human meningeal cells and astrocytes in vitro.

    Directory of Open Access Journals (Sweden)

    Khalil Alkuwaity

    Full Text Available BACKGROUND: Streptococcus agalactiae (Group B Streptococcus, GBS is a leading cause of life-threatening neonatal meningitis and survivors often suffer permanent neurological damage. How this organism interacts with the meninges and subsequently with astrocytes that constitute the underlying cortical glia limitans superficialis is not known. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we demonstrate dose-dependent adherence of GBS over time to human meningioma cells and fetal astrocytes in vitro, which was not influenced by expression of either β-haemolysin/cytolysin (β-h/c toxin, different capsule serotypes or by absence of capsule (p>0.05. Internalization of GBS by both cell types was, however, a slow and an infrequent event (only 0.02-0.4% of associated bacteria were internalised by 9 h. Expression of β-h/c toxin did not play a role in invasion (p>0.05, whereas capsule expression lead to a reduction (p<0.05 in the numbers of intracellular bacteria recovered. GBS strains induced cytotoxicity as demonstrated by the measurement of lactate dehydrogenase (LDH enzyme release by 9 h and by viable staining. Increasing levels of meningioma cell death correlated with bacterial growth and the phenotype of β-h/c toxin production, i.e. from weakly, to normo- to hyper-haemolytic. However, cytotoxicity was significantly greater (p<0.05 towards astrocytes, and infection with initial MOI≥0.003 induced 70-100% LDH release. By comparing wild-type (β-h/c(+ and mutant (ΔcylE β-h/c(- strains and β-h/c toxin extracts and by using the surfactant dipalmitoylphosphatidylcholine in cytotoxicity inhibition experiments, β-h/c toxin was demonstrated as principally responsible for cell death. CONCLUSIONS/SIGNIFICANCE: This study has described key events in the interactions of GBS with meningeal cells and astrocytes in vitro and a major virulence role for β-h/c toxin. Understanding the mechanisms involved will help to identify potential therapies for improving

  18. The multifaceted responses of primary human astrocytes and brain microvascular endothelial cells to the Lyme disease spirochete, Borrelia burgdorferi

    Directory of Open Access Journals (Sweden)

    Catherine A. Brissette

    2013-08-01

    Full Text Available The vector-borne pathogen, Borrelia burgdorferi, causes a multi-system disorder including neurological complications. These neurological disorders, collectively termed neuroborreliosis, can occur in up to 15% of untreated patients. The neurological symptoms are probably a result of a glial-driven, host inflammatory response to the bacterium. However, the specific contributions of individual glial and other support cell types to the pathogenesis of neuroborreliosis are relatively unexplored. The goal of this project was to characterize specific astrocyte and endothelial cell responses to B. burgdorferi. Primary human astrocytes and primary HBMEC (human brain microvascular endothelial cells were incubated with B. burgdorferi over a 72-h period and the transcriptional responses to the bacterium were analyzed by real-time PCR arrays. There was a robust increase in several surveyed chemokine and related genes, including IL (interleukin-8, for both primary astrocytes and HBMEC. Array results were confirmed with individual sets of PCR primers. The production of specific chemokines by both astrocytes and HBMEC in response to B. burgdorferi, including IL-8, CXCL-1, and CXCL-10, were confirmed by ELISA. These results demonstrate that primary astrocytes and HBMEC respond to virulent B. burgdorferi by producing a number of chemokines. These data suggest that infiltrating phagocytic cells, particularly neutrophils, attracted by chemokines expressed at the BBB (blood–brain barrier may be important contributors to the early inflammatory events associated with neuroborreliosis.

  19. Role of astrocytic leptin receptor subtypes on leptin permeation across hCMEC/D3 human brain endothelial cells

    OpenAIRE

    Hsuchou, Hung; Kastin, Abba J; Tu, Hong; Abbott, N Joan; Couraud, Pierre-Olivier; Pan, Weihong

    2010-01-01

    Astrocytic leptin receptors (ObR) can be upregulated in conditions such as adult-onset obesity. To determine whether the levels and subtypes of astrocytic ObR modulate leptin transport, we co-cultured hCMEC/D3 human brain endothelial cells and C6 astrocytoma cells in the Transwell system, and tested leptin permeation from apical to basolateral chambers. In comparison with hCMEC alone, co-culture of C6 cells reduced the permeability of paracellular markers and leptin. Unexpectedly, ObRb overex...

  20. Light and electron microscopic localization of GABAA-receptors on cultured cerebellar granule cells and astrocytes using immunohistochemical techniques

    DEFF Research Database (Denmark)

    Hansen, G H; Hösli, E; Belhage, B;

    1991-01-01

    GABAA-receptors were localized in explant cultures of rat cerebellum and in dissociated primary cultures of rat cerebellar granule cells and rat cerebellar astrocytes using the monoclonal antibody bd-17 directed against the beta-subunit of the GABAA/benzodiazepine/chloride channel complex...... of GABAA-receptors was observed in the plasma membrane of both the cell bodies and processes in dissociated primary cultures of cerebellar granule cells using an indirect preembedding immunogold staining technique which in contrast to the classical PAP technique allows quantitative estimations...... in dissociated primary cultures of cerebellar astrocytes....

  1. Generation of primary cultures of bovine brain endothelial cells and setup of cocultures with rat astrocytes

    DEFF Research Database (Denmark)

    Helms, Hans C; Brodin, Birger

    2014-01-01

    In vitro models of the blood-brain barrier are useful tools to study blood-brain barrier function as well as drug permeation from the systemic circulation to the brain parenchyma. However, a large number of the available in vitro models fail to reflect the tightness of the in vivo blood-brain...... barrier. The present protocol describes the setup of an in vitro coculture model based on primary cultures of endothelial cells from bovine brain microvessels and primary cultures of rat astrocytes. The model displays a high electrical tightness and expresses blood-brain barrier marker proteins....

  2. A hit and run approach to inducible direct reprogramming of astrocytes to neural stem cells

    Directory of Open Access Journals (Sweden)

    Maria ePoulou

    2016-04-01

    Full Text Available Temporal and spatial control of gene expression can be achieved using an inducible system as a fundamental tool for regulated transcription in basic, applied and eventually in clinical research. We describe a novel hit and run inducible direct reprogramming approach. In a single step, two days post-transfection, transiently transfected Sox2FLAG under the Leu3p-αIPM inducible control (iSox2 triggers the activation of endogenous Sox2, redirecting primary astrocytes into abundant distinct nestin-positive radial glia cells. This technique introduces a unique novel tool for safe, rapid and efficient reprogramming amendable to regenerative medicine.

  3. Acceleration of astrocytic differentiation in neural stem cells surviving X-irradiation.

    Science.gov (United States)

    Ozeki, Ayumi; Suzuki, Keiji; Suzuki, Masatoshi; Ozawa, Hiroki; Yamashita, Shunichi

    2012-03-28

    Neural stem cells (NSCs) are highly susceptible to DNA double-strand breaks; however, little is known about the effects of radiation in cells surviving radiation. Although the nestin-positive NSCs predominantly became glial fibrillary acidic protein (GFAP)-positive in differentiation-permissive medium, little or no cells were GFAP positive in proliferation-permissive medium. We found that more than half of the cells surviving X-rays became GFAP positive in proliferation-permissive medium. Moreover, localized irradiation stimulated differentiation of cells outside the irradiated area. These results indicate for the first time that ionizing radiation is able to stimulate astrocyte-specific differentiation of surviving NSCs, whose process is mediated both by the direct activation of nuclear factor-κB and by the indirect bystander effect induced by X-irradiation.

  4. Insulin Promotes Glycogen Storage and Cell Proliferation in Primary Human Astrocytes

    OpenAIRE

    Martin Heni; Hennige, Anita M.; Andreas Peter; Dorothea Siegel-Axel; Anna-Maria Ordelheide; Norbert Krebs; Fausto Machicao; Andreas Fritsche; Hans-Ulrich Häring; Harald Staiger

    2011-01-01

    INTRODUCTION: In the human brain, there are at least as many astrocytes as neurons. Astrocytes are known to modulate neuronal function in several ways. Thus, they may also contribute to cerebral insulin actions. Therefore, we examined whether primary human astrocytes are insulin-responsive and whether their metabolic functions are affected by the hormone. METHODS: Commercially available Normal Human Astrocytes were grown in the recommended medium. Major players in the insulin signaling pathwa...

  5. The balance of Id3 and E47 determines neural stem/precursor cell differentiation into astrocytes.

    Science.gov (United States)

    Bohrer, Christian; Pfurr, Sabrina; Mammadzada, Könül; Schildge, Sebastian; Plappert, Leandra; Hils, Miriam; Pous, Lauriane; Rauch, Katharina S; Dumit, Verónica I; Pfeifer, Dietmar; Dengjel, Jörn; Kirsch, Matthias; Schachtrup, Kristina; Schachtrup, Christian

    2015-11-12

    Adult neural stem/precursor cells (NSPCs) of the subventricular zone (SVZ) are an endogenous source for neuronal replacement in CNS disease. However, adult neurogenesis is compromised after brain injury in favor of a glial cell fate, which is mainly attributed to changes in the NSPC environment. Yet, it is unknown how this unfavorable extracellular environment translates into a transcriptional program altering NSPC differentiation. Here, we show that genetic depletion of the transcriptional regulator Id3 decreased the number of astrocytes generated from SVZ-derived adult NSPCs in the cortical lesion area after traumatic brain injury. Cortical brain injury resulted in rapid BMP-2 and Id3 up-regulation in the SVZ stem cell niche. Id3(-/-) adult NSPCs failed to differentiate into BMP-2-induced astrocytes, while NSPCs deficient for the Id3-controlled transcription factor E47 readily differentiated into astrocytes in the absence of BMP-2. Mechanistically, E47 repressed the expression of several astrocyte-specific genes in adult NSPCs. These results identify Id3 as the BMP-2-induced transcriptional regulator, promoting adult NSPC differentiation into astrocytes upon CNS injury and reveal a molecular link between environmental changes and NSPC differentiation in the CNS after injury. PMID:26438726

  6. Microglia-derived interleukin-6 and leukaemia inhibitory factor promote astrocytic differentiation of neural stem/progenitor cells.

    Science.gov (United States)

    Nakanishi, Masaya; Niidome, Tetsuhiro; Matsuda, Satoru; Akaike, Akinori; Kihara, Takeshi; Sugimoto, Hachiro

    2007-02-01

    Neural stem/progenitor cells (NSPCs) proliferate and differentiate depending on their intrinsic properties and local environment. It has been recognized that astrocytes promote neurogenic differentiation of NSPCs, suggesting the importance of cell-cell interactions between glial cells and NSPCs. Recent studies have demonstrated that microglia, one type of glial cells, play an important role in neurogenesis. However, little is known about how activated microglia control the proliferation and differentiation of NSPCs. In this study, we investigated the possibility that microglia-derived soluble factors regulate the behaviour of NSPCs. To this end, NSPCs and microglial cultures were obtained from rat embryonic day 16 subventricular zone (SVZ) and rat postnatal 1 day cortex, respectively, and the conditioned medium from microglia was prepared. Microglial-conditioned medium had no significant effect on the proliferation of NSPCs. In contrast, it increased the percentage of cells positive for a marker of astrocytes, glial fibrillary acidic protein (GFAP) during differentiation. The induction of astrocytic differentiation by microglial-conditioned medium was reduced by the inhibition of the Janus kinase/signal transducer and activation of transcription (JAK/STAT) and mitogen-activated protein kinase (MAPK) pathways. Furthermore, microglia-derived interleukin (IL)-6 and leukaemia inhibitory factor (LIF) were identified as essential molecules for this astrocytic differentiation using neutralizing antibodies and recombinant cytokines. Our results suggest that microglia as well as astrocytes contribute to the integrity of the local environment of NSPCs, and at least IL-6 and LIF released by activated microglia promote astrocytic differentiation of NSPCs via the activation of the JAK/STAT and MAPK pathways.

  7. A human astrocytoma cell line is highly susceptible to infection with Trypanosoma cruzi

    Directory of Open Access Journals (Sweden)

    Juan Camilo Vargas-Zambrano

    2013-04-01

    Full Text Available Astrocytes play a vital role in neuronal protection, homeostasis, vascular interchange and the local immune response. Some viruses and parasites can cross the blood-brain barrier and infect glia. Trypanosoma cruzi, the aetiological agent of Chagas disease, can seriously compromise the central nervous system, mainly in immune-suppressed individuals, but also during the acute phase of the infection. In this report, the infective capacity of T. cruzi in a human astrocyte tumour-derived cell line was studied. Astrocytes exposed to trypomastigotes (1:10 ratio produced intracellular amastigotes and new trypomastigotes emerged by day 4 post-infection (p.i.. At day 6 p.i., 93% of the cells were infected. Using flow cytometry, changes were observed in both the expression of major histocompatibility complex class I and II molecules and the chemokine secretion pattern of astrocytes exposed to the parasite. Blocking the low-density lipoprotein receptor on astrocytes did not reduce parasite intracellular infection. Thus, T. cruzi can infect astrocytes and modulate the immune response during central nervous system infection.

  8. Extracellular matrix of cultured glial cells: Selective expression of chondroitin 4-sulfate by type-2 astrocytes and their progenitors

    International Nuclear Information System (INIS)

    We have studied the extracellular matrix composition of cultured glial cells by immunocytochemistry with different monoclonal and polyclonal antibodies. Double immunofluorescence experiments and metabolic labeling with [3H]glucosamine performed in different types of cerebellar and cortical cultures showed that bipotential progenitors for type-2 astrocytes and for oligodendrocytes synthesize chondroitin sulfate (CS) and deposit this proteoglycan in their extracellular matrix. The distribution of the various [3H]glucosamine-labeled glycosaminoglycans between the intracellular and the extracellular space was different. CS was present both within the cells and in the culture medium, although in different amounts. Bi-potential progenitors became also O4-positive during their development in vitro. At the stage of O4-positivity they were still stained with antibodies against CS. However, when the progenitor cells were maintained in serum-free medium and differentiated into Gal-C-positive oligodendrocytes, they became CS-negative. In the presence of fetal calf serum in the culture medium, the bipotential progenitors differentiated into GFAP-positive type-2 astrocytes. These cells still expressed CS: their Golgi area and their surface were stained with anti-CS antibodies. Staining with monoclonal antibodies specific for different types of CS (4-sulfate, 6-sulfate, and unsulfated) revealed that both bipotential progenitors and type-2 astrocytes synthesized only chondroitin 4-sulfate. Type-1 astrocytes were negative for both the polyclonal and the monoclonal anti-CS antibodies. Finally, type-2 astrocytes and their progenitors were weakly stained with anti-laminin antibodies and unstained with anti-fibronectin. Type-1 astrocytes were positive for both anti-laminin and anti-fibronectin antibodies and appeared to secrete fibronectin in the extracellular space

  9. Radiosensitivity of mesothelioma cell lines

    International Nuclear Information System (INIS)

    The present study was carried out in order to examine the radiosensitivity of malignant pleural mesothelioma cell lines. Cell kinetics, radiation-induced delay of the cell cycle and DNA ploidy of the cell lines were also determined. For comparison an HeLa and a human foetal fibroblast cell line were simultaneously explored. Six previously cytogenetically and histologically characterized mesothelioma tumor cell lines were applied. A rapid tiazolyl blue microtiter (MTT) assay was used to analyze radiosensitivity and cell kinetics and DNA ploidy of the cultured cells were determined by flow cytometry. The survival fraction after a dose of 2 Gy (SF2), parameters α and β of the linear quadratic model (LQ-model) and mean inactivation dose (DMID) were also estimated. The DNA index of four cell lines equaled 1.0 and two cell lines equaled 1.5 and 1.6. Different mesothelioma cell lines showed a great variation in radiosensitivity. Mean survival fraction after a radiation dose of 2 Gy (SF2) was 0.60 and ranged from 0.36 to 0.81 and mean α value was 0.26 (range 0.48-0.083). The SF2 of the most sensitive diploid mesothelioma cell line was 0.36: Less than that of the foetal fibroblast cell line (0.49). The survival fractions (0.81 and 0.74) of the two most resistant cell lines, which also were aneuploid, were equal to that of the HeLa cell line (0.78). The α/β ratios of the most sensitive cell lines were almost an order of magnitude greater than those of the two most resistant cell lines. Radiation-induced delay of the most resistant aneuploid cell line was similar to that of HeLa cells but in the most sensitive (diploid cells) there was practically no entry into the G1 phase following the 2 Gy radiation dose during 36 h. (orig.)

  10. CLO : The cell line ontology

    NARCIS (Netherlands)

    Sarntivijai, Sirarat; Lin, Yu; Xiang, Zuoshuang; Meehan, Terrence F.; Diehl, Alexander D.; Vempati, Uma D.; Schuerer, Stephan C.; Pang, Chao; Malone, James; Parkinson, Helen; Liu, Yue; Takatsuki, Terue; Saijo, Kaoru; Masuya, Hiroshi; Nakamura, Yukio; Brush, Matthew H.; Haendel, Melissa A.; Zheng, Jie; Stoeckert, Christian J.; Peters, Bjoern; Mungall, Christopher J.; Carey, Thomas E.; States, David J.; Athey, Brian D.; He, Yongqun

    2014-01-01

    Background: Cell lines have been widely used in biomedical research. The community-based Cell Line Ontology (CLO) is a member of the OBO Foundry library that covers the domain of cell lines. Since its publication two years ago, significant updates have been made, including new groups joining the CLO

  11. Astrocytes and Microglia as Non-cell Autonomous Players in the Pathogenesis of ALS

    Science.gov (United States)

    Hyeon, Seung Jae; Im, Hyeonjoo; Ryu, Hyun; Kim, Yunha

    2016-01-01

    Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder that leads to a progressive muscle wasting and paralysis. The pathological phenotypes are featured by severe motor neuron death and glial activation in the lumbar spinal cord. Proposed ALS pathogenic mechanisms include glutamate cytotoxicity, inflammatory pathway, oxidative stress, and protein aggregation. However, the exact mechanisms of ALS pathogenesis are not fully understood yet. Recently, a growing body of evidence provides a novel insight on the importance of glial cells in relation to the motor neuronal damage via the non-cell autonomous pathway. Accordingly, the aim of the current paper is to overview the role of astrocytes and microglia in the pathogenesis of ALS and to better understand the disease mechanism of ALS. PMID:27790057

  12. Impaired APP activity and altered Tau splicing in embryonic stem cell-derived astrocytes obtained from an APPsw transgenic minipig

    DEFF Research Database (Denmark)

    Hall, Vanessa Jane; Lindblad, Maiken Marie; Jakobsen, Jannik E.;

    2015-01-01

    analyze in vitro-produced stem cells and their derivatives from a large mammalian model of the disease created by overexpression of a single mutant human gene (APPsw). We produced hemizygous and homozygous radial glial-like cells following culture and differentiation of embryonic stem cells (ESCs......) isolated from embryos obtained from mated hemizygous minipigs. These cells were confirmed to co-express varying neural markers, including NES, GFAP and BLBP, typical of type one radial glial cells (RGs) from the subgranular zone. These cells had altered expression of CCND1 and NOTCH1 and decreased...... expression of several ribosomal RNA genes. We found that these cells were able to differentiate into astrocytes upon directed differentiation. The astrocytes produced had decreased α- and β-secretase activity, increased γ-secretase activity and altered splicing of tau. This indicates novel aspects of early...

  13. Astrocyte cultures derived from human brain tissue express angiotensinogen mRNA

    International Nuclear Information System (INIS)

    The authors have identified human cultured cell lines that are useful for studying angiotensinogen gene expression and its regulation in the central nervous system. A model cell system of human central nervous system origin expressing angiotensinogen has not previously been available. Expression of angiotensinogen mRNA appears to be a basal property of noninduced human astrocytes, since astrocytic cell lines derived from human glioblastomas or nonneoplastic human brain tissue invariably produced angiotensinogen mRNA. In situ hybridization histochemistry revealed that angiotensinogen mRNA production was not limited to a subpopulation of astrocytes because >99% of cells in these cultures contained angiotensinogen mRNA. These cell lines will be useful in studies of the molecular mechanisms controlling angiotensin synthesis and the role of biologically active angiotensin in the human brain by allowing the authors to examine regulation of expression of the renin-angiotensin system in human astrocyte cultures

  14. Astrocyte cultures derived from human brain tissue express angiotensinogen mRNA

    Energy Technology Data Exchange (ETDEWEB)

    Milsted, A.; Barna, B.P.; Ransohoff, R.M.; Brosnihan, K.B.; Ferrario, C.M. (Cleveland Clinic Foundation, OH (USA))

    1990-08-01

    The authors have identified human cultured cell lines that are useful for studying angiotensinogen gene expression and its regulation in the central nervous system. A model cell system of human central nervous system origin expressing angiotensinogen has not previously been available. Expression of angiotensinogen mRNA appears to be a basal property of noninduced human astrocytes, since astrocytic cell lines derived from human glioblastomas or nonneoplastic human brain tissue invariably produced angiotensinogen mRNA. In situ hybridization histochemistry revealed that angiotensinogen mRNA production was not limited to a subpopulation of astrocytes because >99% of cells in these cultures contained angiotensinogen mRNA. These cell lines will be useful in studies of the molecular mechanisms controlling angiotensin synthesis and the role of biologically active angiotensin in the human brain by allowing the authors to examine regulation of expression of the renin-angiotensin system in human astrocyte cultures.

  15. Common astrocytic programs during brain development, injury and cancer

    OpenAIRE

    Silver, Daniel J.; Steindler, Dennis A.

    2009-01-01

    In addition to radial glial cells of neurohistogenesis, immature astrocytes with stem-cell-like properties cordon off emerging functional patterns in the developing brain. Astrocytes also can be stem cells during adult neurogenesis, and a proposed potency of injury-associated reactive astrocytes has recently been substantiated. Astrocytic cells might additionally be involved in cancer stem cell-associated gliomagenesis. Thus, there are distinguishing roles for stem-cell-like astrocytes during...

  16. Multi-walled carbon nanotubes affect drug transport across cell membrane in rat astrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Chen Xiao [School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Hangkong Road 13, 430030, Wuhan (China); Schluesener, Hermann J, E-mail: mornsmile@yahoo.com [Institute of Brain Research, University of Tuebingen, Calwerstrasse 3, D-72076, Tuebingen (Germany)

    2010-03-12

    The impact of carbon nanotubes on the cell membrane is an aspect of particular importance and interest in the study of carbon nanotubes' interactions with living systems. One of the many functions of the cell membrane is to execute substance transport into and out of the cell. We investigated the influence of multi-walled carbon nanotubes (MWCNTs) on the transport of several compounds across in the cell membrane of rat astrocytes using flow cytometry. These compounds are fluorescein diacetate, carboxyfluorescein diacetate, rhodamine 123 and doxorubicin, which are prosubstrate/substrates of multidrug transporter proteins. Results showed that MWCNTs significantly inhibited cellular uptake of doxorubicin but not the other drugs and the mode of loading made a significant difference in doxorubicin uptake. Retention of fluorescein, carboxyfluorescein and rhodamine 123 was remarkably higher in MWCNT-exposed cells after an efflux period. A kinetics study also demonstrated slower efflux of intracellular fluorescein and rhodamine 123. Data presented in this paper suggest that MWCNTs could affect drug transport across cell membranes. The implications of the findings are discussed.

  17. Multi-walled carbon nanotubes affect drug transport across cell membrane in rat astrocytes

    Science.gov (United States)

    Chen, Xiao; Schluesener, Hermann J.

    2010-03-01

    The impact of carbon nanotubes on the cell membrane is an aspect of particular importance and interest in the study of carbon nanotubes' interactions with living systems. One of the many functions of the cell membrane is to execute substance transport into and out of the cell. We investigated the influence of multi-walled carbon nanotubes (MWCNTs) on the transport of several compounds across in the cell membrane of rat astrocytes using flow cytometry. These compounds are fluorescein diacetate, carboxyfluorescein diacetate, rhodamine 123 and doxorubicin, which are prosubstrate/substrates of multidrug transporter proteins. Results showed that MWCNTs significantly inhibited cellular uptake of doxorubicin but not the other drugs and the mode of loading made a significant difference in doxorubicin uptake. Retention of fluorescein, carboxyfluorescein and rhodamine 123 was remarkably higher in MWCNT-exposed cells after an efflux period. A kinetics study also demonstrated slower efflux of intracellular fluorescein and rhodamine 123. Data presented in this paper suggest that MWCNTs could affect drug transport across cell membranes. The implications of the findings are discussed.

  18. Multi-walled carbon nanotubes affect drug transport across cell membrane in rat astrocytes

    International Nuclear Information System (INIS)

    The impact of carbon nanotubes on the cell membrane is an aspect of particular importance and interest in the study of carbon nanotubes' interactions with living systems. One of the many functions of the cell membrane is to execute substance transport into and out of the cell. We investigated the influence of multi-walled carbon nanotubes (MWCNTs) on the transport of several compounds across in the cell membrane of rat astrocytes using flow cytometry. These compounds are fluorescein diacetate, carboxyfluorescein diacetate, rhodamine 123 and doxorubicin, which are prosubstrate/substrates of multidrug transporter proteins. Results showed that MWCNTs significantly inhibited cellular uptake of doxorubicin but not the other drugs and the mode of loading made a significant difference in doxorubicin uptake. Retention of fluorescein, carboxyfluorescein and rhodamine 123 was remarkably higher in MWCNT-exposed cells after an efflux period. A kinetics study also demonstrated slower efflux of intracellular fluorescein and rhodamine 123. Data presented in this paper suggest that MWCNTs could affect drug transport across cell membranes. The implications of the findings are discussed.

  19. Determining the Oxidation States of Manganese in NT2 Cells and Cultured Astrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Gunter,K.; Aschner, M.; Miller, L.; Eliseev, R.; Salter, J.; Andersen, K.; Gunter, T.

    2006-01-01

    Excessive brain manganese (Mn) can produce a syndrome called 'manganism', which correlates with loss of striatal dopamine and cell death in the striatum and globus pallidus. The prevalent hypothesis for the cause of this syndrome has been oxidation of cell components by the strong oxidizing agent, Mn{sup 3+}, either formed by oxidation of intracellular Mn{sup 2+} or transported into the cell as Mn{sup 3+}. We have recently used X-ray absorption near edge structure spectroscopy (XANES) to determine the oxidation states of manganese complexes in brain and liver mitochondria and in nerve growth factor (NGF)-induced and non-induced PC12 cells. No evidence was found for stabilization or accumulation of Mn{sup 3+} complexes because of oxidation of Mn{sup 2+} by reactive oxygen species in these tissues. Here we extend these studies of manganese oxidation state to cells of brain origin, human neuroteratocarcinoma (NT2) cells and primary cultures of rat astrocytes. Again we find no evidence for stabilization or accumulation of any Mn{sup 3+} complex derived from oxidation of Mn{sup 2+} under a range of conditions.

  20. Multilineage potential of stable human mesenchymal stem cell line derived from fetal marrow.

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    Atsushi Nagai

    Full Text Available Human bone marrow contains two major cell types, hematopoietic stem cells (HSCs and mesenchymal stem cells (MSCs. MSCs possess self-renewal capacity and pluripotency defined by their ability to differentiate into osteoblasts, chondrocytes, adipocytes and muscle cells. MSCs are also known to differentiate into neurons and glial cells in vitro, and in vivo following transplantation into the brain of animal models of neurological disorders including ischemia and intracerebral hemorrhage (ICH stroke. In order to obtain sufficient number and homogeneous population of human MSCs, we have clonally isolated permanent and stable human MSC lines by transfecting primary cell cultures of fetal human bone marrow MSCs with a retroviral vector encoding v-myc gene. One of the cell lines, HM3.B10 (B10, was found to differentiate into neural cell types including neural stem cells, neurons, astrocytes and oligodendrocytes in vitro as shown by expression of genetic markers for neural stem cells (nestin and Musashi1, neurons (neurofilament protein, synapsin and MAP2, astrocytes (glial fibrillary acidic protein, GFAP and oligodendrocytes (myelin basic protein, MBP as determined by RT-PCR assay. In addition, B10 cells were found to differentiate into neural cell types as shown by immunocytochical demonstration of nestin (for neural stem cells, neurofilament protein and beta-tubulin III (neurons GFAP (astrocytes, and galactocerebroside (oligodendrocytes. Following brain transplantation in mouse ICH stroke model, B10 human MSCs integrate into host brain, survive, differentiate into neurons and astrocytes and induce behavioral improvement in the ICH animals. B10 human MSC cell line is not only a useful tool for the studies of organogenesis and specifically for the neurogenesis, but also provides a valuable source of cells for cell therapy studies in animal models of stroke and other neurological disorders.

  1. Immunochemical expression of proliferative cell nuclear antigen in aging cultured astrocytes

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    M. C. Vanzani

    2003-08-01

    Full Text Available Cell differentiation degree and mitotic activity were sequentially assessed by immunoperoxidase labeling of glial fibrillary acidic protein (GFAP and proliferative cell nuclear antigen (PCNA, respectively, in rat brain cultured astrocytes maintained up to 60 days in vitro (DIV of first subculture, or weekly passaged until their 12th subculture. Cell count was performed through a 0.01 mm2 section reticule and morphometric analysis with a stereological grid. The number of double immunoreactive cells peaked by 2 DIV to achieve its lowest value at 60 DIV. At 24 hs of cell seeding of successive passages, such values peaked by the 6th subculture to gradually decrease thereafter. Increasing cell hypertrophy was found during the long-term first subculture but not after passaging. At the end of the observation period, doubly immunolabeled astrocytes were still recorded, thus evidencing retention of proliferative potential despite aging.El grado de diferenciación celular y la actividad mitótica fueron secuencialmente determinados mediante marcación por inmunoperoxidasa de la proteína gliofibrilar ácida (GFAP y del antígeno nuclear de proliferación celular (PCNA, respectivamente, en cultivos astrocitarios obtenidos de encéfalo de rata y mantenidos hasta 60 días in vitro (DIV de su primer subcultivo, o mediante pasajes semanales hasta el 12do subcultivo. El conteo celular se realizó mediante una retícula de 0.01-mm2 de sección y el análisis morfométrico con una grilla estereológica. El número de células doblemente inmunorreactivas alcanzó valores máximos a los 2 DIV para descender a los menores a los 60 DIV. A las 24 hs de sembrado celular de los sucesivos pasajes, esos valores ascendieron hacia el 6to subcultivo para luego declinar. En cuanto a la hipertrofia celular, se observó en todo el curso del primer subcultivo, pero no durante los posteriores pasajes. Al final del período de observación, todavía se continuaban detectando

  2. Pluripotent stem cell lines

    OpenAIRE

    Yu, Junying; Thomson, James A.

    2008-01-01

    The derivation of human embryonic stem cells 10 years ago ignited an explosion of public interest in stem cells, yet this achievement depended on prior decades of research on mouse embryonic carcinoma cells and embryonic stem cells. In turn, the recent derivation of mouse and human induced pluripotent stem cells depended on the prior studies on mouse and human embryonic stem cells. Both human embryonic stem cells and induced pluripotent stem cells can self-renew indefinitely in vitro while ma...

  3. Neural Stem Cell Transplantation Induces Stroke Recovery by Upregulating Glutamate Transporter GLT-1 in Astrocytes

    Science.gov (United States)

    Russo, Gianluca Luigi; Peruzzotti-Jametti, Luca; Rossi, Silvia; Sandrone, Stefano; Butti, Erica; De Ceglia, Roberta; Bergamaschi, Andrea; Motta, Caterina; Gallizioli, Mattia; Studer, Valeria; Colombo, Emanuela; Farina, Cinthia; Comi, Giancarlo; Politi, Letterio Salvatore; Muzio, Luca; Villani, Claudia; Invernizzi, Roberto William; Hermann, Dirk Matthias; Centonze, Diego

    2016-01-01

    Ischemic stroke is the leading cause of disability, but effective therapies are currently widely lacking. Recovery from stroke is very much dependent on the possibility to develop treatments able to both halt the neurodegenerative process as well as to foster adaptive tissue plasticity. Here we show that ischemic mice treated with neural precursor cell (NPC) transplantation had on neurophysiological analysis, early after treatment, reduced presynaptic release of glutamate within the ipsilesional corticospinal tract (CST), and an enhanced NMDA-mediated excitatory transmission in the contralesional CST. Concurrently, NPC-treated mice displayed a reduced CST degeneration, increased axonal rewiring, and augmented dendritic arborization, resulting in long-term functional amelioration persisting up to 60 d after ischemia. The enhanced functional and structural plasticity relied on the capacity of transplanted NPCs to localize in the peri-ischemic and ischemic area, to promote the upregulation of the glial glutamate transporter 1 (GLT-1) on astrocytes and to reduce peri-ischemic extracellular glutamate. The upregulation of GLT-1 induced by transplanted NPCs was found to rely on the secretion of VEGF by NPCs. Blocking VEGF during the first week after stroke reduced GLT-1 upregulation as well as long-term behavioral recovery in NPC-treated mice. Our results show that NPC transplantation, by modulating the excitatory–inhibitory balance and stroke microenvironment, is a promising therapy to ameliorate disability, to promote tissue recovery and plasticity processes after stroke. SIGNIFICANCE STATEMENT Tissue damage and loss of function occurring after stroke can be constrained by fostering plasticity processes of the brain. Over the past years, stem cell transplantation for repair of the CNS has received increasing interest, although underlying mechanism remain elusive. We here show that neural stem/precursor cell transplantation after ischemic stroke is able to foster

  4. Role of astrocytic leptin receptor subtypes on leptin permeation across hCMEC/D3 human brain endothelial cells.

    Science.gov (United States)

    Hsuchou, Hung; Kastin, Abba J; Tu, Hong; Joan Abbott, N; Couraud, Pierre-Olivier; Pan, Weihong

    2010-12-01

    Astrocytic leptin receptors (ObR) can be up-regulated in conditions such as adult-onset obesity. To determine whether the levels and subtypes of astrocytic ObR modulate leptin transport, we co-cultured hCMEC/D3 human brain endothelial cells and C6 astrocytoma cells in the Transwell system, and tested leptin permeation from apical to basolateral chambers. In comparison with hCMEC alone, co-culture of C6 cells reduced the permeability of paracellular markers and leptin. Unexpectedly, ObRb over-expression in C6 cells increased leptin permeation whereas ObRa over-expression showed no effect when compared with the control group of pcDNA-transfected C6 cells. By contrast, the paracellular permeability to the sodium fluorescein control was unchanged by over-expression of ObR subtypes. Leptin remained intact after crossing the monolayer as shown by HPLC and acid precipitation, and this was not affected by C6 cell co-culture or the over-expression of different ObR subtypes. Thus, increased expression of ObRb (and to a lesser extent ObRe) in C6 cells specifically increased the permeation of leptin across the hCMEC monolayer. Consistent with the evidence that the most apparent regulatory changes of ObR during obesity and inflammation occur in astrocytes, the results indicate that astrocytes actively regulate leptin transport across the blood-brain barrier, a mechanism independent of reduction of paracellular permeability. PMID:20977476

  5. Group B streptococcal infection and activation of human astrocytes.

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    Terri D Stoner

    Full Text Available Streptococcus agalactiae (Group B Streptococcus, GBS is the leading cause of life-threatening meningitis in human newborns in industrialized countries. Meningitis results from neonatal infection that occurs when GBS leaves the bloodstream (bacteremia, crosses the blood-brain barrier (BBB, and enters the central nervous system (CNS, where the bacteria contact the meninges. Although GBS is known to invade the BBB, subsequent interaction with astrocytes that physically associate with brain endothelium has not been well studied.We hypothesize that human astrocytes play a unique role in GBS infection and contribute to the development of meningitis. To address this, we used a well- characterized human fetal astrocyte cell line, SVG-A, and examined GBS infection in vitro. We observed that all GBS strains of representative clinically dominant serotypes (Ia, Ib, III, and V were able to adhere to and invade astrocytes. Cellular invasion was dependent on host actin cytoskeleton rearrangements, and was specific to GBS as Streptococcus gordonii failed to enter astrocytes. Analysis of isogenic mutant GBS strains deficient in various cell surface organelles showed that anchored LTA, serine-rich repeat protein (Srr1 and fibronectin binding (SfbA proteins all contribute to host cell internalization. Wild-type GBS also displayed an ability to persist and survive within an intracellular compartment for at least 12 h following invasion. Moreover, GBS infection resulted in increased astrocyte transcription of interleukin (IL-1β, IL-6 and VEGF.This study has further characterized the interaction of GBS with human astrocytes, and has identified the importance of specific virulence factors in these interactions. Understanding the role of astrocytes during GBS infection will provide important information regarding BBB disruption and the development of neonatal meningitis.

  6. CD133 is not present on neurogenic astrocytes in the adult subventricular zone, but on embryonic neural stem cells, ependymal cells, and glioblastoma cells.

    Science.gov (United States)

    Pfenninger, Cosima V; Roschupkina, Teona; Hertwig, Falk; Kottwitz, Denise; Englund, Elisabet; Bengzon, Johan; Jacobsen, Sten Eirik; Nuber, Ulrike A

    2007-06-15

    Human brain tumor stem cells have been enriched using antibodies against the surface protein CD133. An antibody recognizing CD133 also served to isolate normal neural stem cells from fetal human brain, suggesting a possible lineage relationship between normal neural and brain tumor stem cells. Whether CD133-positive brain tumor stem cells can be derived from CD133-positive neural stem or progenitor cells still requires direct experimental evidence, and an important step toward such investigations is the identification and characterization of normal CD133-presenting cells in neurogenic regions of the embryonic and adult brain. Here, we present evidence that CD133 is a marker for embryonic neural stem cells, an intermediate radial glial/ependymal cell type in the early postnatal stage, and for ependymal cells in the adult brain, but not for neurogenic astrocytes in the adult subventricular zone. Our findings suggest two principal possibilities for the origin of brain tumor stem cells: a derivation from CD133-expressing cells, which are normally not present in the adult brain (embryonic neural stem cells and an early postnatal intermediate radial glial/ependymal cell type), or from CD133-positive ependymal cells in the adult brain, which are, however, generally regarded as postmitotic. Alternatively, brain tumor stem cells could be derived from proliferative but CD133-negative neurogenic astrocytes in the adult brain. In the latter case, brain tumor development would involve the production of CD133. PMID:17575139

  7. The RNA helicase DDX1 is involved in restricted HIV-1 Rev function in human astrocytes

    International Nuclear Information System (INIS)

    Productive infection by human immunodeficiency virus type I (HIV-1) in the central nervous system (CNS) involves mainly macrophages and microglial cells. A frequency of less than 10% of human astrocytes is estimated to be infectable with HIV-1. Nonetheless, this relatively low percentage of infected astrocytes, but associated with a large total number of astrocytic cells in the CNS, makes human astrocytes a critical part in the analyses of potential HIV-1 reservoirs in vivo. Investigations in astrocytic cell lines and primary human fetal astrocytes revealed that limited HIV-1 replication in these cells resulted from low-level viral entry, transcription, viral protein processing, and virion maturation. Of note, a low ratio of unspliced versus spliced HIV-1-specific RNA was also investigated, as Rev appeared to act aberrantly in astrocytes, via loss of nuclear and/or nucleolar localization and diminished Rev-mediated function. Host cellular machinery enabling Rev function has become critical for elucidation of diminished Rev activity, especially for those factors leading to RNA metabolism. We have recently identified a DEAD-box protein, DDX1, as a Rev cellular co-factor and now have explored its potential importance in astrocytes. Cells were infected with HIV-1 pseudotyped with envelope glycoproteins of amphotropic murine leukemia viruses (MLV). Semi-quantitative reverse transcriptase-polymerase chain reactions (RT-PCR) for unspliced, singly-spliced, and multiply-spliced RNA clearly showed a lower ratio of unspliced/singly-spliced over multiply-spliced HIV-1-specific RNA in human astrocytes as compared to Rev-permissive, non-glial control cells. As well, the cellular localization of Rev in astrocytes was cytoplasmically dominant as compared to that of Rev-permissive, non-glial controls. This endogenous level of DDX1 expression in astrocytes was demonstrated directly to lead to a shift of Rev sub-cellular distribution dominance from nuclear and/or nucleolar to

  8. Impaired APP activity and altered Tau splicing in embryonic stem cell-derived astrocytes obtained from an APPsw transgenic minipig

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    Vanessa J. Hall

    2015-10-01

    Full Text Available Animal models of familial juvenile onset of Alzheimer's disease (AD often fail to produce diverse pathological features of the disease by modification of single gene mutations that are responsible for the disease. They can hence be poor models for testing and development of novel drugs. Here, we analyze in vitro-produced stem cells and their derivatives from a large mammalian model of the disease created by overexpression of a single mutant human gene (APPsw. We produced hemizygous and homozygous radial glial-like cells following culture and differentiation of embryonic stem cells (ESCs isolated from embryos obtained from mated hemizygous minipigs. These cells were confirmed to co-express varying neural markers, including NES, GFAP and BLBP, typical of type one radial glial cells (RGs from the subgranular zone. These cells had altered expression of CCND1 and NOTCH1 and decreased expression of several ribosomal RNA genes. We found that these cells were able to differentiate into astrocytes upon directed differentiation. The astrocytes produced had decreased α- and β-secretase activity, increased γ-secretase activity and altered splicing of tau. This indicates novel aspects of early onset mechanisms related to cell renewal and function in familial AD astrocytes. These outcomes also highlight that radial glia could be a potentially useful population of cells for drug discovery, and that altered APP expression and altered tau phosphorylation can be detected in an in vitro model of the disease. Finally, it might be possible to use large mammal models to model familial AD by insertion of only a single mutation.

  9. Compartmentalized coculture of rat brain endothelial cells and astrocytes: a syngenic model to study the blood-brain barrier.

    Science.gov (United States)

    Demeuse, Ph; Kerkhofs, A; Struys-Ponsar, C; Knoops, B; Remacle, C; van den Bosch de Aguilar, Ph

    2002-11-15

    The specific structure of the blood-brain barrier (BBB) is based on the partnership of brain endothelial cells and astrocytes. In the last decade, cocultures of these two cell types have been developed as in vitro models. However, these studies did not allow close contacts between both cell types. We report here a syngenic coculture model using rat endothelial cells on one side of a polyethylene terephtalate filter and rat astrocytes on the other. Endothelial cells retain their typical morphology and are factor VIII and OX 26 positive. We optimized the diameter of the membrane pores to establish very close contacts between the cells through the membrane pores without mixing the two cell types. Transmission electron microscopy showed evidence of tight junction formation between the endothelial cells and few pinocytic vesicles. The cocultures reached high electrical resistances up to 1000 Omegacm(2) showing their ability to limit the passage of ions. A 15-fold increase in gamma-glutamyl transpeptidase activity was measured in the endothelial cells in coculture compared to endothelial cell monoculture. Our syngenic coculture represents a useful in vitro model of the rat BBB that may prove to be valuable for studying the passage of substances across the barrier as well as other aspects of the BBB function. PMID:12393158

  10. Vitro Culture and Immunohistochemical Identification of Astrocytes of Infantile Optic Nerve

    Institute of Scientific and Technical Information of China (English)

    Jianliang Zheng; Yuqing Lan; Jie Zhang; Yan Guo; Yan Luo

    2000-01-01

    Objective: To culture astrocytes of optic nerve and to establish the cell lines for further study of healing process after optic nerve trauma.Methods: Optic nerve astrocytes of infantile and adults with sudden death were cultured by tissue inoculation or tissue digestion with 0. 25% Trypsin and 0. 06% EDTA. The second and fourth passage cells were stained with HE and anti- GFAP, S-100 protein, Vimentin, and CD34 antibodies.Results: The trypsinized astrocytes of infantile optic nerve reached confluence in 7 days,but the astrocytes of adults weren't successfully cultured. The cultured cells were in polygonal shape with processes; the cytoplasm was abundant and pink; the cells had light-blue nuclei. These cells were positive in GFAP, S-100 protein and vimentin staining, and negative in CD34 staining.Conclusions: The results showed that astrocytes of infantile optic nerve can be successfully cultured and trypsinization is a better method than tissue inoculation. The culture of infantile astrocytes is easier than that of adult astrocytes. Immunohistochemistry were used to determine the source and type of those cultured cells.

  11. Astrocytes from adult Wistar rats aged in vitro show changes in glial functions.

    Science.gov (United States)

    Souza, Débora Guerini; Bellaver, Bruna; Raupp, Gustavo Santos; Souza, Diogo Onofre; Quincozes-Santos, André

    2015-11-01

    Astrocytes, the most versatile cells of the central nervous system, play an important role in the regulation of neurotransmitter homeostasis, energy metabolism, antioxidant defenses and the anti-inflammatory response. Recently, our group characterized cortical astrocyte cultures from adult Wistar rats. In line with that work, we studied glial function using an experimental in vitro model of aging astrocytes (30 days in vitro after reaching confluence) from newborn (NB), adult (AD) and aged (AG) Wistar rats. We evaluated metabolic parameters, such as the glucose uptake, glutamine synthetase (GS) activity, and glutathione (GSH) content, as well as the GFAP, GLUT-1 and xCT expression. AD and AG astrocytes take up less glucose than NB astrocytes and had decreased GLUT1 expression levels. Furthermore, AD and AG astrocytes exhibited decreased GS activity compared to NB cells. Simultaneously, AD and AG astrocytes showed an increase in GSH levels, along with an increase in xCT expression. NB, AD and AG astrocytes presented similar morphology; however, differences in GFAP levels were observed. Taken together, these results improve the knowledge of cerebral senescence and represent an innovative tool for brain studies of aging. PMID:26210720

  12. A co-culture model of the hippocampal neurogenic niche reveals differential effects of astrocytes, endothelial cells and pericytes on proliferation and differentiation of adult murine precursor cells

    Directory of Open Access Journals (Sweden)

    Fanny Ehret

    2015-11-01

    Full Text Available The niche concept of stem cell biology proposes a functional unit between the precursor cells and their local microenvironment, to which several cell types might contribute by cell–cell contacts, extracellular matrix, and humoral factors. We here established three co-culture models (with cell types separated by membrane for both adherent monolayers and neurospheres to address the potential influence of different niche cell types in the neurogenic zone of the adult hippocampus of mice. Astrocytes and endothelial cells enhanced precursor cell proliferation and neurosphere formation. Endothelial factors also led to a prolonged increase in proliferation after growth factor withdrawal, which otherwise induces differentiation. All niche cell types enhanced cell survival in monolayer cultures, endothelial cells also stimulated neuronal differentiation. A parallel trend elicited by astrocytes did not reach conventional statistical significance. Pericytes had variable effects here. We did not observe changes in differentiation in neurosphere co-cultures. In summary, our data indicate that in precursor cell culture protocols survival could be improved by adding as yet unknown factors physiologically contributed by astrocytes and endothelial cells. Our findings also underscore the complexity of the niche and the differential impact of factors from the different sources on distinct aspects of neuronal development. With the help of the models presented here, identification of these factors and their specific biological activity can now be initiated.

  13. Cell lines and Salmonella

    NARCIS (Netherlands)

    Jonge R de; Hendriks H; Garssen J; Universteit Utrecht, afdeling; MGB; LPI

    2001-01-01

    In human gastrointestinal disease caused by Salmonella, transepithelial migration of neutrophils follows the attachment of bacteria to epithelial tissue. This migration of neutrophils is stimulated by the release of chemokines, including interleukin-8 (Il -8), from the epithelial cells. We have dev

  14. Astrocytes: Key Regulators of Neuroinflammation.

    Science.gov (United States)

    Colombo, Emanuela; Farina, Cinthia

    2016-09-01

    Astrocytes are crucial regulators of innate and adaptive immune responses in the injured central nervous system. Depending on timing and context, astrocyte activity may exacerbate inflammatory reactions and tissue damage, or promote immunosuppression and tissue repair. Recent literature has unveiled key factors and intracellular signaling pathways that govern astrocyte behavior during neuroinflammation. Here we have re-visited in vivo studies on astrocyte signaling in neuroinflammatory models focusing on evidences obtained from the analysis of transgenic mice where distinct genes involved in ligand binding, transcriptional regulation and cell communication have been manipulated in astrocytes. The integration of in vivo observations with in vitro data clarifies precise signaling steps, highlights the crosstalk among pathways and identifies shared effector mechanisms in neuroinflammation.

  15. Regional Susceptibility to Domoic Acid in Primary Astrocyte Cells Cultured from the Brain Stem and Hippocampus

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    Olga M. Pulido

    2008-02-01

    Full Text Available Domoic acid is a marine biotoxin associated with harmful algal blooms and is the causative agent of amnesic shellfish poisoning in marine animals and humans. It is also an excitatory amino acid analog to glutamate and kainic acid which acts through glutamate receptors eliciting a very rapid and potent neurotoxic response. The hippocampus, among other brain regions, has been identified as a specific target site having high sensitivity to DOM toxicity. Histopathology evidence indicates that in addition to neurons, the astrocytes were also injured. Electron microscopy data reported in this study further supports the light microscopy findings. Furthermore, the effect of DOM was confirmed by culturing primary astrocytes from the hippocampus and the brain stem and subsequently exposing them to domoic acid. The RNA was extracted and used for biomarker analysis. The biomarker analysis was done for the early response genes including c-fos, c-jun, c-myc, Hsp-72; specific marker for the astrocytes- GFAP and the glutamate receptors including GluR 2, NMDAR 1, NMDAR 2A and B. Although, the astrocyte-GFAP and c-fos were not affected, c-jun and GluR 2 were down-regulated. The microarray analysis revealed that the chemokines / cytokines, tyrosine kinases (Trk, and apoptotic genes were altered. The chemokines that were up-regulated included - IL1-a, IL-1B, IL-6, the small inducible cytokine, interferon protein IP-10, CXC chemokine LIX, and IGF binding proteins. The Bax, Bcl-2, Trk A and Trk B were all downregulated. Interestingly, only the hippocampal astrocytes were affected. Our findings suggest that astrocytes may present a possible target for pharmacological interventions for the prevention and treatment of amnesic shellfish poisoning and for other brain pathologies involving excitotoxicity

  16. Canine distemper virus persistence in demyelinating encephalitis by swift intracellular cell-to-cell spread in astrocytes is controlled by the viral attachment protein

    OpenAIRE

    Wyss-Fluehmann, Gaby; Zurbriggen, Andreas; Vandevelde, Marc; Plattet, Philippe

    2010-01-01

    The mechanism of viral persistence, the driving force behind the chronic progression of inflammatory demyelination in canine distemper virus (CDV) infection, is associated with non-cytolytic viral cell-to-cell spread. Here, we studied the molecular mechanisms of viral spread of a recombinant fluorescent protein-expressing virulent CDV in primary canine astrocyte cultures. Time-lapse video microscopy documented that CDV spread was very efficient using cell processes contacting remote target ce...

  17. Thyroid cell lines in research on goitrogenesis.

    Science.gov (United States)

    Gerber, H; Peter, H J; Asmis, L; Studer, H

    1991-12-01

    Thyroid cell lines have contributed a lot to the understanding of goitrogenesis. The cell lines mostly used in thyroid research are briefly discussed, namely the rat thyroid cell lines FRTL and FRTL-5, the porcine thyroid cell lines PORTHOS and ARTHOS, The sheep thyroid cell lines OVNIS 5H and 6H, the cat thyroid cell lines PETCAT 1 to 4 and ROMCAT, and the human thyroid cell lines FTC-133 and HTh 74. Chinese hamster ovary (CHO) cells and COS-7 cells, stably transfected with TSH receptor cDNA and expressing a functional TSH receptor, are discussed as examples for non-thyroidal cells, transfected with thyroid genes. PMID:1726925

  18. HIV-1 Entry and Trans-Infection of Astrocytes Involves CD81 Vesicles

    Science.gov (United States)

    Gray, Lachlan R.; Turville, Stuart G.; HItchen, Tina L.; Cheng, Wan-Jung; Ellett, Anne M.; Salimi, Hamid; Roche, Michael J.; Wesselingh, Steve L.; Gorry, Paul R.; Churchill, Melissa J.

    2014-01-01

    Astrocytes are extensively infected with HIV-1 in vivo and play a significant role in the development of HIV-1-associated neurocognitive disorders. Despite their extensive infection, little is known about how astrocytes become infected, since they lack cell surface CD4 expression. In the present study, we investigated the fate of HIV-1 upon infection of astrocytes. Astrocytes were found to bind and harbor virus followed by biphasic decay, with HIV-1 detectable out to 72 hours. HIV-1 was observed to associate with CD81-lined vesicle structures. shRNA silencing of CD81 resulted in less cell-associated virus but no loss of co-localization between HIV-1 and CD81. Astrocytes supported trans-infection of HIV-1 to T-cells without de novo virus production, and the virus-containing compartment required 37°C to form, and was trypsin-resistant. The CD81 compartment observed herein, has been shown in other cell types to be a relatively protective compartment. Within astrocytes, this compartment may be actively involved in virus entry and/or spread. The ability of astrocytes to transfer virus, without de novo viral synthesis suggests they are capable of sequestering and protecting virus and thus, they could potentially facilitate viral dissemination in the CNS. PMID:24587404

  19. Effects of folic acid on in vitro astrocytic differentiation of neural stem cells from neonatal rats

    Institute of Scientific and Technical Information of China (English)

    Xumei Zhang; Guowei Huang; Zhihong Tian; Guanglei Wang; Dalin Ren

    2009-01-01

    ary acidic protein/BrdU-positive ceils (P<0.05), and significantly decreased Ngn1 protein expression (P<0.05).CONCLUSION: Folic acid promotes astrocytic differentiation of NSCs, which might be related to downregulation of Ngnl protein expression.

  20. Neuronal modulation of calcium channel activity in cultured rat astrocytes.

    OpenAIRE

    Corvalan, V; Cole, R; de Vellis, J.; Hagiwara, S.

    1990-01-01

    The patch-clamp technique was used to study whether cocultivation of neurons and astrocytes modulates the expression of calcium channel activity in astrocytes. Whole-cell patch-clamp recordings from rat brain astrocytes cocultured with rat embryonic neurons revealed two types of voltage-dependent inward currents carried by Ca2+ and blocked by either Cd2+ or Co2+ that otherwise were not detected in purified astrocytes. This expression of calcium channel activity in astrocytes was neuron depend...

  1. Ornithine and Homocitrulline Impair Mitochondrial Function, Decrease Antioxidant Defenses and Induce Cell Death in Menadione-Stressed Rat Cortical Astrocytes: Potential Mechanisms of Neurological Dysfunction in HHH Syndrome.

    Science.gov (United States)

    Zanatta, Ângela; Rodrigues, Marília Danyelle Nunes; Amaral, Alexandre Umpierrez; Souza, Débora Guerini; Quincozes-Santos, André; Wajner, Moacir

    2016-09-01

    Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is caused by deficiency of ornithine translocase leading to predominant tissue accumulation and high urinary excretion of ornithine (Orn), homocitrulline (Hcit) and ammonia. Although affected patients commonly present neurological dysfunction manifested by cognitive deficit, spastic paraplegia, pyramidal and extrapyramidal signs, stroke-like episodes, hypotonia and ataxia, its pathogenesis is still poorly known. Although astrocytes are necessary for neuronal protection. Therefore, in the present study we investigated the effects of Orn and Hcit on cell viability (propidium iodide incorporation), mitochondrial function (thiazolyl blue tetrazolium bromide-MTT-reduction and mitochondrial membrane potential-ΔΨm), antioxidant defenses (GSH) and pro-inflammatory response (NFkB, IL-1β, IL-6 and TNF-α) in unstimulated and menadione-stressed cortical astrocytes that were previously shown to be susceptible to damage by neurotoxins. We first observed that Orn decreased MTT reduction, whereas both amino acids decreased GSH levels, without altering cell viability and the pro-inflammatory factors in unstimulated astrocytes. Furthermore, Orn and Hcit decreased cell viability and ΔΨm in menadione-treated astrocytes. The present data indicate that the major compounds accumulating in HHH syndrome impair mitochondrial function and reduce cell viability and the antioxidant defenses in cultured astrocytes especially when stressed by menadione. It is presumed that these mechanisms may be involved in the neuropathology of this disease. PMID:27161368

  2. Ornithine and Homocitrulline Impair Mitochondrial Function, Decrease Antioxidant Defenses and Induce Cell Death in Menadione-Stressed Rat Cortical Astrocytes: Potential Mechanisms of Neurological Dysfunction in HHH Syndrome.

    Science.gov (United States)

    Zanatta, Ângela; Rodrigues, Marília Danyelle Nunes; Amaral, Alexandre Umpierrez; Souza, Débora Guerini; Quincozes-Santos, André; Wajner, Moacir

    2016-09-01

    Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is caused by deficiency of ornithine translocase leading to predominant tissue accumulation and high urinary excretion of ornithine (Orn), homocitrulline (Hcit) and ammonia. Although affected patients commonly present neurological dysfunction manifested by cognitive deficit, spastic paraplegia, pyramidal and extrapyramidal signs, stroke-like episodes, hypotonia and ataxia, its pathogenesis is still poorly known. Although astrocytes are necessary for neuronal protection. Therefore, in the present study we investigated the effects of Orn and Hcit on cell viability (propidium iodide incorporation), mitochondrial function (thiazolyl blue tetrazolium bromide-MTT-reduction and mitochondrial membrane potential-ΔΨm), antioxidant defenses (GSH) and pro-inflammatory response (NFkB, IL-1β, IL-6 and TNF-α) in unstimulated and menadione-stressed cortical astrocytes that were previously shown to be susceptible to damage by neurotoxins. We first observed that Orn decreased MTT reduction, whereas both amino acids decreased GSH levels, without altering cell viability and the pro-inflammatory factors in unstimulated astrocytes. Furthermore, Orn and Hcit decreased cell viability and ΔΨm in menadione-treated astrocytes. The present data indicate that the major compounds accumulating in HHH syndrome impair mitochondrial function and reduce cell viability and the antioxidant defenses in cultured astrocytes especially when stressed by menadione. It is presumed that these mechanisms may be involved in the neuropathology of this disease.

  3. A “Hit and Run” Approach to Inducible Direct Reprogramming of Astrocytes to Neural Stem Cells

    Science.gov (United States)

    Poulou, Maria; Mandalos, Nikolaos P.; Karnavas, Theodoros; Saridaki, Marannia; McKay, Ronald D. G.; Remboutsika, Eumorphia

    2016-01-01

    Temporal and spatial control of gene expression can be achieved using an inducible system as a fundamental tool for regulated transcription in basic, applied and eventually in clinical research. We describe a novel “hit and run” inducible direct reprogramming approach. In a single step, 2 days post-transfection, transiently transfected Sox2FLAG under the Leu3p-αIPM inducible control (iSox2) triggers the activation of endogenous Sox2, redirecting primary astrocytes into abundant distinct nestin-positive radial glia cells. This technique introduces a unique novel tool for safe, rapid and efficient reprogramming amendable to regenerative medicine. PMID:27148066

  4. Id4 and FABP7 are preferentially expressed in cells with astrocytic features in oligodendrogliomas and oligoastrocytomas

    Directory of Open Access Journals (Sweden)

    Nicholas M Kelly

    2005-07-01

    Full Text Available Abstract Background Oligodendroglioma (ODG and oligoastrocytoma (OAC are diffusely infiltrating primary brain tumors whose pathogenesis remains unclear. We previously identified a group of genes whose expression was inversely correlated with survival in a cohort of patients with glioblastoma (GBM, and some of these genes are also reportedly expressed in ODG and OAC. We examined the expression patterns and localization of these survival-associated genes in ODG and OAC in order to analyze their possible roles in the oncogenesis of these two tumor types. Methods We used UniGene libraries derived from GBM and ODG specimens to examine the expression levels of the transcripts for each of the 50 GBM survival-associated genes. We used immunohistochemistry and cDNA microarrays to examine expression of selected survival-associated genes and Id4, a gene believed to control the timing of oligodendrocyte development. The expression of FABP7 and Id4 and the survival of patients with ODG and OAC were also analyzed. Results Transcripts of most survival-associated genes as well as Id4 were present in both GBM and ODG tumors, whereas protein expression of Id4 and one of the survival-associated genes, brain-type fatty acid-binding protein (FABP7, was present in cells with astrocytic features, including reactive and neoplastic astrocytes, but not in neoplastic oligodendrocytes. Id4 was co-expressed with FABP7 in microgemistocytes in ODG and in neoplastic astrocytes in OAC. Id4 and FABP7 expression, however, did not correlate with the clinical outcome of patients with ODG or OAC tumors. Conclusion Expression of Id4 and some of our previously identified GBM survival-associated genes is present in developing or mature oligodendrocytes. However, protein expression of Id4 and FABP7 in GBM, ODG, and OAC suggests that this group of functionally important genes might demonstrate two patterns of expression in these glioma subtypes: one group is universally expressed in

  5. A hit and run approach to inducible direct reprogramming of astrocytes to neural stem cells

    OpenAIRE

    Maria ePoulou; Nikolaos eMandalos; Theodoros eKarnavas; Marannia eSaridaki; Ronald G. McKay; Eumorphia eRemboutsika

    2016-01-01

    Temporal and spatial control of gene expression can be achieved using an inducible system as a fundamental tool for regulated transcription in basic, applied and eventually in clinical research. We describe a novel hit and run inducible direct reprogramming approach. In a single step, two days post-transfection, transiently transfected Sox2FLAG under the Leu3p-αIPM inducible control (iSox2) triggers the activation of endogenous Sox2, redirecting primary astrocytes into abundant distinct nest...

  6. Dynamic reactive astrocytes after focal ischemia

    Institute of Scientific and Technical Information of China (English)

    Shinghua Ding

    2014-01-01

    Astrocytes are specialized and most numerous glial cell type in the central nervous system and play important roles in physiology. Astrocytes are also critically involved in many neural disor-ders including focal ischemic stroke, a leading cause of brain injury and human death. One of the prominent pathological features of focal ischemic stroke is reactive astrogliosis and glial scar for-mation associated with morphological changes and proliferation. This review paper discusses the recent advances in spatial and temporal dynamics of morphology and proliferation of reactive astrocytes after ischemic stroke based on results from experimental animal studies. As reactive astrocytes exhibit stem cell-like properties, knowledge of dynamics of reactive astrocytes and glial scar formation will provide important insights for astrocyte-based cell therapy in stroke.

  7. Development of a Novel Method for the Purification and Culture of Rodent Astrocytes

    OpenAIRE

    Foo, Lynette C.; Allen, Nicola J.; Bushong, Eric A.; Ventura, P. Britten; Chung, Won-Suk; Zhou, Lu; Cahoy, John D.; Daneman, Richard; Zong, Hui; Ellisman, Mark H.; Barres, Ben A.

    2011-01-01

    The inability to purify and culture astrocytes has long hindered studies of their function. Whereas astrocyte progenitor cells can be cultured from neonatal brain, culture of mature astrocytes from postnatal brain has not been possible. Here we report a new method to prospectively purify astrocytes by immunopanning. These astrocytes undergo apoptosis in culture, but vascular cells and HBEGF promote their survival in serum-free culture. We found that some developing astrocytes normally undergo...

  8. Lentivirus-Mediated Knockdown of Astrocyte Elevated Gene-1 Inhibits Growth and Induces Apoptosis through MAPK Pathways in Human Retinoblastoma Cells.

    Directory of Open Access Journals (Sweden)

    Ying Chang

    Full Text Available To explore expression and function of astrocyte elevated gene-1 (AEG-1 in human retinoblastoma (RB.The expression of AEG-1 in histological sections of human RBs and in RB cell lines was examined using immunohistochemical staining and RT-PCR and Western blotting respectively. We knocked down AEG-1 gene levels by AEG-1-siRNA lentivirus transfection of human RB cell lines SO-RB50 and Y79, and using an MTT assay, we assessed the role of AEG-1 on RB cell proliferation. The biological significance of lentivirus transfection induced AEG-1 down-regulation was examined by assessing the apoptosis rate in the transfected RB cells by Annexin V-APC staining and flow cytometry. We additionally measured the expression of Bcl-2, Bax, cleaved-caspase-3 and caspase-3, and the phosphorylation and non-phosphorylation alternation of MAPKs.AEG-1 expression was detected to be strongly positive in the histological slides of 35 out of 54 (65% patients with RB. AEG-1 expression increased significantly (P<0.05 with tumor stage. In the RB cell lines SO-RB50, Y79 and WERI-RB1 as compared with retinal pigment epithelium cells, expression of AEG-1 mRNA and AEG-1 protein was significantly higher. In AEG-1-siRNA lentivirus transfected cell cultures as compared with negative control lentivirus transfected cell cultures, levels of AEG-1 mRNA and of AEG-1 protein (P<0.05 and cell growth rates (P<0.01 were significantly lower, and apoptosis rate (P<0.001, Bax/Bcl-2 ratio and cleaved-caspase-3 protein level were significantly increased. The P-ERK/ERK ratio was significantly decreased in the AEG-1-siRNA lentivirus transfected cell lines.Expression of AEG-1 was associated with RB, in histological slides of patients and in cell culture experiments. Lentivirus transfection induced knockdown of AEG-1 had a tumor suppressive effect, potentially by tumor cell apoptosis induction through inhibition of ERK.

  9. Light and electron microscopic localization of GABAA-receptors on cultured cerebellar granule cells and astrocytes using immunohistochemical techniques

    DEFF Research Database (Denmark)

    Hansen, Gert Helge; Hösli, E; Belhage, B;

    1991-01-01

    . At the light microscope level specific staining of GABAA-receptors was localized in various types of neurones in explant cultures of rat cerebellum using the indirect peroxidase-antiperoxidase (PAP) technique, whereas no specific staining was found in astrocytes. At the electron microscope level labeling...... of GABAA-receptors was observed in the plasma membrane of both the cell bodies and processes in dissociated primary cultures of cerebellar granule cells using an indirect preembedding immunogold staining technique which in contrast to the classical PAP technique allows quantitative estimations...... to be performed. Quantification of the labeling intensity revealed a higher concentration of GABAA-receptors per microns plasma membrane in the cell bodies than in the processes. In discrete areas an extremely high density of the GABAA-receptors was observed. No specific labeling of GABAA-receptors was observed...

  10. Injury and repair of astrocyte after ionizing radiation

    International Nuclear Information System (INIS)

    Astrocyte is the most glial cell in the central nervous system. In the present experiment, radiation injury to the central nervous system (CNS) triggers a large network of cellular changes including neuron, glial cell and endothelial cell in morphology and metabolism and function. Astrocyte changes rapidly after ionizing radiation. There is a relationship between astrocyte and the pathologic process and function recover of damaged brain tissue following CNS injury. This suggests that astrocyte plays an important role in cure of clinical radiation injury

  11. Astrocytes in the tempest of multiple sclerosis.

    Science.gov (United States)

    Miljković, Djordje; Timotijević, Gordana; Mostarica Stojković, Marija

    2011-12-01

    Astrocytes are the most abundant cell population within the CNS of mammals. Their glial role is perfectly performed in the healthy CNS as they support functions of neurons. The omnipresence of astrocytes throughout the white and grey matter and their intimate relation with blood vessels of the CNS, as well as numerous immunity-related actions that these cells are capable of, imply that astrocytes should have a prominent role in neuroinflammatory disorders, such as multiple sclerosis (MS). The role of astrocytes in MS is rather ambiguous, as they have the capacity to both stimulate and restrain neuroinflammation and tissue destruction. In this paper we present some of the proved and the proposed functions of astrocytes in neuroinflammation and discuss the effect of MS therapeutics on astrocytes. PMID:21443873

  12. Zirconium oxide ceramic foam: a promising supporting biomaterial for massive production of glial cell line-derived neurotrophic factor*

    OpenAIRE

    Liu, Zhong-Wei; Li, Wen-qiang; Wang, Jun-kui; Ma, Xian-cang; Liang, Chen; Liu, Peng; Chu, Zheng; Dang, Yong-hui

    2014-01-01

    This study investigated the potential application of a zirconium oxide (ZrO2) ceramic foam culturing system to the production of glial cell line-derived neurotrophic factor (GDNF). Three sets of ZrO2 ceramic foams with different pore densities of 10, 20, and 30 pores per linear inch (PPI) were prepared to support a 3D culturing system. After primary astrocytes were cultured in these systems, production yields of GDNF were evaluated. The biomaterial biocompatibility, cell proliferation and act...

  13. Sphingosine 1 Phosphate at the Blood Brain Barrier: Can the Modulation of S1P Receptor 1 Influence the Response of Endothelial Cells and Astrocytes to Inflammatory Stimuli?

    Directory of Open Access Journals (Sweden)

    Simona F Spampinato

    Full Text Available The ability of the Blood Brain Barrier (BBB to maintain proper barrier functions, keeping an optimal environment for central nervous system (CNS activity and regulating leukocytes' access, can be affected in CNS diseases. Endothelial cells and astrocytes are the principal BBB cellular constituents and their interaction is essential to maintain its function. Both endothelial cells and astrocytes express the receptors for the bioactive sphingolipid S1P. Fingolimod, an immune modulatory drug whose structure is similar to S1P, has been approved for treatment in multiple sclerosis (MS: fingolimod reduces the rate of MS relapses by preventing leukocyte egress from the lymph nodes. Here, we examined the ability of S1P and fingolimod to act on the BBB, using an in vitro co-culture model that allowed us to investigate the effects of S1P on endothelial cells, astrocytes, and interactions between the two. Acting selectively on endothelial cells, S1P receptor signaling reduced cell death induced by inflammatory cytokines. When acting on astrocytes, fingolimod treatment induced the release of a factor, granulocyte macrophage colony-stimulating factor (GM-CSF that reduced the effects of cytokines on endothelium. In an in vitro BBB model incorporating shear stress, S1P receptor modulation reduced leukocyte migration across the endothelial barrier, indicating a novel mechanism that might contribute to fingolimod efficacy in MS treatment.

  14. Identification of soluble CD14 as an endogenous agonist for toll-like receptor 2 on human astrocytes by genome-scale functional screening of glial cell derived proteins

    NARCIS (Netherlands)

    Bsibsi, M.; Bajramovic, J.J.; Duijvenvoorden, E. van; Persoon, C.; Ravid, R.; Noort, J.M. van; Vogt, M.H.J.

    2007-01-01

    Human astrocytes express a limited repertoire of Toll-like receptor (TLR) family members including TLR1-4, which are expressed on the cell surface. Also, TLR3 but not TLR4 activation on astrocytes induces expression of several factors involved in neuroprotection and down-regulation of inflammation r

  15. Trafficking of astrocytic vesicles in hippocampal slices

    International Nuclear Information System (INIS)

    The increasingly appreciated role of astrocytes in neurophysiology dictates a thorough understanding of the mechanisms underlying the communication between astrocytes and neurons. In particular, the uptake and release of signaling substances into/from astrocytes is considered as crucial. The release of different gliotransmitters involves regulated exocytosis, consisting of the fusion between the vesicle and the plasma membranes. After fusion with the plasma membrane vesicles may be retrieved into the cytoplasm and may continue to recycle. To study the mobility implicated in the retrieval of secretory vesicles, these structures have been previously efficiently and specifically labeled in cultured astrocytes, by exposing live cells to primary and secondary antibodies. Since the vesicle labeling and the vesicle mobility properties may be an artifact of cell culture conditions, we here asked whether the retrieving exocytotic vesicles can be labeled in brain tissue slices and whether their mobility differs to that observed in cell cultures. We labeled astrocytic vesicles and recorded their mobility with two-photon microscopy in hippocampal slices from transgenic mice with fluorescently tagged astrocytes (GFP mice) and in wild-type mice with astrocytes labeled by Fluo4 fluorescence indicator. Glutamatergic vesicles and peptidergic granules were labeled by the anti-vesicular glutamate transporter 1 (vGlut1) and anti-atrial natriuretic peptide (ANP) antibodies, respectively. We report that the vesicle mobility parameters (velocity, maximal displacement and track length) recorded in astrocytes from tissue slices are similar to those reported previously in cultured astrocytes.

  16. Trafficking of astrocytic vesicles in hippocampal slices

    Energy Technology Data Exchange (ETDEWEB)

    Potokar, Maja; Kreft, Marko [Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloska 4, 1000 Ljubljana (Slovenia); Celica Biomedical Center, Technology Park 24, 1000 Ljubljana (Slovenia); Lee, So-Young; Takano, Hajime; Haydon, Philip G. [Department of Neuroscience, Room 215, Stemmler Hall, University of Pennsylvania, School of Medicine, Philadelphia, PA 19104 (United States); Zorec, Robert, E-mail: Robert.Zorec@mf.uni-lj.si [Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloska 4, 1000 Ljubljana (Slovenia); Celica Biomedical Center, Technology Park 24, 1000 Ljubljana (Slovenia)

    2009-12-25

    The increasingly appreciated role of astrocytes in neurophysiology dictates a thorough understanding of the mechanisms underlying the communication between astrocytes and neurons. In particular, the uptake and release of signaling substances into/from astrocytes is considered as crucial. The release of different gliotransmitters involves regulated exocytosis, consisting of the fusion between the vesicle and the plasma membranes. After fusion with the plasma membrane vesicles may be retrieved into the cytoplasm and may continue to recycle. To study the mobility implicated in the retrieval of secretory vesicles, these structures have been previously efficiently and specifically labeled in cultured astrocytes, by exposing live cells to primary and secondary antibodies. Since the vesicle labeling and the vesicle mobility properties may be an artifact of cell culture conditions, we here asked whether the retrieving exocytotic vesicles can be labeled in brain tissue slices and whether their mobility differs to that observed in cell cultures. We labeled astrocytic vesicles and recorded their mobility with two-photon microscopy in hippocampal slices from transgenic mice with fluorescently tagged astrocytes (GFP mice) and in wild-type mice with astrocytes labeled by Fluo4 fluorescence indicator. Glutamatergic vesicles and peptidergic granules were labeled by the anti-vesicular glutamate transporter 1 (vGlut1) and anti-atrial natriuretic peptide (ANP) antibodies, respectively. We report that the vesicle mobility parameters (velocity, maximal displacement and track length) recorded in astrocytes from tissue slices are similar to those reported previously in cultured astrocytes.

  17. The NRTIs Lamivudine, Stavudine and Zidovudine Have Reduced HIV-1 Inhibitory Activity in Astrocytes

    Science.gov (United States)

    Gray, Lachlan R.; Tachedjian, Gilda; Ellett, Anne M.; Roche, Michael J.; Cheng, Wan-Jung; Guillemin, Gilles J.; Brew, Bruce J.; Turville, Stuart G.; Wesselingh, Steve L.; Gorry, Paul R.; Churchill, Melissa J.

    2013-01-01

    HIV-1 establishes infection in astrocytes and macroage-lineage cells of the central nervous system (CNS). Certain antiretroviral drugs (ARVs) can penetrate the CNS, and are therefore often used in neurologically active combined antiretroviral therapy (Neuro-cART) regimens, but their relative activity in the different susceptible CNS cell populations is unknown. Here, we determined the HIV-1 inhibitory activity of CNS-penetrating ARVs in astrocytes and macrophage-lineage cells. Primary human fetal astrocytes (PFA) and the SVG human astrocyte cell line were used as in vitro models for astrocyte infection, and monocyte-derived macrophages (MDM) were used as an in vitro model for infection of macrophage-lineage cells. The CNS-penetrating ARVs tested were the nucleoside reverse transcriptase inhibitors (NRTIs) abacavir (ABC), lamivudine (3TC), stavudine (d4T) and zidovudine (ZDV), the non-NRTIs efavirenz (EFV), etravirine (ETR) and nevirapine (NVP), and the integrase inhibitor raltegravir (RAL). Drug inhibition assays were performed using single-round HIV-1 entry assays with luciferase viruses pseudotyped with HIV-1 YU-2 envelope or vesicular stomatitis virus G protein (VSV-G). All the ARVs tested could effectively inhibit HIV-1 infection in macrophages, with EC90s below concentrations known to be achievable in the cerebral spinal fluid (CSF). Most of the ARVs had similar potency in astrocytes, however the NRTIs 3TC, d4T and ZDV had insufficient HIV-1 inhibitory activity in astrocytes, with EC90s 12-, 187- and 110-fold greater than achievable CSF concentrations, respectively. Our data suggest that 3TC, d4T and ZDV may not adequately target astrocyte infection in vivo, which has potential implications for their inclusion in Neuro-cART regimens. PMID:23614033

  18. Micropatterned substrates for studying astrocytes in culture

    Directory of Open Access Journals (Sweden)

    William Lee

    2009-12-01

    Full Text Available Recent studies of the physiological roles of astrocytes have ignited renewed interest in the functional significance of these glial cells in the central nervous system. Many of the newly discovered astrocytic functions were initially demonstrated and characterized in cell culture systems. We discuss the use of microculture techniques and micropatterning of cell-adhesive substrates in studies of astrocytic Ca2+ excitability and bidirectional neuron-astrocyte signaling. This culturing approach aims to reduce the level of complexity of the system by limiting the interacting partners and by controlling the localization of cells. It provides tight control over experimental conditions allowing detailed characterization of cellular functions and intercellular communication. Although such a reductionist approach yields some difference in observations between astrocytic properties in culture and in situ, general phenomena discovered in cell culture systems, however, have also been found in vivo.

  19. Tibolone protects astrocytic cells from glucose deprivation through a mechanism involving estrogen receptor beta and the upregulation of neuroglobin expression.

    Science.gov (United States)

    Avila-Rodriguez, Marco; Garcia-Segura, Luis Miguel; Hidalgo-Lanussa, Oscar; Baez, Eliana; Gonzalez, Janneth; Barreto, George E

    2016-09-15

    Tibolone, a synthetic steroid used for the prevention of osteoporosis and the treatment of climacteric symptoms in post-menopausal women, may exert tissue selective estrogenic actions acting on estrogen receptors (ERs). We previously showed that tibolone protects human T98G astroglial cells against glucose deprivation (GD). In this study we have explored whether the protective effect of tibolone on these cells is mediated by ERs. Experimental studies showed that both ERα and ERβ were involved in the protection by tibolone on GD cells, being ERβ preferentially involved on these actions over ERα. Tibolone increased viability of GD cells by a mechanism fully blocked by an ERβ antagonist and partially blocked by an ERα antagonist. Furthermore, ERβ inhibition prevented the effect of tibolone on nuclear fragmentation, ROS and mitochondrial membrane potential in GD cells. The protective effect of tibolone was mediated by neuroglobin. Tibolone upregulated neuroglobin in T98G cells and primary mouse astrocytes by a mechanism involving ERβ and neuroglobin silencing prevented the protective action of tibolone on GD cells. In summary, tibolone protects T98G cells by a mechanism involving ERβ and the upregulation of neuroglobin. PMID:27250720

  20. Interlab Cell Line Collection: Bioresource of Established Human and Animal Cell Lines

    OpenAIRE

    Parodi, Barbara; Aresu, Ottavia; Visconti, Paola; Manniello, Maria Assunta; Strada, Paolo

    2015-01-01

    The Interlab Cell Line Collection (ICLC) was established in 1994 as a core facility of the National Institute of Cancer Research. It supplies: human and animal cell lines; Short Tandem Repeat (STR) profiling of human cell lines; quality control service; mycoplasma detection and eradication service; safe deposit service and patent deposit service of cell lines and hybridomas. The catalogue of services is on-line, and the cell lines are distributed all over the world. 

  1. Knockdown of astrocyte elevated gene-1 inhibits tumor growth and modifies microRNAs expression profiles in human colorectal cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Sujun [East Department of Gastroenterology, Institute of Geriatrics, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong 510080 (China); Southern Medical University, Guangzhou, Guangdong 510515 (China); Wu, Binwen, E-mail: wubinwengd@aliyun.com [East Department of Gastroenterology, Institute of Geriatrics, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong 510080 (China); Li, Dongfeng; Zhou, Weihong; Deng, Gang; Zhang, Kaijun; Li, Youjia [East Department of Gastroenterology, Institute of Geriatrics, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong 510080 (China)

    2014-02-14

    Highlights: • AEG-1 expression in CRC cell lines and down-regulation or upregulation of AEG-1 in vitro. • Knockdown of AEG-1 inhibits cell proliferation, colony formation and invasion. • Upregulation of AEG-1 enhances proliferation, invasion and colony formation. • Knockdown of AEG-1 accumulates G0/G1-phase cells and promotes apoptosis in CRC cells. • AEG-1 knockdown increases 5-FU cytotoxicity. - Abstract: Astrocyte elevated gene-1 (AEG-1), upregulated in various types of malignancies including colorectal cancer (CRC), has been reported to be associated with the carcinogenesis. MicroRNAs (miRNAs) are widely involved in the initiation and progression of cancer. However, the functional significance of AEG-1 and the relationship between AEG-1 and microRNAs in human CRC remains unclear. The aim of this study was to investigate whether AEG-1 could serve as a potential therapeutic target of human CRC and its possible mechanism. We adopted a strategy of ectopic overexpression or RNA interference to upregulate or downregulate expression of AEG-1 in CRC models. Their phenotypic changes were analyzed by Western blot, MTT and transwell matrix penetration assays. MicroRNAs expression profiles were performed using microarray analysis followed by validation using qRT-PCR. Knockdown of AEG-1 could significantly inhibit colon cancer cell proliferation, colony formation, invasion and promotes apoptosis. Conversely, upregulation of AEG-1 could significantly enhance cell proliferation, invasion and reduced apoptisis. AEG-1 directly contributes to resistance to chemotherapeutic drug. Targeted downregulation of AEG-1 might improve the expression of miR-181a-2{sup ∗}, -193b and -193a, and inversely inhibit miR-31 and -9{sup ∗}. Targeted inhibition of AEG-1 can lead to modification of key elemental characteristics, such as miRNAs, which may become a potential effective therapeutic strategy for CRC.

  2. Knockdown of astrocyte elevated gene-1 inhibits tumor growth and modifies microRNAs expression profiles in human colorectal cancer cells

    International Nuclear Information System (INIS)

    Highlights: • AEG-1 expression in CRC cell lines and down-regulation or upregulation of AEG-1 in vitro. • Knockdown of AEG-1 inhibits cell proliferation, colony formation and invasion. • Upregulation of AEG-1 enhances proliferation, invasion and colony formation. • Knockdown of AEG-1 accumulates G0/G1-phase cells and promotes apoptosis in CRC cells. • AEG-1 knockdown increases 5-FU cytotoxicity. - Abstract: Astrocyte elevated gene-1 (AEG-1), upregulated in various types of malignancies including colorectal cancer (CRC), has been reported to be associated with the carcinogenesis. MicroRNAs (miRNAs) are widely involved in the initiation and progression of cancer. However, the functional significance of AEG-1 and the relationship between AEG-1 and microRNAs in human CRC remains unclear. The aim of this study was to investigate whether AEG-1 could serve as a potential therapeutic target of human CRC and its possible mechanism. We adopted a strategy of ectopic overexpression or RNA interference to upregulate or downregulate expression of AEG-1 in CRC models. Their phenotypic changes were analyzed by Western blot, MTT and transwell matrix penetration assays. MicroRNAs expression profiles were performed using microarray analysis followed by validation using qRT-PCR. Knockdown of AEG-1 could significantly inhibit colon cancer cell proliferation, colony formation, invasion and promotes apoptosis. Conversely, upregulation of AEG-1 could significantly enhance cell proliferation, invasion and reduced apoptisis. AEG-1 directly contributes to resistance to chemotherapeutic drug. Targeted downregulation of AEG-1 might improve the expression of miR-181a-2∗, -193b and -193a, and inversely inhibit miR-31 and -9∗. Targeted inhibition of AEG-1 can lead to modification of key elemental characteristics, such as miRNAs, which may become a potential effective therapeutic strategy for CRC

  3. Astrocyte, the star avatar: redefined

    Indian Academy of Sciences (India)

    Pankaj Seth; Nitin Koul

    2008-09-01

    Until recently, the neuroscience community held the belief that glial cells such as astrocytes and oligodendrocytes functioned solely as “support” cells of the brain. In this role, glial cells simply provide physical support and housekeeping functions for the more important cells of the brain, the neurons. However, this view has changed radically in recent years with the discovery of previously unrecognized and surprising functions for this underappreciated cell type. In the past decade or so, emerging evidence has provided new insights into novel glial cell activities such as control of synapse formation and function, communication, cerebrovascular tone regulation, immune regulation and adult neurogenesis. Such advances in knowledge have effectively elevated the role of the astrocyte to one that is more important than previously realized. This review summarizes the past and present knowledge of glial cell functions that has evolved over the years, and has resulted in a new appreciation of astrocytes and their value in studying the neurobiology of human brain cells and their functions. In this review, we highlight recent advances in the role of glial cells in physiology, pathophysiology and, most importantly, in adult neurogenesis and “stemness”, with special emphasis on astrocytes.

  4. Hypoxia inducible factor-2α regulates the development of retinal astrocytic network by maintaining adequate supply of astrocyte progenitors.

    Directory of Open Access Journals (Sweden)

    Li-Juan Duan

    Full Text Available Here we investigate the role of hypoxia inducible factor (HIF-2α in coordinating the development of retinal astrocytic and vascular networks. Three Cre mouse lines were used to disrupt floxed Hif-2α, including Rosa26(CreERT2, Tie2(Cre, and GFAP(Cre. Global Hif-2α disruption by Rosa26(CreERT2 led to reduced astrocytic and vascular development in neonatal retinas, whereas endothelial disruption by Tie2(Cre had no apparent effects. Hif-2α deletion in astrocyte progenitors by GFAP(Cre significantly interfered with the development of astrocytic networks, which failed to reach the retinal periphery and were incapable of supporting vascular development. Perplexingly, the abundance of strongly GFAP(+ mature astrocytes transiently increased at P0 before they began to lag behind the normal controls by P3. Pax2(+ and PDGFRα(+ astrocytic progenitors and immature astrocytes were dramatically diminished at all stages examined. Despite decreased number of astrocyte progenitors, their proliferation index or apoptosis was not altered. The above data can be reconciled by proposing that HIF-2α is required for maintaining the supply of astrocyte progenitors by slowing down their differentiation into non-proliferative mature astrocytes. HIF-2α deficiency in astrocyte progenitors may accelerate their differentiation into astrocytes, a change which greatly interferes with the replenishment of astrocyte progenitors due to insufficient time for proliferation. Rapidly declining progenitor supply may lead to premature cessation of astrocyte development. Given that HIF-2α protein undergoes oxygen dependent degradation, an interesting possibility is that retinal blood vessels may regulate astrocyte differentiation through their oxygen delivery function. While our findings support the consensus that retinal astrocytic template guides vascular development, they also raise the possibility that astrocytic and vascular networks may mutually regulate each other

  5. Comparative effects on rat primary astrocytes and C6 rat glioma cells cultures after 24-h exposure to silver nanoparticles (AgNPs)

    International Nuclear Information System (INIS)

    The aim of this work was to compare the effects of 24-h exposure of rat primary astrocytes and C6 rat glioma cells to 7.8 nm AgNPs. Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor and current treatments lead to diverse side-effects; for this reason, it is imperative to investigate new approaches, including those alternatives provided by nanotechnology, like nanomaterials (NMs) such as silver nanoparticles. Herein, we found that C6 rat glioma cells, but no primary astrocytes, decreased cell viability after AgNPs treatment; however, both cell types diminished their proliferation. The decrease of glioma C6 cells proliferation was related with necrosis, while in primary astrocytes, the decreased proliferation was associated with the induction of apoptosis. The ionic control (AgNO3) exerted a different profile than AgNPs; the bulk form did not modify the basal effect in each determination, whereas cisplatin, a well-known antitumoral drug used as a comparative control, promoted cytotoxicity in both cell types at specific concentrations. Our findings prompt the need to determine the fine molecular and cellular mechanisms involved in the differential biological responses to AgNPs in order to develop new tools or alternatives based on nanotechnology that may contribute to the understanding, impact and use of NMs in specific targets, like glioblastoma cells

  6. Comparative effects on rat primary astrocytes and C6 rat glioma cells cultures after 24-h exposure to silver nanoparticles (AgNPs)

    Energy Technology Data Exchange (ETDEWEB)

    Salazar-García, Samuel; Silva-Ramírez, Ana Sonia; Ramirez-Lee, Manuel A.; Rosas-Hernandez, Hector [Universidad Autonoma de San Luis Potosi, Facultad de Ciencias Quimicas (Mexico); Rangel-López, Edgar [Instituto Nacional de Neurologia y Neurocirugia Manuel Velasco Suárez, Laboratorio de Aminoacidos Excitadores (Mexico); Castillo, Claudia G. [Facultad de Medicina, Universidad Autonoma de San Luis Potosi (Mexico); Santamaría, Abel [Instituto Nacional de Neurologia y Neurocirugia Manuel Velasco Suárez, Laboratorio de Aminoacidos Excitadores (Mexico); Martinez-Castañon, Gabriel A. [Universidad Autonoma de San Luis Potosi, Facultad de Estomatologia (Mexico); Gonzalez, Carmen, E-mail: cgonzalez.uaslp@gmail.com, E-mail: gonzalez.castillocarmen@fcq.uaslp.mx [Universidad Autonoma de San Luis Potosi, Facultad de Ciencias Quimicas (Mexico)

    2015-11-15

    The aim of this work was to compare the effects of 24-h exposure of rat primary astrocytes and C6 rat glioma cells to 7.8 nm AgNPs. Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor and current treatments lead to diverse side-effects; for this reason, it is imperative to investigate new approaches, including those alternatives provided by nanotechnology, like nanomaterials (NMs) such as silver nanoparticles. Herein, we found that C6 rat glioma cells, but no primary astrocytes, decreased cell viability after AgNPs treatment; however, both cell types diminished their proliferation. The decrease of glioma C6 cells proliferation was related with necrosis, while in primary astrocytes, the decreased proliferation was associated with the induction of apoptosis. The ionic control (AgNO{sub 3}) exerted a different profile than AgNPs; the bulk form did not modify the basal effect in each determination, whereas cisplatin, a well-known antitumoral drug used as a comparative control, promoted cytotoxicity in both cell types at specific concentrations. Our findings prompt the need to determine the fine molecular and cellular mechanisms involved in the differential biological responses to AgNPs in order to develop new tools or alternatives based on nanotechnology that may contribute to the understanding, impact and use of NMs in specific targets, like glioblastoma cells.

  7. Functions of astrocytes and their potential as therapeutic targets

    OpenAIRE

    Kimelberg, Harold K.; NEDERGAARD, Maiken

    2010-01-01

    Astrocytes are often referred to, and historically have been regarded as, support cells of the mammalian CNS. Work over the last decade suggests otherwise, that astrocytes may in fact play a more active role in higher neural processing than previously recognized. Because astrocytes can potentially serve as novel therapeutic targets, it is critical to understand how astrocytes execute their diverse supportive tasks while maintaining neuronal health. To that end, this review will focus on the s...

  8. Epigenetic Regulation of HIV-1 Latency in Astrocytes

    OpenAIRE

    Narasipura, Srinivas D.; Kim, Stephanie; Al-Harthi, Lena

    2014-01-01

    HIV infiltrates the brain at early times postinfection and remains latent within astrocytes and macrophages. Because astrocytes are the most abundant cell type in the brain, we evaluated epigenetic regulation of HIV latency in astrocytes. We have shown that class I histone deacetylases (HDACs) and a lysine-specific histone methyltransferase, SU(VAR)3-9, play a significant role in silencing of HIV transcription in astrocytes. Our studies add to a growing body of evidence demonstrating that ast...

  9. Astrocytes in multiple sclerosis.

    Science.gov (United States)

    Ludwin, Samuel K; Rao, Vijayaraghava Ts; Moore, Craig S; Antel, Jack P

    2016-08-01

    Recent experimental and clinical studies on astrocytes are unraveling the capabilities of these multi-functional cells in normal homeostasis, and in central nervous system (CNS) disease. This review focuses on understanding their behavior in all aspects of the initiation, evolution, and resolution of the multiple sclerosis (MS) lesion. Astrocytes display remarkable flexibility and variability of their physical structure and biochemical output, each aspect finely tuned to the specific stage and location of the disease, participating in both pathogenic and beneficial changes seen in acute and progressive forms. As examples, chemo-attractive or repulsive molecules may facilitate the entry of destructive immune cells but may also aid in the recruitment of oligodendrocyte precursors, essential for repair. Pro-inflammatory cytokines may attack pathogenic cells and also destroy normal oligodendrocytes, myelin, and axons. Protective trophic factors may also open the blood-brain barrier and modulate the extracellular matrix to favor recruitment and persistence of CNS-specific immune cells. A chronic glial scar may confer structural support following tissue loss and inhibit ingress of further noxious insults and also inhibit migration of reparative cells and molecules into the damaged tissue. Continual study into these processes offers the therapeutic opportunities to enhance the beneficial capabilities of these cells while limiting their destructive effects. PMID:27207458

  10. Astrocytes release ATP through lysosomal exocytosis

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    @@ Astrocytes, the most abundant type of glial cells in the brain, have been found to release signaling molecules, including adenosine triphosphate(ATP), the most important energy carrier inside the cell as well as a universal extracellular signaling molecule.

  11. Critical role of astrocytic interleukin-17 A in post-stroke survival and neuronal differentiation of neural precursor cells in adult mice.

    Science.gov (United States)

    Lin, Y; Zhang, J-C; Yao, C-Y; Wu, Y; Abdelgawad, A F; Yao, S-L; Yuan, S-Y

    2016-01-01

    The brain and the immune system interact in complex ways after ischemic stroke, and the long-term effects of immune response associated with stroke remain controversial. As a linkage between innate and adaptive immunity, interleukin-17 A (IL-17 A) secreted from gamma delta (γδ) T cells has detrimental roles in the pathogenesis of acute ischemic stroke. However, to date, the long-term actions of IL-17 A after stroke have not been investigated. Here, we found that IL-17 A showed two distinct peaks of expression in the ischemic hemisphere: the first occurring within 3 days and the second on day 28 after stroke. Our data also showed that astrocyte was the major cellular source of IL-17 A that maintained and augmented subventricular zone (SVZ) neural precursor cells (NPCs) survival, neuronal differentiation, and subsequent synaptogenesis and functional recovery after stroke. IL-17 A also promoted neuronal differentiation in cultured NPCs from the ischemic SVZ. Furthermore, our in vitro data revealed that in primary astrocyte cultures activated astrocytes released IL-17 A via p38 mitogen-activated protein kinase (MAPK). Culture media from reactive astrocytes increased neuronal differentiation of NSCs in vitro. Blockade of IL-17 A with neutralizing antibody prevented this effect. In addition, after screening for multiple signaling pathways, we revealed that the p38 MAPK/calpain 1 signaling pathway was involved in IL-17 A-mediated neurogenesis in vivo and in vitro. Thus, our results reveal a previously uncharacterized property of astrocytic IL-17 A in the maintenance and augment of survival and neuronal differentiation of NPCs, and subsequent synaptogenesis and spontaneous recovery after ischemic stroke. PMID:27336717

  12. Functional Oxygen Sensitivity of Astrocytes.

    Science.gov (United States)

    Angelova, Plamena R; Kasymov, Vitaliy; Christie, Isabel; Sheikhbahaei, Shahriar; Turovsky, Egor; Marina, Nephtali; Korsak, Alla; Zwicker, Jennifer; Teschemacher, Anja G; Ackland, Gareth L; Funk, Gregory D; Kasparov, Sergey; Abramov, Andrey Y; Gourine, Alexander V

    2015-07-22

    In terrestrial mammals, the oxygen storage capacity of the CNS is limited, and neuronal function is rapidly impaired if oxygen supply is interrupted even for a short period of time. However, oxygen tension monitored by the peripheral (arterial) chemoreceptors is not sensitive to regional CNS differences in partial pressure of oxygen (PO2 ) that reflect variable levels of neuronal activity or local tissue hypoxia, pointing to the necessity of a functional brain oxygen sensor. This experimental animal (rats and mice) study shows that astrocytes, the most numerous brain glial cells, are sensitive to physiological changes in PO2 . Astrocytes respond to decreases in PO2 a few millimeters of mercury below normal brain oxygenation with elevations in intracellular calcium ([Ca(2+)]i). The hypoxia sensor of astrocytes resides in the mitochondria in which oxygen is consumed. Physiological decrease in PO2 inhibits astroglial mitochondrial respiration, leading to mitochondrial depolarization, production of free radicals, lipid peroxidation, activation of phospholipase C, IP3 receptors, and release of Ca(2+) from the intracellular stores. Hypoxia-induced [Ca(2+)]i increases in astrocytes trigger fusion of vesicular compartments containing ATP. Blockade of astrocytic signaling by overexpression of ATP-degrading enzymes or targeted astrocyte-specific expression of tetanus toxin light chain (to interfere with vesicular release mechanisms) within the brainstem respiratory rhythm-generating circuits reveals the fundamental physiological role of astroglial oxygen sensitivity; in low-oxygen conditions (environmental hypoxia), this mechanism increases breathing activity even in the absence of peripheral chemoreceptor oxygen sensing. These results demonstrate that astrocytes are functionally specialized CNS oxygen sensors tuned for rapid detection of physiological changes in brain oxygenation. Significance statement: Most, if not all, animal cells possess mechanisms that allow them to

  13. Isolation and Characterization of Ischemia-Derived Astrocytes (IDAs) with Ability to Transactivate Quiescent Astrocytes

    OpenAIRE

    Villarreal, Alejandro; Rosciszewski, Gerardo; Murta, Veronica; Cadena, Vanesa; Usach, Vanina; Dodes-Traian, Martin M.; Setton-Avruj, Patricia; Barbeito, Luis H.; Ramos, Alberto J.

    2016-01-01

    Reactive gliosis involving activation and proliferation of astrocytes and microglia, is a widespread but largely complex and graded glial response to brain injury. Astroglial population has a previously underestimated high heterogeneity with cells differing in their morphology, gene expression profile, and response to injury. Here, we identified a subset of reactive astrocytes isolated from brain focal ischemic lesions that show several atypical characteristics. Ischemia-derived astrocytes (I...

  14. Mitochondrial Damage and Apoptosis Induced by Adenosine Deaminase Inhibition and Deoxyadenosine in Human Neuroblastoma Cell Lines.

    Science.gov (United States)

    Garcia-Gil, Mercedes; Tozzi, Maria Grazia; Balestri, Francesco; Colombaioni, Laura; Camici, Marcella

    2016-07-01

    The treatment with deoxycoformycin, a strong adenosine deaminase inhibitor, in combination with deoxyadenosine, causes apoptotic cell death of two human neuroblastoma cell lines, SH-SY5Y and LAN5. Herein we demonstrate that, in SH-SY5Y cells, this combination rapidly decreases mitochondrial reactive oxygen species and, in parallel, increases mitochondrial mass, while, later, induces nuclear fragmentation, and activation of caspase-8, -9, and -3. In previous papers we have shown that a human astrocytoma cell line, subjected to the same treatment, undergoes apoptotic death as well. Therefore, both astrocytoma and neuroblastoma cell lines undergo apoptotic death following the combined treatment with deoxycoformycin and deoxyadenosine, but several differences have been found in the mode of action, possibly reflecting a different functional and metabolic profile of the two cell lines. Overall this work indicates that the neuroblastoma cell lines, like the line of astrocytic origin, are very sensitive to purine metabolism perturbation thus suggesting new therapeutic approaches to nervous system tumors. J. Cell. Biochem. 117: 1671-1679, 2016. © 2015 Wiley Periodicals, Inc. PMID:26659614

  15. Effects of intrathecal injection of glial cell inhibitor on spinal cord astrocytes following chronic compression of dorsal root ganglia in rats

    Institute of Scientific and Technical Information of China (English)

    Xianhong Zhang; Wen Shen; Mingde Wang; Yinming Zeng

    2009-01-01

    obtained to count astrocytes under an inverted microscope. RESULTS: No significant differences in astrocyte count were detected between the fluorocitrate and model groups. Cell bodies were small with a few processes in the fluorocitrate group, compared with the model group. The astrocyte count decreased significantly in the minocycline group and the minocycline and fluorocitrate group compared with the sham operation, model, PBS and fluorocitrate groups (P < 0.01). The decrease in astrocyte count was mainly found in layers Ⅲ-Ⅳ of the spinal dorsal horn. Cell body volume was smaller and process numbers were fewer in the minocycline group and the minocycline and fluorocitrate group, compared with the model and PBS groups. CONCLUSION: Fluorocitrate can inhibit astrocyte activation, but does not affect astrocyte proliferation. However, minocycline can inhibit the activation and proliferation of astrocytes.

  16. Neuron-glia interactions through the Heartless FGF receptor signaling pathway mediate morphogenesis of Drosophila astrocytes.

    Science.gov (United States)

    Stork, Tobias; Sheehan, Amy; Tasdemir-Yilmaz, Ozge E; Freeman, Marc R

    2014-07-16

    Astrocytes are critically important for neuronal circuit assembly and function. Mammalian protoplasmic astrocytes develop a dense ramified meshwork of cellular processes to form intimate contacts with neuronal cell bodies, neurites, and synapses. This close neuron-glia morphological relationship is essential for astrocyte function, but it remains unclear how astrocytes establish their intricate morphology, organize spatial domains, and associate with neurons and synapses in vivo. Here we characterize a Drosophila glial subtype that shows striking morphological and functional similarities to mammalian astrocytes. We demonstrate that the Fibroblast growth factor (FGF) receptor Heartless autonomously controls astrocyte membrane growth, and the FGFs Pyramus and Thisbe direct astrocyte processes to ramify specifically in CNS synaptic regions. We further show that the shape and size of individual astrocytes are dynamically sculpted through inhibitory or competitive astrocyte-astrocyte interactions and Heartless FGF signaling. Our data identify FGF signaling through Heartless as a key regulator of astrocyte morphological elaboration in vivo.

  17. Human Cell Line and Tissue Sample Authentication

    OpenAIRE

    Ewing, Margaret M.; McLaren, Robert S.; Hebble, Kathryn D.; Ready, Kim; Storts, Douglas R.; Hooper, Kyle

    2013-01-01

    Background: Short Tandem Repeat (STR) genotyping analysis is a proven technology for uniquely identifying virtually all human samples. STR genotyping was adopted as the preferred technology for identification of human tissue culture cell lines by the ATCC Standards Development Organization (ASN-0002: Authentication of Human Cell Lines: Standardization of STR Profiling). We developed new automation-compatible protocols/systems for generating STR profiles from human cell lines or tissue samples...

  18. Copper Metabolism of Astrocytes

    OpenAIRE

    Ralf Dringen; Scheiber, Ivo F.; Julian FB Mercer

    2013-01-01

    This short review will summarize the current knowledge on the uptake, storage, and export of copper ions by astrocytes and will address the potential roles of astrocytes in copper homeostasis in the normal and diseased brain. Astrocytes in culture efficiently accumulate copper by processes that include both the copper transporter Ctr1 and Ctr1-independent mechanisms. Exposure of astrocytes to copper induces an increase in cellular glutathione (GSH) content as well as synthesis of metallothion...

  19. Effects of triptolide on hippocampal microglial cells and astrocytes in the APP/PS1 double transgenic mouse model of Alzheimer’s disease

    Institute of Scientific and Technical Information of China (English)

    Jian-ming Li; Yan Zhang; Liang Tang; Yong-heng Chen; Qian Gao; Mei-hua Bao; Ju Xiang; De-liang Lei

    2016-01-01

    The principal pathology of Alzheimer’s disease includes neuronal extracellular deposition of amyloid-beta peptides and formation of senile pl aques, which in turn induce neuroinlfammation in the brain. Triptolide, a natural extract from the vine-like herb Tripterygium wilfordiiHook F, has potent anti-inlfammatory and immunosuppressive efifcacy. Therefore, we determined if triptolide can inhibit activation and proliferation of microglial cells and astrocytes in the APP/PS1 double transgenic mouse model of Alzheimer’s disease. We used 1 or 5 μg/kg/d triptolide to treat APP/PS1 double transgenic mice (aged 4–4.5 months) for 45 days. Unbiased stereology analysis found that triptolide dose-dependent-ly reduced the total number of microglial cells, and transformed microglial cells into the resting state. Further, triptolide (5 μg/kg/d) also reduced the total number of hippocampal astrocytes. Our in vivo test results indicate that triptolide suppresses activation and proliferation of microglial cells and astrocytes in the hippocampus of APP/PS1 double transgenic mice with Alzheimer’s disease.

  20. Molecular Characterization of Putative Chordoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Silke Brüderlein

    2010-01-01

    Full Text Available Immortal tumor cell lines are an important model system for cancer research, however, misidentification and cross-contamination of cell lines are a common problem. Seven chordoma cell lines are reported in the literature, but none has been characterized in detail. We analyzed gene expression patterns and genomic copy number variations in five putative chordoma cell lines (U-CH1, CCL3, CCL4, GB60, and CM319. We also created a new chordoma cell line, U-CH2, and provided genotypes for cell lines for identity confirmation. Our analyses revealed that CCL3, CCL4, and GB60 are not chordoma cell lines, and that CM319 is a cancer cell line possibly derived from chordoma, but lacking expression of key chordoma biomarkers. U-CH1 and U-CH2 both have gene expression profiles, copy number aberrations, and morphology consistent with chordoma tumors. These cell lines also harbor genetic changes, such as loss of p16, MTAP, or PTEN, that make them potentially useful models for studying mechanisms of chordoma pathogenesis and for evaluating targeted therapies.

  1. Dopamine receptor activation increases glial cell line-derived neurotrophic factor in experimental stroke.

    Science.gov (United States)

    Kuric, Enida; Wieloch, Tadeusz; Ruscher, Karsten

    2013-09-01

    Treatment with levodopa enhances functional recovery after experimental stroke but its mechanisms of action are elusive. Reactive astrocytes in the ischemic hemisphere are involved in mechanisms promoting recovery and also express dopamine 1 (D1) and dopamine 2 (D2) receptors. Here we investigated if the activation of astrocytic dopamine receptors (D1 and D2) regulates the expression of glial cell line-derived neurotrophic factor (GDNF) after combined in vitro hypoxia/aglycemia (H/A) and studied the expression of GDNF in the ischemic brain after treatment with levodopa/benserazide following transient occlusion of the middle cerebral artery (tMCAO) in the rat. Twenty-four hours after H/A, GDNF levels were upregulated in exposed astrocytes compared to normoxic control cultures and further elevated by the addition of the selective D1 receptor agonist (R)-(+)-SKF-38393 hydrochloride while D1 receptor antagonism by R(+)-SCH-23390 hydrochloride significantly reduced GDNF. No effect on GDNF levels was observed by the application of the D2 receptor agonist R(-)-2,10,11-trihydroxy-N-propyl-noraporphine hydrobromide hydrate or S-(-)-eticlopride hydrochloride (D2 receptor antagonist). After tMCAO, GDNF was upregulated in D1 expressing reactive astrocytes in the peri-infarct area. In addition, treatment with levodopa/benserazide significantly increased GDNF levels in the infarct core and peri-infarct area after tMCAO without affecting the expression of glial fibrillar acidic protein (GFAP), an intermediate filament and marker of reactive gliosis. After stroke, GDNF levels increase in the ischemic hemisphere in rats treated with levodopa, implicating GDNF in the mechanisms of tissue reorganization and plasticity and in l-DOPA enhanced recovery of lost brain function. Our results support levodopa treatment as a potential recovery enhancing therapy in stroke patients.

  2. HLA expression in hepatocellular carcinoma cell lines.

    Science.gov (United States)

    Wadee, A A; Paterson, A; Coplan, K A; Reddy, S G

    1994-08-01

    The present study undertook to investigate the biological significance of human leucocyte antigen expression in hepatocellular carcinoma and to elucidate the role of potential modulating agents on human leucocyte antigen expression. These studies used several hepatic tumour-derived cell lines as in vitro model systems. The cell lines included PLC/PRF/5 (Alexander cell line), Hep3B, HepG2, TONG PHC, HA22T/VGH, HA59T/VGH and Mahlavu. The cell lines K562 and Raji were used as negative and positive controls, respectively. K562, a B lymphoid-derived cell line, was shown to express negligible amounts of human leucocyte antigens, while Raji, an erythromyeloid-derived cell line, expressed both class I and class II human leucocyte antigens as well as their respective invariant chains, beta 2-microglobulin and Ii. Using an ELISA, experiments performed on these cell lines confirmed the natural expression of class I and class II antigens by the HA22T/VGH and HA59T/VGH cell lines, whereas PLC/PRF/5 displayed class II surface antigens only. The effects of modulating agents such as interferon-gamma sodium butyrate and clofazimine on human leucocyte antigen expression were investigated using the HA22T/VGH, HA59T/VGH and TONG PHC cell lines. These agents increased class II and class II human leucocyte antigen expression on HA22T/VGH and TONG PHC cells, but had no effect on the HA59T/VGH cell line. The results suggest a potential use for these agents as modulators of human leucocyte antigen expression by human heptocellular cell lines.

  3. Treadmill exercise ameliorates symptoms of attention deficit/hyperactivity disorder through reducing Purkinje cell loss and astrocytic reaction in spontaneous hypertensive rats

    OpenAIRE

    Yun, Hyo-Soon; Park, Mi-Sook; Ji, Eun-Sang; Kim, Tae-Woon; Ko, Il-Gyu; Kim, Hyun-Bae; Kim, Hong

    2014-01-01

    Attention deficit/hyperactivity disorder (ADHD) is a neurobehavioral disorder of cognition. We investigated the effects of treadmill exercise on Purkinje cell and astrocytic reaction in the cerebellum of the ADHD rat. Adult male spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKYR) weighing 210± 10 g were used. The animals were randomly divided into four groups (n= 15): control group, ADHD group, ADHD and methylphenidate (MPH)-treated group, ADHD and treadmill exercise group. The...

  4. Bone marrow-derived mesenchymal stem cells improve diabetes-induced cognitive impairment by exosome transfer into damaged neurons and astrocytes

    OpenAIRE

    Masako Nakano; Kanna Nagaishi; Naoto Konari; Yuki Saito; Takako Chikenji; Yuka Mizue; Mineko Fujimiya

    2016-01-01

    The incidence of dementia is higher in diabetic patients, but no effective treatment has been developed. This study showed that rat bone marrow mesenchymal stem cells (BM-MSCs) can improve the cognitive impairments of STZ-diabetic mice by repairing damaged neurons and astrocytes. The Morris water maze test demonstrated that cognitive impairments induced by diabetes were significantly improved by intravenous injection of BM-MSCs. In the CA1 region of the hippocampus, degeneration of neurons an...

  5. In Situ Probing of Cholesterol in Astrocytes at the Single Cell Level using Laser Desorption Ionization Mass Spectrometric Imaging with Colloidal Silver

    Energy Technology Data Exchange (ETDEWEB)

    Perdian, D.C.; Cha, Sangwon; Oh, Jisun; Sakaguchi, Donald S.; Yeung, Edward S.; and Lee, Young Jin

    2010-03-18

    Mass spectrometric imaging has been utilized to localize individual astrocytes and to obtain cholesterol populations at the single-cell level in laser desorption ionization (LDI) with colloidal silver. The silver ion adduct of membrane-bound cholesterol was monitored to detect individual cells. Good correlation between mass spectrometric and optical images at different cell densities indicates the ability to perform single-cell studies of cholesterol abundance. The feasibility of quantification is confirmed by the agreement between the LDI-MS ion signals and the results from a traditional enzymatic fluorometric assay. We propose that this approach could be an effective tool to study chemical populations at the cellular level.

  6. Effects of xenogeneic acellular bone on the astrocyte cells%脱细胞异种骨对星形胶质细胞的影响

    Institute of Scientific and Technical Information of China (English)

    李丽华; 张余; 黄金龙; 李梅; 夏虹; 侯丽

    2014-01-01

    Objective To observe the effects of xenogeneic acellular bone on the proliferation , migration and characteristic of astrocyte cells .Methods The xenogeneic bone was prepared by epoxy crosslinking technology after chemical and physical processing .Different concentrations (0.05, 0.1, 0.2 g/mL) of xenogeneic bone extracted liquid were added into the culture mediums with astrocyte cells .Scanning electron microscope ( SEM) was used for morphologic observation of astrocyte cells .MTT method was used detect the proliferation of the astrocyte cells , and to optimize the con-centration of xenogeneic bone extracted liquid .Transwell method and flow cytometry were used to access the cell migration and cell cycle , respectively .Results The astrocyte cells were polygonal , and were in a good state under SEM .The opti-mized concentration of xenogeneic bone extracted liquid was 0.05 g/mL, which had a positive effect on cell proliferation . In this concentration , the number of transwell cells was increased ( P0.05).Conclusion Xenogeneic acellular bone has low influence on astrocyte cells , providing good biosecurity to astro-cyte cells.%目的:探讨脱细胞异种骨对星形胶质细胞增殖、迁移及细胞特性的影响。方法采用环氧交联技术制备脱细胞异种骨,将不同浸提浓度(0.05、0.1、0.2 g/mL)的异种骨浸提液加入含星形胶质细胞的培养基中。扫描电镜观察细胞形态;MTT法检测细胞增殖情况,确定最佳浸提浓度;Transwell小室检测细胞迁移情况;流式细胞仪检测细胞周期。结果扫描电镜示星形胶质细胞生长状态良好,呈星状结构;不同浸提浓度的异种骨浸提液中,0.05 g/mL的浸提液对细胞增殖有促进作用,0.05 g/mL为最佳浸提浓度;经该浸提浓度异种骨浸提液处理的细胞,迁移数量明显增多(P<0.05),但细胞各周期比例无明显变化(P>0.05)。结论经脱细胞脱基质处理的异种骨,

  7. Loose excitation-secretion coupling in astrocytes.

    Science.gov (United States)

    Vardjan, Nina; Parpura, Vladimir; Zorec, Robert

    2016-05-01

    Astrocytes play an important housekeeping role in the central nervous system. Additionally, as secretory cells, they actively participate in cell-to-cell communication, which can be mediated by membrane-bound vesicles. The gliosignaling molecules stored in these vesicles are discharged into the extracellular space after the vesicle membrane fuses with the plasma membrane. This process is termed exocytosis, regulated by SNARE proteins, and triggered by elevations in cytosolic calcium levels, which are necessary and sufficient for exocytosis in astrocytes. For astrocytic exocytosis, calcium is sourced from the intracellular endoplasmic reticulum store, although its entry from the extracellular space contributes to cytosolic calcium dynamics in astrocytes. Here, we discuss calcium management in astrocytic exocytosis and the properties of the membrane-bound vesicles that store gliosignaling molecules, including the vesicle fusion machinery and kinetics of vesicle content discharge. In astrocytes, the delay between the increase in cytosolic calcium activity and the discharge of secretions from the vesicular lumen is orders of magnitude longer than that in neurons. This relatively loose excitation-secretion coupling is likely tailored to the participation of astrocytes in modulating neural network processing. PMID:26358496

  8. Nitric Oxide in Astrocyte-Neuron Signaling

    Energy Technology Data Exchange (ETDEWEB)

    Nianzhen Li

    2002-06-27

    Astrocytes, a subtype of glial cell, have recently been shown to exhibit Ca{sup 2+} elevations in response to neurotransmitters. A Ca{sup 2+} elevation can propagate to adjacent astrocytes as a Ca{sup 2+} wave, which allows an astrocyte to communicate with its neighbors. Additionally, glutamate can be released from astrocytes via a Ca{sup 2+}-dependent mechanism, thus modulating neuronal activity and synaptic transmission. In this dissertation, the author investigated the roles of another endogenous signal, nitric oxide (NO), in astrocyte-neuron signaling. First the author tested if NO is generated during astrocytic Ca{sup 2+} signaling by imaging NO in purified murine cortical astrocyte cultures. Physiological concentrations of a natural messenger, ATP, caused a Ca{sup 2+}-dependent NO production. To test the roles of NO in astrocytic Ca{sup 2+} signaling, the author applied NO to astrocyte cultures via addition of a NO donor, S-nitrosol-N-acetylpenicillamine (SNAP). NO induced an influx of external Ca{sup 2+}, possibly through store-operated Ca{sup 2+} channels. The NO-induced Ca{sup 2+} signaling is cGMP-independent since 8-Br-cGMP, an agonistic analog of cGMP, did not induce a detectable Ca{sup 2+} change. The consequence of this NO-induced Ca{sup 2+} influx was assessed by simultaneously monitoring of cytosolic and internal store Ca{sup 2+} using fluorescent Ca{sup 2+} indicators x-rhod-1 and mag-fluo-4. Blockage of NO signaling with the NO scavenger PTIO significantly reduced the refilling percentage of internal stores following ATP-induced Ca{sup 2+} release, suggesting that NO modulates internal store refilling. Furthermore, locally photo-release of NO to a single astrocyte led to a Ca{sup 2+} elevation in the stimulated astrocyte and a subsequent Ca{sup 2+} wave to neighbors. Finally, the author tested the role of NO inglutamate-mediated astrocyte-neuron signaling by recording the astrocyte-evoked glutamate-dependent neuronal slow inward current (SIC

  9. Extensive astrocyte infection is prominent in human immunodeficiency virus-associated dementia.

    Science.gov (United States)

    Churchill, Melissa J; Wesselingh, Steven L; Cowley, Daniel; Pardo, Carlos A; McArthur, Justin C; Brew, Bruce J; Gorry, Paul R

    2009-08-01

    Astrocyte infection with human immunodeficiency virus (HIV) is considered rare, so astrocytes are thought to play a secondary role in HIV neuropathogenesis. By combining double immunohistochemistry, laser capture microdissection, and highly sensitive multiplexed polymerase chain reaction to detect HIV DNA in single astrocytes in vivo, we showed that astrocyte infection is extensive in subjects with HIV-associated dementia, occurring in up to 19% of GFAP+ cells. In addition, astrocyte infection frequency correlated with the severity of neuropathological changes and proximity to perivascular macrophages. Our data indicate that astrocytes can be extensively infected with HIV, and suggest an important role for HIV-infected astrocytes in HIV neuropathogenesis.

  10. Artificial astrocytes improve neural network performance.

    Directory of Open Access Journals (Sweden)

    Ana B Porto-Pazos

    Full Text Available Compelling evidence indicates the existence of bidirectional communication between astrocytes and neurons. Astrocytes, a type of glial cells classically considered to be passive supportive cells, have been recently demonstrated to be actively involved in the processing and regulation of synaptic information, suggesting that brain function arises from the activity of neuron-glia networks. However, the actual impact of astrocytes in neural network function is largely unknown and its application in artificial intelligence remains untested. We have investigated the consequences of including artificial astrocytes, which present the biologically defined properties involved in astrocyte-neuron communication, on artificial neural network performance. Using connectionist systems and evolutionary algorithms, we have compared the performance of artificial neural networks (NN and artificial neuron-glia networks (NGN to solve classification problems. We show that the degree of success of NGN is superior to NN. Analysis of performances of NN with different number of neurons or different architectures indicate that the effects of NGN cannot be accounted for an increased number of network elements, but rather they are specifically due to astrocytes. Furthermore, the relative efficacy of NGN vs. NN increases as the complexity of the network increases. These results indicate that artificial astrocytes improve neural network performance, and established the concept of Artificial Neuron-Glia Networks, which represents a novel concept in Artificial Intelligence with implications in computational science as well as in the understanding of brain function.

  11. Ezh2 Expression in Astrocytes Induces Their Dedifferentiation Toward Neural Stem Cells

    NARCIS (Netherlands)

    Sher, Falak; Boddeke, Erik; Copray, Sjef

    2011-01-01

    Recently, we have demonstrated the expression of the polycomb group protein Ezh2 in embryonic and adult neural stem cells. Although Ezh2 remained highly expressed when neural stem cells differentiate into oligodendrocyte precursor cells, it is downregulated during the differentiation into neurons or

  12. GFAP promoter directs lacZ expression specifically in a rat hepatic stellate cell line

    Institute of Scientific and Technical Information of China (English)

    Gunter Maubach; Michelle Chin Chia Lim; Chun-Yan Zhang; Lang Zhuo

    2006-01-01

    AIM: The GFAP was traditionally considered to be a biomarker for neural glia (mainly astrocytes and nonmyelinating Schwann cells). Genetically, a 2.2-kb human GFAP promoter has been successfully used to target astrocytes in vitro and in vivo. More recently, GFAP was also established as one of the several makers for identifying hepatic stellate cells (HSC). In this project,possible application of the same 2.2-kb human GFAP promoter for targeting HSC was investigated.METHODS: The GFAP-lacZ transgene was transfected into various cell lines (HSC, hepatocyte, and other nonHSC cell types). The transgene expression specificity was determined by X-gal staining of the β-galactosidase activity. And the responsiveness of the transgene was tested with a typical pro-fibrotic cytokine TGF-β1. The expression of endogenous GFAP gene was assessed by real-time RT-PCR, providing a reference for the transgene expression.RESULTS: The results demonstrated for the first time that the 2.2 kb hGFAP promoter was not only capable of directing HSC-specific expression, but also responding to a known pro-fibrogenic cytokine TGF-β1 by upregulation in a dose- and time-dependent manner, similar to the endogenous GFAP.CONCLUSION: In conclusion, these findings suggested novel utilities for using the GFAP promoter to specifically manipulate HSC for therapeutic purpose.

  13. Generation and properties of a new human ventral mesencephalic neural stem cell line

    Energy Technology Data Exchange (ETDEWEB)

    Villa, Ana; Liste, Isabel; Courtois, Elise T.; Seiz, Emma G.; Ramos, Milagros [Center of Molecular Biology ' Severo Ochoa' , Autonomous University of Madrid-C.S.I.C., Campus Cantoblanco 28049-Madrid (Spain); Meyer, Morten [Department of Anatomy and Neurobiology, Institute of Medical Biology, University of Southern Denmark, Winslowparken 21,st, DK-500, Odense C (Denmark); Juliusson, Bengt; Kusk, Philip [NsGene A/S, Ballerup (Denmark); Martinez-Serrano, Alberto, E-mail: amserrano@cbm.uam.es [Center of Molecular Biology ' Severo Ochoa' , Autonomous University of Madrid-C.S.I.C., Campus Cantoblanco 28049-Madrid (Spain)

    2009-07-01

    Neural stem cells (NSCs) are powerful research tools for the design and discovery of new approaches to cell therapy in neurodegenerative diseases like Parkinson's disease. Several epigenetic and genetic strategies have been tested for long-term maintenance and expansion of these cells in vitro. Here we report the generation of a new stable cell line of human neural stem cells derived from ventral mesencephalon (hVM1) based on v-myc immortalization. The cells expressed neural stem cell and radial glia markers like nestin, vimentin and 3CB2 under proliferation conditions. After withdrawal of growth factors, proliferation and expression of v-myc were dramatically reduced and the cells differentiated into astrocytes, oligodendrocytes and neurons. hVM1 cells yield a large number of dopaminergic neurons (about 12% of total cells are TH{sup +}) after differentiation, which also produce dopamine. In addition to proneural genes (NGN2, MASH1), differentiated cells show expression of several genuine mesencephalic dopaminergic markers such as: LMX1A, LMX1B, GIRK2, ADH2, NURR1, PITX3, VMAT2 and DAT, indicating that they retain their regional identity. Our data indicate that this cell line and its clonal derivatives may constitute good candidates for the study of development and physiology of human dopaminergic neurons in vitro, and to develop tools for Parkinson's disease cell replacement preclinical research and drug testing.

  14. Generation and properties of a new human ventral mesencephalic neural stem cell line

    International Nuclear Information System (INIS)

    Neural stem cells (NSCs) are powerful research tools for the design and discovery of new approaches to cell therapy in neurodegenerative diseases like Parkinson's disease. Several epigenetic and genetic strategies have been tested for long-term maintenance and expansion of these cells in vitro. Here we report the generation of a new stable cell line of human neural stem cells derived from ventral mesencephalon (hVM1) based on v-myc immortalization. The cells expressed neural stem cell and radial glia markers like nestin, vimentin and 3CB2 under proliferation conditions. After withdrawal of growth factors, proliferation and expression of v-myc were dramatically reduced and the cells differentiated into astrocytes, oligodendrocytes and neurons. hVM1 cells yield a large number of dopaminergic neurons (about 12% of total cells are TH+) after differentiation, which also produce dopamine. In addition to proneural genes (NGN2, MASH1), differentiated cells show expression of several genuine mesencephalic dopaminergic markers such as: LMX1A, LMX1B, GIRK2, ADH2, NURR1, PITX3, VMAT2 and DAT, indicating that they retain their regional identity. Our data indicate that this cell line and its clonal derivatives may constitute good candidates for the study of development and physiology of human dopaminergic neurons in vitro, and to develop tools for Parkinson's disease cell replacement preclinical research and drug testing.

  15. Superantigen presenting capacity of human astrocytes

    DEFF Research Database (Denmark)

    Hassan-Zahraee, M; Ladiwala, U; Lavoie, P M;

    2000-01-01

    We found that human fetal astrocytes (HFA) are able to support superantigen (SAG) staphylococcal enterotoxin B (SEB) and toxic shock syndrome toxin-1 (TSST-1)-induced activation of immediately ex vivo allogenic human CD4 T cells. Using radiolabelled toxins, we demonstrate that both SEB and TSST-1...... bind with high affinity to MHC class II antigen expressing astrocytes; binding is displaceable with excess cold toxin. Competition experiments further indicate that TSST-1 and SEB at least partially compete with each other for binding to astrocytes suggesting they bind to the same HLA-DR region...

  16. Mutant ubiquitin attenuates interleukin-1β- and tumor necrosis factor-α-induced pro-inflammatory signaling in human astrocytic cells.

    Directory of Open Access Journals (Sweden)

    Kyungsun Choi

    Full Text Available A frameshift mutation of ubiquitin called ubiquitin(+1 (UBB(+1 was found in the aging and Alzheimer's disease brains and thought to be associated with neuronal dysfuction and degeneration. Even though ubiquitylation has been known to regulate vital cellular functions mainly through proteasome-dependent degradation of polyubiquitinated substrates, proteolysis-independent roles of ubiquitylation have emerged as key mechanisms in various signaling cascades. In this study, we have investigated the effect of UBB(+1 on proinflammatory signaling such as interleukin-1β (IL-1β and tumor necrosis factor-α (TNF-α in human astrocytes. Treatment with TNF-α and IL-1β induced expression of CCL2 and CXCL8 by human astrocytic cells; while ectopic expression of UBB(+1 significantly abrogated the proinflammatory cytokine-induced expression of chemokines. Ectopic expression of UBB(+1 suppressed TNF-α- and IL-1β-induced activation of NF-κB and JNK signaling pathway. Furthermore, we have demonstrated that polyubiquitylation of TRAFs and subsequent phosphorylation of TAK1 were significantly inhibited by stable expression of UBB(+1. Collectively, these results suggest that UBB(+1 may affect proinflammatory signaling in the central nervous system via inhibitory mechanisms of ubiquitin-dependent signaling in human astrocytes.

  17. Standards for Cell Line Authentication and Beyond

    Science.gov (United States)

    Cole, Kenneth D.; Plant, Anne L.

    2016-01-01

    Different genomic technologies have been applied to cell line authentication, but only one method (short tandem repeat [STR] profiling) has been the subject of a comprehensive and definitive standard (ASN-0002). Here we discuss the power of this document and why standards such as this are so critical for establishing the consensus technical criteria and practices that can enable progress in the fields of research that use cell lines. We also examine other methods that could be used for authentication and discuss how a combination of methods could be used in a holistic fashion to assess various critical aspects of the quality of cell lines. PMID:27300367

  18. Difference in Membrane Repair Capacity Between Cancer Cell Lines and a Normal Cell Line.

    Science.gov (United States)

    Frandsen, Stine Krog; McNeil, Anna K; Novak, Ivana; McNeil, Paul L; Gehl, Julie

    2016-08-01

    Electroporation-based treatments and other therapies that permeabilize the plasma membrane have been shown to be more devastating to malignant cells than to normal cells. In this study, we asked if a difference in repair capacity could explain this observed difference in sensitivity. Membrane repair was investigated by disrupting the plasma membrane using laser followed by monitoring fluorescent dye entry over time in seven cancer cell lines, an immortalized cell line, and a normal primary cell line. The kinetics of repair in living cells can be directly recorded using this technique, providing a sensitive index of repair capacity. The normal primary cell line of all tested cell lines exhibited the slowest rate of dye entry after laser disruption and lowest level of dye uptake. Significantly, more rapid dye uptake and a higher total level of dye uptake occurred in six of the seven tested cancer cell lines (p electroporation. Viability in the primary normal cell line (98 % viable cells) was higher than in the three tested cancer cell lines (81-88 % viable cells). These data suggest more effective membrane repair in normal, primary cells and supplement previous explanations why electroporation-based therapies and other therapies permeabilizing the plasma membrane are more effective on malignant cells compared to normal cells in cancer treatment. PMID:27312328

  19. Establishment of a hamster lymphoma cell line

    Directory of Open Access Journals (Sweden)

    Abe,Shinji

    1974-08-01

    Full Text Available The establishment of a hamster lymphoma cell line was attempted. Simple mincing and trypsinization of lymphoma tissue resulted in a high degree of cell degeneration. The ascitic tumor cells produced by intraperitoneal transplantation of lymphoma tissue gave a better result. These ascitic cells grew and were cultured successively in medium consisting of RPMI 1640 and 20% fetal calf serum. Cells were round and grew in suspension. Accelerated cell growth was observed one month after starting the culture. In the stained preparations, cells were lymphoblastic. Cells were transplantable into new-born hamsters and produced tumors, but not in young adult hamsters.

  20. Lipopolysaccharide-Induced Apoptosis of Astrocytes: Therapeutic Intervention by Minocycline.

    Science.gov (United States)

    Sharma, Arpita; Patro, Nisha; Patro, Ishan K

    2016-05-01

    Astrocytes are most abundant glial cell type in the brain and play a main defensive role in central nervous system against glutamate-induced toxicity by virtue of numerous transporters residing in their membranes and an astrocyte-specific enzyme glutamine synthetase (GS). In view of that, a dysregulation in the astrocytic activity following an insult may result in glutamate-mediated toxicity accompanied with astrocyte and microglial activation. The present study suggests that the lipopolysaccharide (LPS)-induced inflammation results in significant astrocytic apoptosis compared to other cell types in hippocampus and minocycline could not efficiently restrict the glutamate-mediated toxicity and apoptosis of astrocytes. Upon LPS exposure 76 % astrocytes undergo degeneration followed by 44 % oligodendrocytes, 26 % neurons and 10 % microglia. The pronounced astrocytic apoptosis resulted from the LPS-induced glutamate excitotoxicity leading to their hyperactivation as evident from their hypertrophied morphology, glutamate transporter 1 upregulation and downregulation of GS. Therapeutic minocycline treatment to LPS-infused rats efficiently restricted the inflammatory response and degeneration of other cell types but could not significantly combat with the apoptosis of astrocytes. Our study demonstrates a novel finding on cellular degeneration in the hippocampus revealing more of astrocytic death and suggests a more careful consideration on the protective efficacy of minocycline. PMID:26188416

  1. Susceptibility of cell lines to avian viruses

    Directory of Open Access Journals (Sweden)

    Simoni Isabela Cristina

    1999-01-01

    Full Text Available The susceptibility of the five cell lines - IB-RS-2, RK-13, Vero, BHK-21, CER - to reovirus S1133 and infectious bursal disease virus (IBDV vaccine GBV-8 strain was studied to better define satisfactory and sensitive cell culture systems. Cultures were compared for presence of CPE, virus titers and detection of viral RNA. CPE and viral RNA were detected in CER and BHK-21 cells after reovirus inoculation and in RK-13 cell line after IBDV inoculation and with high virus titers. Virus replication by production of low virus titers occurred in IB-RS-2 and Vero cells with reovirus and in BHK-21 cell line with IBDV.

  2. Anions Govern Cell Volume: A Case Study of Relative Astrocytic and Neuronal Swelling in Spreading Depolarization.

    Directory of Open Access Journals (Sweden)

    Niklas Hübel

    Full Text Available Cell volume changes are ubiquitous in normal and pathological activity of the brain. Nevertheless, we know little about the dynamics of cell and tissue swelling, and the differential changes in the volumes of neurons and glia during pathological states such as spreading depolarizations (SD under ischemic and non-ischemic conditions, and epileptic seizures. By combining the Hodgkin-Huxley type spiking dynamics, dynamic ion concentrations, and simultaneous neuronal and astroglial volume changes into a comprehensive model, we elucidate why glial cells swell more than neurons in SD and the special case of anoxic depolarization (AD, and explore the relative contributions of the two cell types to tissue swelling. Our results demonstrate that anion channels, particularly Cl-, are intrinsically connected to cell swelling and blocking these currents prevents changes in cell volume. The model is based on a simple and physiologically realistic description. We introduce model extensions that are either derived purely from first physical principles of electroneutrality, osmosis, and conservation of particles, or by a phenomenological combination of these principles and known physiological facts. This work provides insights into numerous studies related to neuronal and glial volume changes in SD that otherwise seem contradictory, and is broadly applicable to swelling in other cell types and conditions.

  3. Isolation and Characterization of Ischemia-Derived Astrocytes (IDAs) with Ability to Transactivate Quiescent Astrocytes

    Science.gov (United States)

    Villarreal, Alejandro; Rosciszewski, Gerardo; Murta, Veronica; Cadena, Vanesa; Usach, Vanina; Dodes-Traian, Martin M.; Setton-Avruj, Patricia; Barbeito, Luis H.; Ramos, Alberto J.

    2016-01-01

    Reactive gliosis involving activation and proliferation of astrocytes and microglia, is a widespread but largely complex and graded glial response to brain injury. Astroglial population has a previously underestimated high heterogeneity with cells differing in their morphology, gene expression profile, and response to injury. Here, we identified a subset of reactive astrocytes isolated from brain focal ischemic lesions that show several atypical characteristics. Ischemia-derived astrocytes (IDAs) were isolated from early ischemic penumbra and core. IDA did not originate from myeloid precursors, but rather from pre-existing local progenitors. Isolated IDA markedly differ from primary astrocytes, as they proliferate in vitro with high cell division rate, show increased migratory ability, have reduced replicative senescence and grow in the presence of macrophages within the limits imposed by the glial scar. Remarkably, IDA produce a conditioned medium that strongly induced activation on quiescent primary astrocytes and potentiated the neuronal death triggered by oxygen-glucose deprivation. When re-implanted into normal rat brains, eGFP-IDA migrated around the injection site and induced focal reactive gliosis. Inhibition of gamma secretases or culture on quiescent primary astrocytes monolayers facilitated IDA differentiation to astrocytes. We propose that IDA represent an undifferentiated, pro-inflammatory, highly replicative and migratory astroglial subtype emerging from the ischemic microenvironment that may contribute to the expansion of reactive gliosis. Main Points: Ischemia-derived astrocytes (IDA) were isolated from brain ischemic tissue IDA show reduced replicative senescence, increased cell division and spontaneous migration IDA potentiate death of oxygen-glucose deprived cortical neurons IDA propagate reactive gliosis on quiescent astrocytes in vitro and in vivo Inhibition of gamma secretases facilitates IDA differentiation to astrocytes PMID:27313509

  4. Copper Metabolism of Astrocytes

    Directory of Open Access Journals (Sweden)

    Ralf eDringen

    2013-03-01

    Full Text Available This short review will summarize the current knowledge on the uptake, storage and export of copper ions by astrocytes and will address the potential roles of astrocytes in copper homeostasis in the normal and diseased brain. Astrocytes in culture efficiently accumulate copper by processes that include both the copper transporter Ctr1 and Ctr1-independent mechanisms. Exposure of astrocytes to copper induces an increase in cellular glutathione (GSH content as well as synthesis of metallothioneins, suggesting that excess of copper is stored as complex with GSH and in metallothioneins. Furthermore, exposure of astrocytes to copper accelerates the release of GSH and of glycolytically generated lactate. Astrocytes are able to export copper and express the Menkes protein ATP7A. This protein undergoes reversible, copper-dependent trafficking between the trans-Golgi network and vesicular structures. The ability of astrocytes to efficiently take up, store and export copper suggests that astrocytes play a key role in the supply of neurons with copper and that astrocytes should be considered as target for therapeutic inventions that aim to correct disturbances in brain copper homeostasis.

  5. Dynamic reactive astrocytes after focal ischemia

    OpenAIRE

    Ding, Shinghua

    2014-01-01

    Astrocytes are specialized and most numerous glial cell type in the central nervous system and play important roles in physiology. Astrocytes are also critically involved in many neural disorders including focal ischemic stroke, a leading cause of brain injury and human death. One of the prominent pathological features of focal ischemic stroke is reactive astrogliosis and glial scar formation associated with morphological changes and proliferation. This review paper discusses the recent advan...

  6. Stargazing: Monitoring subcellular dynamics of brain astrocytes.

    Science.gov (United States)

    Benjamin Kacerovsky, J; Murai, K K

    2016-05-26

    Astrocytes are major non-neuronal cell types in the central nervous system that regulate a variety of processes in the brain including synaptic transmission, neurometabolism, and cerebrovasculature tone. Recent discoveries have revealed that astrocytes perform very specialized and heterogeneous roles in brain homeostasis and function. Exactly how astrocytes fulfill such diverse roles in the brain remains to be fully understood and is an active area of research. In this review, we focus on the complex subcellular anatomical features of protoplasmic gray matter astrocytes in the mature, healthy brain that likely empower these cells with the ability to detect and respond to changes in neuronal and synaptic activity. In particular, we discuss how intricate processes on astrocytes allow these cells to communicate with neurons and their synapses and strategically deliver specific cellular organelles such as mitochondria and ribosomes to active compartments within the neuropil. Understanding the properties of these structural elements will lead to a better understanding of how astrocytes function in the healthy and diseased brain. PMID:26162237

  7. Human Pluripotent Stem Cell-Derived Radial Glia Recapitulate Developmental Events and Provide Real-Time Access to Cortical Neurons and Astrocytes

    Science.gov (United States)

    Peng, Chian-Yu; Pan, Liuliu; Kessler, John A.

    2015-01-01

    Studies of human cerebral cortex development are limited by difficulties in accessing and manipulating human neural tissue at specific development stages. We have derived human radial glia (hRG), which are responsible for most cerebral cortex neurogenesis, from human pluripotent stem cells. These hRG display the hallmark morphological, cellular, and molecular features of radial glia in vitro. They can be passaged and generate layer-specific subtypes of cortical neurons in a temporal and passage-dependent fashion. In later passages, they adopt a distinct progenitor phenotype that gives rise to cortical astrocytes and GABAergic interneurons. These hRG are also capable of following developmental cues to engraft, differentiate, migrate, and integrate into the embryonic mouse cortex when injected into E14 lateral ventricles. Moreover, hRG-derived cells can be cryopreserved at specific stages and retain their stage-specific phenotypes and competence when revived. Our study demonstrates that cultured hRG maintain a cell-intrinsic clock that regulates the progressive generation of stage-specific neuronal and glial subtypes. It also describes an easily accessible cell source for studying hRG lineage specification and progression and an on-demand supply of specific cortical neuron subtypes and astrocytes. PMID:25834120

  8. Primary cultures of astrocytes

    DEFF Research Database (Denmark)

    Lange, Sofie C; Bak, Lasse Kristoffer; Waagepetersen, Helle S;

    2012-01-01

    . Such cultures have been an invaluable tool for studying roles of astrocytes in physiological and pathological states. Many central astrocytic functions in metabolism, amino acid neurotransmission and calcium signaling were discovered using this tissue culture preparation and most of these observations were...

  9. Virus Discovery Using Tick Cell Lines

    Science.gov (United States)

    Bell-Sakyi, Lesley; Attoui, Houssam

    2016-01-01

    While ticks have been known to harbor and transmit pathogenic arboviruses for over 80 years, the application of high-throughput sequencing technologies has revealed that ticks also appear to harbor a diverse range of endogenous tick-only viruses belonging to many different families. Almost nothing is known about these viruses; indeed, it is unclear in most cases whether the identified viral sequences are derived from actual replication-competent viruses or from endogenous virus elements incorporated into the ticks’ genomes. Tick cell lines play an important role in virus discovery and isolation through the identification of novel viruses chronically infecting such cell lines and by acting as host cells to aid in determining whether or not an entire replication-competent, infective virus is present in a sample. Here, we review recent progress in tick-borne virus discovery and comment on the actual and potential applications for tick cell lines in this emerging research area. PMID:27679414

  10. Co-culture with microglia promotes neural stem cells differentiation into astrocytes

    Institute of Scientific and Technical Information of China (English)

    GU Feng; WANG Juan; FU Li; MA Yong-jie

    2011-01-01

    Background Neural stem cells (NSCs) are a self-renewing and multipotent population of the central nervous system (CNS),which are active during development and maintain homeostasis and tissue integrity throughout life.Microglias are an immune cell population resident in the CNS,which have crucial physiological functions in the developing and adult CNS.This study aimed to investigate that whether microglia co-cultured with NSCs could promote astrogliogenesis from NSCs.Methods Microglia and NSCs were co-cultured in 24-well insert plates.NSCs were plated in the bottom of the well and microglia in the insert.Fluorescent staining,Western blotting and RT-PCR were used to determine the effect of microglia on NSCs differentiation.Results Co-culture of microglia and NSCs promoted astrogliogenesis from NSCs.Several key genes,such as Notch 1,Notch 2,Notch 3,Hes 5,and NRSFwera downregulated,while the critical genes Id1 and Id2 were upregulated.BMP2 and FGF2 were upregulated.Conclusion Microglias act as a regulator of NSCs astrogliogenesis.

  11. Astrocytic Vesicle Mobility in Health and Disease

    Directory of Open Access Journals (Sweden)

    Robert Zorec

    2013-05-01

    Full Text Available Astrocytes are no longer considered subservient to neurons, and are, instead, now understood to play an active role in brain signaling. The intercellular communication of astrocytes with neurons and other non-neuronal cells involves the exchange of molecules by exocytotic and endocytotic processes through the trafficking of intracellular vesicles. Recent studies of single vesicle mobility in astrocytes have prompted new views of how astrocytes contribute to information processing in nervous tissue. Here, we review the trafficking of several types of membrane-bound vesicles that are specifically involved in the processes of (i intercellular communication by gliotransmitters (glutamate, adenosine 5'-triphosphate, atrial natriuretic peptide, (ii plasma membrane exchange of transporters and receptors (EAAT2, MHC-II, and (iii the involvement of vesicle mobility carrying aquaporins (AQP4 in water homeostasis. The properties of vesicle traffic in astrocytes are discussed in respect to networking with neighboring cells in physiologic and pathologic conditions, such as amyotrophic lateral sclerosis, multiple sclerosis, and states in which astrocytes contribute to neuroinflammatory conditions.

  12. Astrocytic vesicle mobility in health and disease.

    Science.gov (United States)

    Potokar, Maja; Vardjan, Nina; Stenovec, Matjaž; Gabrijel, Mateja; Trkov, Saša; Jorgačevski, Jernej; Kreft, Marko; Zorec, Robert

    2013-01-01

    Astrocytes are no longer considered subservient to neurons, and are, instead, now understood to play an active role in brain signaling. The intercellular communication of astrocytes with neurons and other non-neuronal cells involves the exchange of molecules by exocytotic and endocytotic processes through the trafficking of intracellular vesicles. Recent studies of single vesicle mobility in astrocytes have prompted new views of how astrocytes contribute to information processing in nervous tissue. Here, we review the trafficking of several types of membrane-bound vesicles that are specifically involved in the processes of (i) intercellular communication by gliotransmitters (glutamate, adenosine 5'-triphosphate, atrial natriuretic peptide), (ii) plasma membrane exchange of transporters and receptors (EAAT2, MHC-II), and (iii) the involvement of vesicle mobility carrying aquaporins (AQP4) in water homeostasis. The properties of vesicle traffic in astrocytes are discussed in respect to networking with neighboring cells in physiologic and pathologic conditions, such as amyotrophic lateral sclerosis, multiple sclerosis, and states in which astrocytes contribute to neuroinflammatory conditions.

  13. Transcriptomic analyses of primary astrocytes under TNFα treatment

    OpenAIRE

    Birck, Cindy; Koncina, Eric; Heurtaux, Tony; Glaab, Enrico; Michelucci, Alessandro; Heuschling, Paul; Grandbarbe, Luc

    2016-01-01

    Astrocytes, the most abundant glial cell population in the central nervous system, have important functional roles in the brain as blood brain barrier maintenance, synaptic transmission or intercellular communications [1], [2]. Numerous studies suggested that astrocytes exhibit a functional and morphological high degree of plasticity. For example, following any brain injury, astrocytes become reactive and hypertrophic. This phenomenon, also called reactive gliosis, is characterized by a set o...

  14. The Neurogenic Potential of Astrocytes Is Regulated by Inflammatory Signals.

    Science.gov (United States)

    Michelucci, Alessandro; Bithell, Angela; Burney, Matthew J; Johnston, Caroline E; Wong, Kee-Yew; Teng, Siaw-Wei; Desai, Jyaysi; Gumbleton, Nigel; Anderson, Gregory; Stanton, Lawrence W; Williams, Brenda P; Buckley, Noel J

    2016-08-01

    Although the adult brain contains neural stem cells (NSCs) that generate new neurons throughout life, these astrocyte-like populations are restricted to two discrete niches. Despite their terminally differentiated phenotype, adult parenchymal astrocytes can re-acquire NSC-like characteristics following injury, and as such, these 'reactive' astrocytes offer an alternative source of cells for central nervous system (CNS) repair following injury or disease. At present, the mechanisms that regulate the potential of different types of astrocytes are poorly understood. We used in vitro and ex vivo astrocytes to identify candidate pathways important for regulation of astrocyte potential. Using in vitro neural progenitor cell (NPC)-derived astrocytes, we found that exposure of more lineage-restricted astrocytes to either tumor necrosis factor alpha (TNF-α) (via nuclear factor-κB (NFκB)) or the bone morphogenetic protein (BMP) inhibitor, noggin, led to re-acquisition of NPC properties accompanied by transcriptomic and epigenetic changes consistent with a more neurogenic, NPC-like state. Comparative analyses of microarray data from in vitro-derived and ex vivo postnatal parenchymal astrocytes identified several common pathways and upstream regulators associated with inflammation (including transforming growth factor (TGF)-β1 and peroxisome proliferator-activated receptor gamma (PPARγ)) and cell cycle control (including TP53) as candidate regulators of astrocyte phenotype and potential. We propose that inflammatory signalling may control the normal, progressive restriction in potential of differentiating astrocytes as well as under reactive conditions and represent future targets for therapies to harness the latent neurogenic capacity of parenchymal astrocytes. PMID:26138449

  15. Investigation of the selenium metabolism in cancer cell lines

    DEFF Research Database (Denmark)

    Lunøe, Kristoffer; Gabel-Jensen, Charlotte; Stürup, Stefan;

    2011-01-01

    The aim of this work was to compare different selenium species for their ability to induce cell death in different cancer cell lines, while investigating the underlying chemistry by speciation analysis. A prostate cancer cell line (PC-3), a colon cancer cell line (HT-29) and a leukaemia cell line...

  16. Transient acidification and subsequent proinflammatory cytokine stimulation of astrocytes induce distinct activation phenotypes

    OpenAIRE

    Renner, Nicole A.; Sansing, Hope A.; Inglis, Fiona M; Mehra, Smriti; Kaushal, Deepak; Lackner, Andrew A; Andrew G MacLean

    2013-01-01

    The foot processes of astrocytes cover over 60% of the surface of brain microvascular endothelial cells, regulating tight junction integrity. Retraction of astrocyte foot processes has been postulated to be a key mechanism in pathology. Therefore, movement of an astrocyte in response to a proinflammatory cytokine or even limited retraction of processes would result in leaky junctions between endothelial cells. Astrocytes lie at the gateway to the CNS and are instrumental in controlling leukoc...

  17. Astrocyte Aquaporin Dynamics in Health and Disease.

    Science.gov (United States)

    Potokar, Maja; Jorgačevski, Jernej; Zorec, Robert

    2016-01-01

    The family of aquaporins (AQPs), membrane water channels, consists of diverse types of proteins that are mainly permeable to water; some are also permeable to small solutes, such as glycerol and urea. They have been identified in a wide range of organisms, from microbes to vertebrates and plants, and are expressed in various tissues. Here, we focus on AQP types and their isoforms in astrocytes, a major glial cell type in the central nervous system (CNS). Astrocytes have anatomical contact with the microvasculature, pia, and neurons. Of the many roles that astrocytes have in the CNS, they are key in maintaining water homeostasis. The processes involved in this regulation have been investigated intensively, in particular regulation of the permeability and expression patterns of different AQP types in astrocytes. Three aquaporin types have been described in astrocytes: aquaporins AQP1 and AQP4 and aquaglyceroporin AQP9. The aim here is to review their isoforms, subcellular localization, permeability regulation, and expression patterns in the CNS. In the human CNS, AQP4 is expressed in normal physiological and pathological conditions, but astrocytic expression of AQP1 and AQP9 is mainly associated with a pathological state. PMID:27420057

  18. Targeting astrocytes in bipolar disorder.

    Science.gov (United States)

    Peng, Liang; Li, Baoman; Verkhratsky, Alexei

    2016-06-01

    Astrocytes are homeostatic cells of the central nervous system, which are critical for development and maintenance of synaptic transmission and hence of synaptically connected neuronal ensembles. Astrocytic densities are reduced in bipolar disorder, and therefore deficient astroglial function may contribute to overall disbalance in neurotransmission and to pathological evolution. Classical anti-bipolar drugs (lithium salts, valproic acid and carbamazepine) affect expression of astroglial genes and modify astroglial signalling and homeostatic cascades. Many effects of both antidepressant and anti-bipolar drugs are exerted through regulation of glutamate homeostasis and glutamatergic transmission, through K(+) buffering, through regulation of calcium-dependent phospholipase A2 (that controls metabolism of arachidonic acid) or through Ca(2+) homeostatic and signalling pathways. Sometimes anti-depressant and anti-bipolar drugs exert opposite effects, and some effects on gene expression in drug treated animals are opposite in neurones vs. astrocytes. Changes in the intracellular pH induced by anti-bipolar drugs affect uptake of myo-inositol and thereby signalling via inositoltrisphosphate (InsP3), this being in accord with one of the main theories of mechanism of action for these drugs. PMID:27015045

  19. 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.

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

    International Nuclear Information System (INIS)

    Research highlights: → Four oral squamous cancer cell lines (OSCCL) were analyzed for cancer stem cells (CSCs). → Single cell derived colonies of OSCCL express CSC-marker CD133 differentially. → Monoclonal cell lines showed reduced sensitivity for Paclitaxel. → In situ CD133+ cells are slow cycling (Ki67-) indicating a reduced drug sensitivity. → CD133+ 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+ 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.

  1. Cholinergic properties of neurons differentiated from an embryonal carcinoma cell-line (P19).

    Science.gov (United States)

    Parnas, D; Linial, M

    1995-11-01

    P19 is a mouse-derived embryonal carcinoma cell-line capable of differentiation toward ectodermal, mesodermal and endodermal lineages. Following treatment with retinoic acid these cells differentiate into neurons, astrocytes and fibroblast-like cells. We induced P19 differentiation under conditions which lead to a homogeneous neuronal culture (> 95% neurons). Under these conditions, most cells (approximately 85%) express high levels of the cholinergic markers acetyl cholinesterase and choline acetyltransferase while approximately 10% of cells express the GABAergic marker glutamic acid decarboxylase. While the proportion of the GABAergic neurons is constant at different culture conditions, the cholinergic phenotype is suppressed at high cell densities. The cholinergic nature of P19 neurons is also evident in their ability to form contacts with a muscle cell-line--C2. At day 10 of differentiation cells are capable of depolarization-dependent acetylcholine release. The release is Ca2+ dependent, and drops to baseline levels at 0.5 mM Ca2+. The cells also respond to sub-nM levels of alpha-latrotoxin by acetylcholine release. All major proteins implicated in synapse functionality are expressed prior to day 10 at both at RNA and protein levels. However, the expression pattern of each gene is unique. The genes include cytoskeletal proteins, synaptic vesicle proteins and terminal specific proteins. We suggest that this cell-line can serve as an in-vitro model system for the study of neuronal phenotype acquisition. Under our conditions, the P19 cells can also provide a system in which to study the differentiation of functional cholinergic neurons. PMID:8787867

  2. Optical modulation of astrocyte network using ultrashort pulsed laser

    Science.gov (United States)

    Yoon, Jonghee; Ku, Taeyun; Chong, Kyuha; Ryu, Seung-Wook; Choi, Chulhee

    2012-03-01

    Astrocyte, the most abundant cell type in the central nervous system, has been one of major topics in neuroscience. Even though many tools have been developed for the analysis of astrocyte function, there has been no adequate tool that can modulates astrocyte network without pharmaceutical or genetic interventions. Here we found that ultrashort pulsed laser stimulation can induce label-free activation of astrocytes as well as apoptotic-like cell death in a dose-dependent manner. Upon irradiation with high intensity pulsed lasers, the irradiated cells with short exposure time showed very rapid mitochondria fragmentation, membrane blebbing and cytoskeletal retraction. We applied this technique to investigate in vivo function of astrocyte network in the CNS: in the aspect of neurovascular coupling and blood-brain barrier. We propose that this noninvasive technique can be widely applied for in vivo study of complex cellular network.

  3. Spatial and temporal single-cell volume estimation by a fluorescence imaging technique with application to astrocytes in primary culture

    Science.gov (United States)

    Khatibi, Siamak; Allansson, Louise; Gustavsson, Tomas; Blomstrand, Fredrik; Hansson, Elisabeth; Olsson, Torsten

    1999-05-01

    Cell volume changes are often associated with important physiological and pathological processes in the cell. These changes may be the means by which the cell interacts with its surrounding. Astroglial cells change their volume and shape under several circumstances that affect the central nervous system. Following an incidence of brain damage, such as a stroke or a traumatic brain injury, one of the first events seen is swelling of the astroglial cells. In order to study this and other similar phenomena, it is desirable to develop technical instrumentation and analysis methods capable of detecting and characterizing dynamic cell shape changes in a quantitative and robust way. We have developed a technique to monitor and to quantify the spatial and temporal volume changes in a single cell in primary culture. The technique is based on two- and three-dimensional fluorescence imaging. The temporal information is obtained from a sequence of microscope images, which are analyzed in real time. The spatial data is collected in a sequence of images from the microscope, which is automatically focused up and down through the specimen. The analysis of spatial data is performed off-line and consists of photobleaching compensation, focus restoration, filtering, segmentation and spatial volume estimation.

  4. A Digital Realization of Astrocyte and Neural Glial Interactions.

    Science.gov (United States)

    Hayati, Mohsen; Nouri, Moslem; Haghiri, Saeed; Abbott, Derek

    2016-04-01

    The implementation of biological neural networks is a key objective of the neuromorphic research field. Astrocytes are the largest cell population in the brain. With the discovery of calcium wave propagation through astrocyte networks, now it is more evident that neuronal networks alone may not explain functionality of the strongest natural computer, the brain. Models of cortical function must now account for astrocyte activities as well as their relationships with neurons in encoding and manipulation of sensory information. From an engineering viewpoint, astrocytes provide feedback to both presynaptic and postsynaptic neurons to regulate their signaling behaviors. This paper presents a modified neural glial interaction model that allows a convenient digital implementation. This model can reproduce relevant biological astrocyte behaviors, which provide appropriate feedback control in regulating neuronal activities in the central nervous system (CNS). Accordingly, we investigate the feasibility of a digital implementation for a single astrocyte constructed by connecting a two coupled FitzHugh Nagumo (FHN) neuron model to an implementation of the proposed astrocyte model using neuron-astrocyte interactions. Hardware synthesis, physical implementation on FPGA, and theoretical analysis confirm that the proposed neuron astrocyte model, with significantly low hardware cost, can mimic biological behavior such as the regulation of postsynaptic neuron activity and the synaptic transmission mechanisms. PMID:26390499

  5. A “Hit and Run” Approach to Inducible Direct Reprogramming of Astrocytes to Neural Stem Cells

    OpenAIRE

    Poulou, Maria; Mandalos, Nikolaos P.; Karnavas, Theodoros; Saridaki, Marannia; Ronald D G McKay; Remboutsika, Eumorphia

    2016-01-01

    Temporal and spatial control of gene expression can be achieved using an inducible system as a fundamental tool for regulated transcription in basic, applied and eventually in clinical research. We describe a novel “hit and run” inducible direct reprogramming approach. In a single step, 2 days post-transfection, transiently transfected Sox2FLAG under the Leu3p-αIPM inducible control (iSox2) triggers the activation of endogenous Sox2, redirecting primary astrocytes into abundant distinct nesti...

  6. Don't fence me in: Harnessing the beneficial roles of astrocytes for spinal cord repair

    OpenAIRE

    White, Robin E.; Jakeman, Lyn B.

    2008-01-01

    Astrocytes comprise a heterogeneous cell population that plays a complex role in repair after spinal cord injury. Reactive astrocytes are major contributors to the glial scar that is a physical and chemical barrier to axonal regeneration. Yet, consistent with a supportive role in development, astrocytes secrete neurotrophic factors and protect neurons and glia spared by the injury. In development and after injury, local cues are modulators of astrocyte phenotype and function. When multipotent...

  7. In vivo astrocytic Ca2+ signaling in health and brain disorders

    OpenAIRE

    Ding, Shinghua

    2013-01-01

    Astrocytes are the predominant glial cell type in the CNS. Although astrocytes are electrically nonexcitable, their excitability is manifested by their Ca2+ signaling, which serves as a mediator of neuron–glia bidirectional interactions via tripartite synapses. Studies from in vivo two-photon imaging indicate that in healthy animals, the properties of spontaneous astrocytic Ca2+ signaling are affected by animal species, age, wakefulness and the location of astrocytes in the brain. Intercellul...

  8. Spinal astrocyte gap junctions contribute to oxaliplatin-induced mechanical hypersensitivity

    OpenAIRE

    Yoon, Seo-Yeon; Robinson, Caleb R.; Zhang, Haijun; Dougherty, Patrick M.

    2013-01-01

    Spinal glial cells contribute to the development of many types of inflammatory and neuropathic pain. Here the contribution of spinal astrocytes and astrocyte gap junctions to oxaliplatin-induced mechanical hypersensitivity was explored. The expression of glial fibrillary acidic protein (GFAP) in spinal dorsal horn was significantly increased at day 7 but recovered at day 14 after oxaliplatin treatment, suggesting a transient activation of spinal astrocytes by chemotherapy. Astrocyte-specific ...

  9. Low-level bisphenol A increases production of glial fibrillary acidic protein in differentiating astrocyte progenitor cells through excessive STAT3 and Smad1 activation

    International Nuclear Information System (INIS)

    The effects of bisphenol A (BPA) on the differentiation of serum-free mouse embryo (SFME) cells, the astrocyte progenitor cells in the central nervous system, were examined. SFME cells were exposed to 10 ng/ml leukemia inhibitory factor (LIF) and 10 ng/ml bone morphogenetic protein 2 (BMP2) to increase glial fibrillary acidic protein (GFAP) expression and induce cell differentiation. Various concentrations of BPA (0.1 pg/ml-1 μg/ml) were then added to determine their effects on the cell differentiation. SFME cells were effectively differentiated by LIF and BMP2 in completely serum-free cultures. Cell proliferation following cell differentiation was not significantly affected by low-level BPA. However, GFAP expression was significantly increased in SFME cells in the presence of 1-100 pg/ml BPA. These increases were due to excessive activation of signal transducer and activator of transcription 3 (STAT3) and mothers against decapentaplegic homolog 1 (Smad1) by the low-level BPA

  10. Astrocytic gap junctional communication is reduced in amyloid-β-treated cultured astrocytes, but not in Alzheimer's disease transgenic mice

    Directory of Open Access Journals (Sweden)

    Gerald A Dienel

    2010-08-01

    Full Text Available Alzheimer's disease is characterized by accumulation of amyloid deposits in brain, progressive cognitive deficits and reduced glucose utilization. Many consequences of the disease are attributed to neuronal dysfunction, but roles of astrocytes in its pathogenesis are not well understood. Astrocytes are extensively coupled via gap junctions, and abnormal trafficking of metabolites and signalling molecules within astrocytic syncytia could alter functional interactions among cells comprising the neurovascular unit. To evaluate the influence of amyloid-β on astrocyte gap junctional communication, cultured astrocytes were treated with monomerized amyloid-β1–40 (1 μmol/l for intervals ranging from 2 h to 5 days, and the areas labelled by test compounds were determined by impaling a single astrocyte with a micropipette and diffusion of material into coupled cells. Amyloid-β-treated astrocytes had rapid, sustained 50–70% reductions in the area labelled by Lucifer Yellow, anionic Alexa Fluor® dyes and energy-related compounds, 6-NBDG (a fluorescent glucose analogue, NADH and NADPH. Amyloid-β treatment also caused a transient increase in oxidative stress. In striking contrast with these results, spreading of Lucifer Yellow within astrocytic networks in brain slices from three regions of 8.5–14-month-old control and transgenic Alzheimer's model mice was variable, labelling 10–2000 cells; there were no statistically significant differences in the number of dye-labelled cells among the groups or with age. Thus amyloid-induced dysfunction of gap junctional communication in cultured astrocytes does not reflect the maintenance of dye transfer through astrocytic syncytial networks in transgenic mice; the pathophysiology of Alzheimer's disease is not appropriately represented by the cell culture system.

  11. Sodium signaling and astrocyte energy metabolism

    KAUST Repository

    Chatton, Jean-Yves

    2016-03-31

    The Na+ gradient across the plasma membrane is constantly exploited by astrocytes as a secondary energy source to regulate the intracellular and extracellular milieu, and discard waste products. One of the most prominent roles of astrocytes in the brain is the Na+-dependent clearance of glutamate released by neurons during synaptic transmission. The intracellular Na+ load collectively generated by these processes converges at the Na,K-ATPase pump, responsible for Na+ extrusion from the cell, which is achieved at the expense of cellular ATP. These processes represent pivotal mechanisms enabling astrocytes to increase the local availability of metabolic substrates in response to neuronal activity. This review presents basic principles linking the intracellular handling of Na+ following activity-related transmembrane fluxes in astrocytes and the energy metabolic pathways involved. We propose a role of Na+ as an energy currency and as a mediator of metabolic signals in the context of neuron-glia interactions. We further discuss the possible impact of the astrocytic syncytium for the distribution and coordination of the metabolic response, and the compartmentation of these processes in cellular microdomains and subcellular organelles. Finally, we illustrate future avenues of investigation into signaling mechanisms aimed at bridging the gap between Na+ and the metabolic machinery. © 2016 Wiley Periodicals, Inc.

  12. Sodium signaling and astrocyte energy metabolism.

    Science.gov (United States)

    Chatton, Jean-Yves; Magistretti, Pierre J; Barros, L Felipe

    2016-10-01

    The Na(+) gradient across the plasma membrane is constantly exploited by astrocytes as a secondary energy source to regulate the intracellular and extracellular milieu, and discard waste products. One of the most prominent roles of astrocytes in the brain is the Na(+) -dependent clearance of glutamate released by neurons during synaptic transmission. The intracellular Na(+) load collectively generated by these processes converges at the Na,K-ATPase pump, responsible for Na(+) extrusion from the cell, which is achieved at the expense of cellular ATP. These processes represent pivotal mechanisms enabling astrocytes to increase the local availability of metabolic substrates in response to neuronal activity. This review presents basic principles linking the intracellular handling of Na(+) following activity-related transmembrane fluxes in astrocytes and the energy metabolic pathways involved. We propose a role of Na(+) as an energy currency and as a mediator of metabolic signals in the context of neuron-glia interactions. We further discuss the possible impact of the astrocytic syncytium for the distribution and coordination of the metabolic response, and the compartmentation of these processes in cellular microdomains and subcellular organelles. Finally, we illustrate future avenues of investigation into signaling mechanisms aimed at bridging the gap between Na(+) and the metabolic machinery. GLIA 2016;64:1667-1676. PMID:27027636

  13. Astrocytes Grown in Alvetex(®) Three Dimensional Scaffolds Retain a Non-reactive Phenotype.

    Science.gov (United States)

    Ugbode, Christopher I; Hirst, Warren D; Rattray, Marcus

    2016-08-01

    Protocols which permit the extraction of primary astrocytes from either embryonic or postnatal mice are well established however astrocytes in culture are different to those in the mature CNS. Three dimensional (3D) cultures, using a variety of scaffolds may enable better phenotypic properties to be developed in culture. We present data from embryonic (E15) and postnatal (P4) murine primary cortical astrocytes grown on coated coverslips or a 3D polystyrene scaffold, Alvetex. Growth of both embryonic and postnatal primary astrocytes in the 3D scaffold changed astrocyte morphology to a mature, protoplasmic phenotype. Embryonic-derived astrocytes in 3D expressed markers of mature astrocytes, namely the glutamate transporter GLT-1 with low levels of the chondroitin sulphate proteoglycans, NG2 and SMC3. Embryonic astrocytes derived in 3D show lower levels of markers of reactive astrocytes, namely GFAP and mRNA levels of LCN2, PTX3, Serpina3n and Cx43. Postnatal-derived astrocytes show few protein changes between 2D and 3D conditions. Our data shows that Alvetex is a suitable scaffold for growth of astrocytes, and with appropriate choice of cells allows the maintenance of astrocytes with the properties of mature cells and a non-reactive phenotype. PMID:27099962

  14. In vitro differentiation of human adipose-derived adult stromal cells into neuron-like cells in hippocampal astrocyte conditioned medium

    Institute of Scientific and Technical Information of China (English)

    Xinchun Ye; Hongjun He; Feng Yang; Kepeng Zhao; Jun Yao; Bin Liu

    2006-01-01

    BACKGROUND: At present, researches on differentiating from human adipose-derived adult stromal cells (hADASC) to neuron-like cells are focus on inducing by artificial-synthetic compound solution;however,hippocampal astrocyte conditioned medium(HCAM)can induce in vitro differentiation from hADASC to neuron-like cells is still unclear.OBJECTIVE:To observe whether HCAM can induce in vitro differentiation from hADASC to neuron-like cells.DESIGN:Randomized control study.SETTING:Department of Neurology,Taixing People's Hospital;Central Laboratory,North China Coal Medical College.MATERIALS:Donor of adipose tissue was donated by female volunteers suffering from caesarean section in the department of obstetrics & gynecology in our hospital and aged 20-35 years. Adipose tissue was collected from subcutaneous tissue of abdomen during the operation.In addition.8 male newborn Wistar rats within 24 hours with average body mass of 20 g were provided by Animal Institute of Chinese Academy of Medical Sciences.Rabbit-anti-human Nestin polyclonal antibody.Rabbit-anti-human glial fibriliary acidic protein (GFAP)polyclonal antibody, rabbit-anti-human neuro-specific enolase polyclonal antibody and mouse-anti-human microtubal associated protein 2(MAP-2)polyclonal antibody were provided by Wuhan Boster Company.METHODS:The experiment was carried out in the Central Laboratory of North China Coal Medical College from October 2004 to June 2005.hADASC was cultured with HCAM and its growth and morphological changes were observed under inverted phase contrast microscope.Immunocytochemistry.immunofluorescence and Western blotting were used to evaluate the expressions of Nestin,which was a specific sign of nerve precursor,neuro-specific enolase and MAP-2,which was a specific sign of nerve cell,and GFAP,which was a specific sign of neuroglial cells.MAIN OUTCOME MEASURES:Nestin,which was a specific sign of nerve precursor,neuro-specific enolase and MAP-2,which was a specific sign of nerve cell

  15. Culturing of PC12 Cells, Neuronal Cells, Astrocytes Cultures and Brain Slices in an Open Microfluidic System

    DEFF Research Database (Denmark)

    Al Atraktchi, Fatima Al-Zahraa; Bakmand, Tanya; Rømer Sørensen, Ane;

    The brain is the center of the nervous system, where serious neurodegenerative diseases such as Parkinson’s, Alzheimer’s and Huntington’s are products of functional loss in the neural cells (1). Typical techniques used to investigate these diseases lack precise control of the cellular surroundings...

  16. Selenoprotein S expression in reactive astrocytes following brain injury.

    Science.gov (United States)

    Fradejas, Noelia; Serrano-Pérez, Maria Del Carmen; Tranque, Pedro; Calvo, Soledad

    2011-06-01

    Selenoprotein S (SelS) is an endoplasmic reticulum (ER)-resident protein involved in the unfolded protein response. Besides reducing ER-stress, SelS attenuates inflammation by decreasing pro-inflammatory cytokines. We have recently shown that SelS is responsive to ischemia in cultured astrocytes. To check the possible association of SelS with astrocyte activation, here we investigate the expression of SelS in two models of brain injury: kainic acid (KA) induced excitotoxicity and cortical mechanical lesion. The regulation of SelS and its functional consequences for neuroinflammation, ER-stress, and cell survival were further analyzed using cultured astrocytes from mouse and human. According to our immunofluorescence analysis, SelS expression is prominent in neurons and hardly detectable in astrocytes from control mice. However, brain injury intensely upregulates SelS, specifically in reactive astrocytes. SelS induction by KA was evident at 12 h and faded out after reaching maximum levels at 3-4 days. Analysis of mRNA and protein expression in cultured astrocytes showed SelS upregulation by inflammatory stimuli as well as ER-stress inducers. In turn, siRNA-mediated SelS silencing combined with adenoviral overexpression assays demonstrated that SelS reduces ER-stress markers CHOP and spliced XBP-1, as well as inflammatory cytokines IL-1β and IL-6 in stimulated astrocytes. SelS overexpression increased astrocyte resistance to ER-stress and inflammatory stimuli. Conversely, SelS suppression compromised astrocyte viability. In summary, our results reveal the upregulation of SelS expression in reactive astrocytes, as well as a new protective role for SelS against inflammation and ER-stress that can be relevant to astrocyte function in the context of inflammatory neuropathologies. PMID:21456042

  17. Susceptibility testing of fish cell lines for virus isolation

    DEFF Research Database (Denmark)

    Ariel, Ellen; Skall, Helle Frank; Olesen, Niels Jørgen

    2009-01-01

    compare susceptibility between cell lines and between lineages within a laboratory and between laboratories (Inter-laboratory Proficiency Test). The objective being that the most sensitive cell line and lineages are routinely selected for diagnostic purposes.In comparing cell lines, we simulated "non......-cell-culture-adapted" virus by propagating the virus in heterologous cell lines to the one tested. A stock of test virus was produced and stored at - 80 °C and tests were conducted biannually. This procedure becomes complicated when several cell lines are in use and does not account for variation among lineages. In comparing...... cell lineages, we increased the number of isolates of each virus, propagated stocks in a given cell line and tested all lineages of that line in use in the laboratory. Testing of relative cell line susceptibility between laboratories is carried out annually via the Inter-laboratory Proficiency Test...

  18. Glucose-coated gold nanoparticles transfer across human brain endothelium and enter astrocytes in vitro.

    Directory of Open Access Journals (Sweden)

    Radka Gromnicova

    Full Text Available The blood-brain barrier prevents the entry of many therapeutic agents into the brain. Various nanocarriers have been developed to help agents to cross this barrier, but they all have limitations, with regard to tissue-selectivity and their ability to cross the endothelium. This study investigated the potential for 4 nm coated gold nanoparticles to act as selective carriers across human brain endothelium and subsequently to enter astrocytes. The transfer rate of glucose-coated gold nanoparticles across primary human brain endothelium was at least three times faster than across non-brain endothelia. Movement of these nanoparticles occurred across the apical and basal plasma membranes via the cytosol with relatively little vesicular or paracellular migration; antibiotics that interfere with vesicular transport did not block migration. The transfer rate was also dependent on the surface coating of the nanoparticle and incubation temperature. Using a novel 3-dimensional co-culture system, which includes primary human astrocytes and a brain endothelial cell line hCMEC/D3, we demonstrated that the glucose-coated nanoparticles traverse the endothelium, move through the extracellular matrix and localize in astrocytes. The movement of the nanoparticles through the matrix was >10 µm/hour and they appeared in the nuclei of the astrocytes in considerable numbers. These nanoparticles have the correct properties for efficient and selective carriers of therapeutic agents across the blood-brain barrier.

  19. In vitro study of uptake and synthesis of creatine and its precursors by cerebellar granule cells and astrocytes suggests some hypotheses on the physiopathology of the inherited disorders of creatine metabolism

    Directory of Open Access Journals (Sweden)

    Carducci Claudia

    2012-04-01

    Full Text Available Abstract Background The discovery of the inherited disorders of creatine (Cr synthesis and transport in the last few years disclosed the importance of blood Cr supply for the normal functioning of the brain. These putatively rare diseases share a common pathogenetic mechanism (the depletion of brain Cr and similar phenotypes characterized by mental retardation, language disturbances, seizures and movement disorders. In the effort to improve our knowledge on the mechanisms regulating Cr pool inside the nervous tissue, Cr transport and synthesis and related gene transcripts were explored in primary cultures of rat cerebellar granule cells and astrocytes. Methods Cr uptake and synthesis were explored in vitro by incubating monotypic primary cultures of rat type I astrocytes and cerebellar granule cells with: a D3-Creatine (D3Cr and D3Cr plus β-guanidinopropionate (GPA, an inhibitor of Cr transporter, and b labelled precursors of Guanidinoacetate (GAA and Cr (Arginine, Arg; Glycine, Gly. Intracellular D3Cr and labelled GAA and Cr were assessed by ESI-MS/MS. Creatine transporter (CT1, L-arginine:glycine amidinotransferase (AGAT, and S-adenosylmethionine:guanidinoacetate N-methyltransferase (GAMT gene expression was assessed in the same cells by real time PCR. Results D3Cr signal was extremely high in cells incubated with this isotope (labelled/unlabelled Cr ratio reached about 10 and 122, respectively in cerebellar granule cells and astrocytes and was reduced by GPA. Labelled Arg and Gly were taken up by the cells and incorporated in GAA, whose concentration paralleled that of these precursors both in the extracellular medium and inside the cells (astrocytes. In contrast, the increase of labelled Cr was relatively much more limited since labelled Cr after precursors' supplementation did not exceed 2,7% (cerebellar granule cells and 21% (astrocytes of unlabelled Cr. Finally, AGAT, GAMT and SLC6A8 were expressed in both kind of cells. Conclusions Our

  20. Regulatory networks define phenotypic classes of human stem cell lines

    OpenAIRE

    Müller, Franz-Josef; Louise C. Laurent; Kostka, Dennis; Ulitsky, Igor; Williams, Roy; Lu, Christina; Park, In-Hyun; Rao, Mahendra S.; Shamir, Ron; Philip H. Schwartz; Schmidt, Nils O.; Loring, Jeanne F.

    2008-01-01

    Stem cells are defined as self-renewing cell populations that can differentiate into multiple distinct cell types. However, hundreds of different human cell lines from embryonic, fetal, and adult sources have been called stem cells, even though they range from pluripotent cells, typified by embryonic stem cells, which are capable of virtually unlimited proliferation and differentiation, to adult stem cell lines, which can generate a far more limited repertory of differentiated cell types. The...

  1. Paracrine effect of carbon monoxide - astrocytes promote neuroprotection through purinergic signaling in mice.

    Science.gov (United States)

    Queiroga, Cláudia S F; Alves, Raquel M A; Conde, Sílvia V; Alves, Paula M; Vieira, Helena L A

    2016-08-15

    The neuroprotective role of carbon monoxide (CO) has been studied in a cell-autonomous mode. Herein, a new concept is disclosed - CO affects astrocyte-neuron communication in a paracrine manner to promote neuroprotection. Neuronal survival was assessed when co-cultured with astrocytes that had been pre-treated or not with CO. The CO-pre-treated astrocytes reduced neuronal cell death, and the cellular mechanisms were investigated, focusing on purinergic signaling. CO modulates astrocytic metabolism and extracellular ATP content in the co-culture medium. Moreover, several antagonists of P1 adenosine and P2 ATP receptors partially reverted CO-induced neuroprotection through astrocytes. Likewise, knocking down expression of the neuronal P1 adenosine receptor A2A-R (encoded by Adora2a) reverted the neuroprotective effects of CO-exposed astrocytes. The neuroprotection of CO-treated astrocytes also decreased following prevention of ATP or adenosine release from astrocytic cells and inhibition of extracellular ATP metabolism into adenosine. Finally, the neuronal downstream event involves TrkB (also known as NTRK2) receptors and BDNF. Pharmacological and genetic inhibition of TrkB receptors reverts neuroprotection triggered by CO-treated astrocytes. Furthermore, the neuronal ratio of BDNF to pro-BDNF increased in the presence of CO-treated astrocytes and decreased whenever A2A-R expression was silenced. In summary, CO prevents neuronal cell death in a paracrine manner by targeting astrocytic metabolism through purinergic signaling. PMID:27383770

  2. GABA(A) Increases Calcium in Subventricular Zone Astrocyte-Like Cells Through L- and T-Type Voltage-Gated Calcium Channels

    DEFF Research Database (Denmark)

    Young, Stephanie Z; Platel, Jean-Claude; Nielsen, Jakob V;

    2010-01-01

    induced Ca(2+) increases in 40-50% of SVZ astrocytes. GABA(A)-induced Ca(2+) increases were prevented with nifedipine and mibefradil, blockers of L- and T-type voltage-gated calcium channels (VGCC). The L-type Ca(2+) channel activator BayK 8644 increased the percentage of GABA(A)-responding astrocyte...

  3. Down-Regulation of Neurocan Expression in Reactive Astrocytes Promotes Axonal Regeneration and Facilitates the Neurorestorative Effects of Bone Marrow Stromal Cells in the Ischemic Rat Brain

    Institute of Scientific and Technical Information of China (English)

    LI HONG SHEN; YI LI; QI GAO; SMITA SAVANT-BHONSALE; MICHAEL CHOPP

    2008-01-01

    脑卒中后缺血组织边界形成胶质疤痕,抑制轴突再生.神经蛋白聚糖是一种轴突延长抑制分子,在卒中后胶质疤痕中表达上调.骨髓基质干细胞(BMSCs)可降低胶质疤痕壁的厚度,加速缺血周边区的轴突重塑.为了进一步明确BMSCs在轴突再生中的作用及机制,本文重点研究脑缺血组织中BMSCs对神经蛋白聚糖表达的作用.31只成年雄性Wistar大鼠大脑中动脉阻塞(MCAo)2 h,24 h后从中选择16只给予尾静脉注射3 × 106鼠BMSCs(BMSCs组),15只注射磷酸盐缓冲生理盐水(对照组).缺血后8 d处死实验大鼠,免疫染色表明反应性星形胶质细胞是神经蛋白聚糖的原始来源,且BMSCs组缺血半暗带脑组织的神经聚糖表达明显低于对照组,生长相关蛋白43表达高于对照组,这在蛋白印迹分析中得到确认.为了进一步检测BMSCs在星形胶质细胞神经蛋白聚糖表达中的作用,用激光捕获显微切割法从缺血周边区收集单纯的反应性星形胶质细胞.BMSCs组的神经蛋白聚糖基因表达明显下调(n=4/组).原代培养的星形胶质细胞也表现出相同改变,糖氧剥离的星形胶质细胞再给氧时与BMSCs共培养会抑制神经蛋白聚糖基因的表达上调(n=3/组).本研究表明BMSCs通过下调梗死周边星形胶质细胞中神经蛋白聚糖的表达来促进轴突再生.%The glial scar, a primarily astrocytic structure bordering the infarct tissue inhibits axonal regeneration after stroke. Neurocan, an axonal extension inhibitory molecule, is upregulated in the scar region after stroke. Bone marrow stromal cells (BMSCs) reduce the thickness of glial scar wall and facilitate axonal remodeling in the ischemic boundary zone. To further clarify the role of BMSCs in axonal regeneration and its underlying mechanism, the current study focused on the effect of BMSCs on neurocan expression in the ischemic brain. Thirty-one adult male Wistar rats were subjected to 2 h of middle

  4. A Common Progenitor for Retinal Astrocytes and Oligodendrocytes

    OpenAIRE

    Rompani, Santiago B.; Cepko, Constance L.

    2010-01-01

    Developing neural tissue undergoes a period of neurogenesis followed by a period of gliogenesis. The lineage relationships among glial cell types have not been defined for most areas of the nervous system. Here we use retroviruses to label clones of glial cells in the chick retina. We found that almost every clone had both astrocytes and oligodendrocytes. In addition, we discovered a novel glial cell type, with features intermediate between those of astrocytes and oligodendrocytes, which we h...

  5. Investigation on the suitable pressure for the preservation of astrocyte

    International Nuclear Information System (INIS)

    The effects of pressure on the survival rate of astrocytes in growth medium (DMEM) were investigated at room temperature and at 40C, in an effort to establish the best conditions for the preservation. Survival rate at 40C was found to be higher than that at room temperature. The survival rate of astrocytes preserved for 4 days at 40C increased with increasing pressure up to 1.6 MPa, but decreased with increasing pressure above 1.6 MPa. At 10 MPa, all astrocytes died. The survival rate of cultured astrocytes decreased significantly following pressurization for 2 hours and the subsequent preservation for 2 days at atmospheric pressure. Therefore, it is necessary to maintain pressure when preserving astrocytes. These results indicate that the cells can be stored at 40C under pressurization without freezing and without adding cryoprotective agents. Moreover, it may be possible to use this procedure as a new preservation method when cryopreservation is impractical.

  6. Astrocytes Control Neuronal Excitability in the Nucleus Accumbens

    Directory of Open Access Journals (Sweden)

    Tommaso Fellin

    2007-01-01

    Full Text Available Though accumulating evidence shows that the metabotropic glutamate receptor 5 (mGluR5 mediates some of the actions of extracellular glutamate after cocaine use, the cellular events underlying this action are poorly understood. In this review, we will discuss recent results showing that mGluR5 receptors are key regulators of astrocyte activity. Synaptic release of glutamate activates mGluR5 expressed in perisynaptic astrocytes and generates intense Ca2+ signaling in these cells. Ca2+ oscillations, in turn, trigger the release from astrocytes of the gliotransmitter glutamate, which modulates neuronal excitability by activating NMDA receptors. By integrating these results with the most recent evidence demonstrating the importance of astrocytes in the regulation of neuronal excitability, we propose that astrocytes are involved in mediating some of the mGluR5-dependent drug-induced behaviors.

  7. Metabolic pathways for glucose in astrocytes.

    Science.gov (United States)

    Wiesinger, H; Hamprecht, B; Dringen, R

    1997-09-01

    Cultured astroglial cells are able to utilize the monosaccharides glucose, mannose, or fructose as well as the sugar alcohol sorbitol as energy fuel. Astroglial uptake of the aldoses is carrier-mediated, whereas a non-saturable transport mechanism is operating for fructose and sorbitol. The first metabolic step for all sugars, including fructose being generated by enzymatic oxidation of sorbitol, is phosphorylation by hexokinase. Besides glucose only mannose may serve as substrate for build-up of astroglial glycogen. Whereas glycogen synthase appears to be present in astrocytes as well as neurons, the exclusive localization of glycogen phosphorylase in astrocytes and ependymal cells of central nervous tissue correlates well with the occurrence of glycogen in these cells. The identification of lactic acid rather than glucose as degradation product of astroglial glycogen appears to render the presence of glucose-6-phosphatase in cultured astrocytes an enigma. The colocalization of pyruvate carboxylase, phosphenolpyruvate carboxykinase and fructose-1,6-bisphosphatase points to astrocytes as being the gluconeogenic cell type of the CNS. PMID:9298844

  8. High prevalence of side population in human cancer cell lines

    OpenAIRE

    Boesch, Maximilian; Zeimet, Alain G; Fiegl, Heidi; Wolf, Barbara; Huber, Julia; Klocker, Helmut; Gastl, Guenther; Sopper, Sieghart; Wolf, Dominik

    2016-01-01

    Cancer cell lines are essential platforms for performing cancer research on human cells. We here demonstrate that, across tumor entities, human cancer cell lines harbor minority populations of putative stem-like cells, molecularly defined by dye extrusion resulting in the side population phenotype. These findings establish a heterogeneous nature of human cancer cell lines and argue for their stem cell origin. This should be considered when interpreting research involving these model systems.

  9. A stereological analysis of NPY, POMC, Orexin, GFAP astrocyte, and Iba1 microglia cell number and volume in diet-induced obese male mice.

    Science.gov (United States)

    Lemus, Moyra B; Bayliss, Jacqueline A; Lockie, Sarah H; Santos, Vanessa V; Reichenbach, Alex; Stark, Romana; Andrews, Zane B

    2015-05-01

    The hypothalamic arcuate nucleus (ARC) contains 2 key neural populations, neuropeptide Y (NPY) and proopiomelanocortin (POMC), and, together with orexin neurons in the lateral hypothalamus, plays an integral role in energy homeostasis. However, no studies have examined total neuronal number and volume after high-fat diet (HFD) exposure using sophisticated stereology. We used design-based stereology to estimate NPY and POMC neuronal number and volume, as well as glial fibrillary acidic protein (astrocyte marker) and ionized calcium-binding adapter molecule 1 (microglia marker) cell number in the ARC; as well as orexin neurons in the lateral hypothalamus. Stereological analysis indicated approximately 8000 NPY and approximately 9000 POMC neurons in the ARC, and approximately 7500 orexin neurons in the lateral hypothalamus. HFD exposure did not affect total neuronal number in any population. However, HFD significantly increased average NPY cell volume and affected NPY and POMC cell volume distribution. HFD reduced orexin cell volume but had a bimodal effect on volume distribution with increased cells at relatively small volumes and decreased cells with relatively large volumes. ARC glial fibrillary acidic protein cells increased after 2 months on a HFD, although no significant difference after 6 months on chow diet or HFD was observed. No differences in ARC ionized calcium-binding adapter molecule 1 cell number were observed in any group. Thus, HFD affects ARC NPY or POMC neuronal cell volume number not cell number. Our results demonstrate the importance of stereology to perform robust unbiased analysis of cell number and volume. These data should be an empirical baseline reference to which future studies are compared.

  10. Calcineurin proteolysis in astrocytes: Implications for impaired synaptic function.

    Science.gov (United States)

    Pleiss, Melanie M; Sompol, Pradoldej; Kraner, Susan D; Abdul, Hafiz Mohmmad; Furman, Jennifer L; Guttmann, Rodney P; Wilcock, Donna M; Nelson, Peter T; Norris, Christopher M

    2016-09-01

    Mounting evidence suggests that astrocyte activation, found in most forms of neural injury and disease, is linked to the hyperactivation of the protein phosphatase calcineurin. In many tissues and cell types, calcineurin hyperactivity is the direct result of limited proteolysis. However, little is known about the proteolytic status of calcineurin in activated astrocytes. Here, we developed a polyclonal antibody to a high activity calcineurin proteolytic fragment in the 45-48kDa range (ΔCN) for use in immunohistochemical applications. When applied to postmortem human brain sections, the ΔCN antibody intensely labeled cell clusters in close juxtaposition to amyloid deposits and microinfarcts. Many of these cells exhibited clear activated astrocyte morphology. The expression of ΔCN in astrocytes near areas of pathology was further confirmed using confocal microscopy. Multiple NeuN-positive cells, particularly those within microinfarct core regions, also labeled positively for ΔCN. This observation suggests that calcineurin proteolysis can also occur within damaged or dying neurons, as reported in other studies. When a similar ΔCN fragment was selectively expressed in hippocampal astrocytes of intact rats (using adeno-associated virus), we observed a significant reduction in the strength of CA3-CA1 excitatory synapses, indicating that the hyperactivation of astrocytic calcineurin is sufficient for disrupting synaptic function. Together, these results suggest that proteolytic activation of calcineurin in activated astrocytes may be a central mechanism for driving and/or exacerbating neural dysfunction during neurodegenerative disease and injury. PMID:27212416

  11. Simultaneous neuron- and astrocyte-specific fluorescent marking

    International Nuclear Information System (INIS)

    Systematic and simultaneous analysis of multiple cell types in the brain is becoming important, but such tools have not yet been adequately developed. Here, we aimed to generate a method for the specific fluorescent labeling of neurons and astrocytes, two major cell types in the brain, and we have developed lentiviral vectors to express the red fluorescent protein tdTomato in neurons and the enhanced green fluorescent protein (EGFP) in astrocytes. Importantly, both fluorescent proteins are fused to histone 2B protein (H2B) to confer nuclear localization to distinguish between single cells. We also constructed several expression constructs, including a tandem alignment of the neuron- and astrocyte-expression cassettes for simultaneous labeling. Introducing these vectors and constructs in vitro and in vivo resulted in cell type-specific and nuclear-localized fluorescence signals enabling easy detection and distinguishability of neurons and astrocytes. This tool is expected to be utilized for the simultaneous analysis of changes in neurons and astrocytes in healthy and diseased brains. - Highlights: • We develop a method for the specific fluorescent labeling of neurons and astrocytes. • Neuron-specific labeling is achieved using Scg10 and synapsin promoters. • Astrocyte-specific labeling is generated using the minimal GFAP promoter. • Nuclear localization of fluorescent proteins is achieved with histone 2B protein

  12. Simultaneous neuron- and astrocyte-specific fluorescent marking

    Energy Technology Data Exchange (ETDEWEB)

    Schulze, Wiebke [Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Hayata-Takano, Atsuko [Molecular Research Center for Children' s Mental Development, United Graduate School of Child Development, Osaka University, Kanazawa University, Hamamatsu University School of Medicine, Chiba University and University of Fukui, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Kamo, Toshihiko [Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Nakazawa, Takanobu, E-mail: takanobunakazawa-tky@umin.ac.jp [iPS Cell-based Research Project on Brain Neuropharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Nagayasu, Kazuki [iPS Cell-based Research Project on Brain Neuropharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Kasai, Atsushi; Seiriki, Kaoru [Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Interdisciplinary Program for Biomedical Sciences, Institute for Academic Initiatives, Osaka University, 1-1 Yamadaoka, Suita, Osaka 565-0871 (Japan); Shintani, Norihito [Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Ago, Yukio [Laboratory of Medicinal Pharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Farfan, Camille [Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); and others

    2015-03-27

    Systematic and simultaneous analysis of multiple cell types in the brain is becoming important, but such tools have not yet been adequately developed. Here, we aimed to generate a method for the specific fluorescent labeling of neurons and astrocytes, two major cell types in the brain, and we have developed lentiviral vectors to express the red fluorescent protein tdTomato in neurons and the enhanced green fluorescent protein (EGFP) in astrocytes. Importantly, both fluorescent proteins are fused to histone 2B protein (H2B) to confer nuclear localization to distinguish between single cells. We also constructed several expression constructs, including a tandem alignment of the neuron- and astrocyte-expression cassettes for simultaneous labeling. Introducing these vectors and constructs in vitro and in vivo resulted in cell type-specific and nuclear-localized fluorescence signals enabling easy detection and distinguishability of neurons and astrocytes. This tool is expected to be utilized for the simultaneous analysis of changes in neurons and astrocytes in healthy and diseased brains. - Highlights: • We develop a method for the specific fluorescent labeling of neurons and astrocytes. • Neuron-specific labeling is achieved using Scg10 and synapsin promoters. • Astrocyte-specific labeling is generated using the minimal GFAP promoter. • Nuclear localization of fluorescent proteins is achieved with histone 2B protein.

  13. Expression of Cyclooxygenase-2 in Ovarian Cancer Cell Lines

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    To investigate the expression of cyclooxygenase-2 (COX-2) in ovarian cancer cell lines,RT-PCR and immunocytochemistry were used to detect the expression of COX-2 in 5 ovarian cancer cell lines. The expression of COX-2 mRNA and protein was detected in all 5 cell lines. It is suggested that COX-2 is expressed in ovarian cancer cell lines, which provides a basis for the chemoprevention of ovarian cancer.

  14. Zirconium oxide ceramic foam: a promising supporting biomaterial for massive production of glial cell line-derived neurotrophic factor.

    Science.gov (United States)

    Liu, Zhong-wei; Li, Wen-qiang; Wang, Jun-kui; Ma, Xian-cang; Liang, Chen; Liu, Peng; Chu, Zheng; Dang, Yong-hui

    2014-12-01

    This study investigated the potential application of a zirconium oxide (ZrO2) ceramic foam culturing system to the production of glial cell line-derived neurotrophic factor (GDNF). Three sets of ZrO2 ceramic foams with different pore densities of 10, 20, and 30 pores per linear inch (PPI) were prepared to support a 3D culturing system. After primary astrocytes were cultured in these systems, production yields of GDNF were evaluated. The biomaterial biocompatibility, cell proliferation and activation of cellular signaling pathways in GDNF synthesis and secretion in the culturing systems were also assessed and compared with a conventional culturing system. In this study, we found that the ZrO2 ceramic foam culturing system was biocompatible, using which the GDNF yields were elevated and sustained by stimulated cell proliferation and activation of signaling pathways in astrocytes cultured in the system. In conclusion, the ZrO2 ceramic foam is promising for the development of a GDNF mass production device for Parkinson's disease treatment.

  15. 77 FR 5489 - Identification of Human Cell Lines Project

    Science.gov (United States)

    2012-02-03

    ... National Institute of Standards and Technology Identification of Human Cell Lines Project AGENCY: National... tandem repeat (STR) profiling up to 1500 human cell line samples as part of the Identification of Human Cell Lines Project. All data and corresponding information will be posted in a publically held...

  16. Astrocyte proliferation following stroke in the mouse depends on distance from the infarct.

    Directory of Open Access Journals (Sweden)

    George E Barreto

    Full Text Available Reactive gliosis is a hallmark of brain pathology and the injury response, yet the extent to which astrocytes proliferate, and whether this is central to astrogliosis is still controversial. We determined the fraction of mature astrocytes that proliferate in a mouse stroke model using unbiased stereology as a function of distance from the infarct edge. Cumulatively 11.1±1.2% of Aldh1l1(+ astrocytes within 400 µm in the cortical penumbra incorporate BrdU in the first week following stroke, while the overall number of astrocytes does not change. The number of astrocytes proliferating fell sharply with distance with more than half of all proliferating astrocytes found within 100 µm of the edge of the infarct. Despite extensive cell proliferation primarily of microglia and neutrophils/monocytes in the week following stroke, few mature astrocytes re-enter cell cycle, and these are concentrated close to the infarct boundary.

  17. Neurons diversify astrocytes in the adult brain through sonic hedgehog signaling.

    Science.gov (United States)

    Farmer, W Todd; Abrahamsson, Therése; Chierzi, Sabrina; Lui, Christopher; Zaelzer, Cristian; Jones, Emma V; Bally, Blandine Ponroy; Chen, Gary G; Théroux, Jean-Francois; Peng, Jimmy; Bourque, Charles W; Charron, Frédéric; Ernst, Carl; Sjöström, P Jesper; Murai, Keith K

    2016-02-19

    Astrocytes are specialized and heterogeneous cells that contribute to central nervous system function and homeostasis. However, the mechanisms that create and maintain differences among astrocytes and allow them to fulfill particular physiological roles remain poorly defined. We reveal that neurons actively determine the features of astrocytes in the healthy adult brain and define a role for neuron-derived sonic hedgehog (Shh) in regulating the molecular and functional profile of astrocytes. Thus, the molecular and physiological program of astrocytes is not hardwired during development but, rather, depends on cues from neurons that drive and sustain their specialized properties. PMID:26912893

  18. Human Brain Astrocytes Mediate TRAIL-mediated Apoptosis after Treatment with IFN-γ

    OpenAIRE

    Lee, Jeonggi; Shin, Jeon-Soo; Choi, In-Hong

    2006-01-01

    TNF-related apoptosis inducing ligand (TRAIL) expressions were studied in primary human brain astrocytes in response to pro-inflammatory cytokines. When astrocytes were treated with IL-1β, TNF-α or IFN-γ, TRAIL was induced in cultured fetal astrocytes. In particular, IFN-γ induced the highest levels of TRAIL in cultured astrocytes. When astrocytes were prereated with IFN-γ, they induced apoptosis in TRAIL-sensitive Peer cells. Our results suggest that IFN-γ modulates the expression of TRAIL i...

  19. Astrocyte-to-neuron signaling in response to photostimulation with a femtosecond laser

    Science.gov (United States)

    Zhao, Yuan; Liu, Xiuli; Zhou, Wei; Zeng, Shaoqun

    2010-08-01

    Conventional stimulation techniques used in studies of astrocyte-to-neuron signaling are invasive or dependent on additional electrical devices or chemicals. Here, we applied photostimulation with a femtosecond laser to selectively stimulate astrocytes in the hippocampal neural network, and the neuronal responses were examined. The results showed that, after photostimulation, cell-specific astrocyte-to-neuron signaling was triggered; sometimes the neuronal responses were even synchronous. Since photostimulation with a femtosecond laser is noninvasive, agent-free, and highly precise, this method has been proved to be efficient in activating astrocytes for investigations of astrocytic functions in neural networks.

  20. Roles of zinc and metallothionein-3 in oxidative stress-induced lysosomal dysfunction, cell death, and autophagy in neurons and astrocytes

    Directory of Open Access Journals (Sweden)

    Lee Sook-Jeong

    2010-10-01

    Full Text Available Abstract Zinc dyshomeostasis has been recognized as an important mechanism for cell death in acute brain injury. An increase in the level of free or histochemically reactive zinc in astrocytes and neurons is considered one of the major causes of death of these cells in ischemia and trauma. Although zinc dyshomeostasis can lead to cell death via diverse routes, the major pathway appears to involve oxidative stress. Recently, we found that a rise of zinc in autophagic vacuoles, including autolysosomes, is a prerequisite for lysosomal membrane permeabilization and cell death in cultured brain cells exposed to oxidative stress conditions. The source of zinc in this process is likely redox-sensitive zinc-binding proteins such as metallothioneins, which release zinc under oxidative conditions. Of the metallothioneins, metallothionein-3 is especially enriched in the central nervous system, but its physiologic role in this tissue is not well established. Like other metallothioneins, metallothionein-3 may function as metal detoxicant, but is also known to inhibit neurite outgrowth and, sometimes, promote neuronal death, likely by serving as a source of toxic zinc release. In addition, metallothionein-3 regulates lysosomal functions. In the absence of metallothionein-3, there are changes in lysosome-associated membrane protein-1 and -2, and reductions in certain lysosomal enzymes that result in decreased autophagic flux. This may have dual effects on cell survival. In acute oxidative injury, zinc dyshomeostasis and lysosomal membrane permeabilization are diminished in metallothionein-3 null cells, resulting in less cell death. But over the longer term, diminished lysosomal function may lead to the accumulation of abnormal proteins and cause cytotoxicity. The roles of zinc and metallothionein-3 in autophagy and/or lysosomal function have just begun to be investigated. In light of evidence that autophagy and lysosomes may play significant roles in the

  1. Dual Phases of Respiration Chain Defect-Augmented mROS-Mediated mCa2+ Stress during Oxidative Insult in Normal and ρ0 RBA1 Astrocytes

    Directory of Open Access Journals (Sweden)

    Tsung-I Peng

    2013-01-01

    Full Text Available Mitochondrial respiratory chain (RC deficits, resulting in augmented mitochondrial ROS (mROS generation, underlie pathogenesis of astrocytes. However, mtDNA-depleted cells (ρ0 lacking RC have been reported to be either sensitive or resistant to apoptosis. In this study, we sought to determine the effects of RC-enhanced mitochondrial stress following oxidative insult. Using noninvasive fluorescence probe-coupled laser scanning imaging microscopy, the ability to resist oxidative stress and levels of mROS formation and mitochondrial calcium (mCa2+ were compared between two different astrocyte cell lines, control and ρ0 astrocytes, over time upon oxidative stress. Our results showed that the cytoplasmic membrane becomes permeated with YO-PRO-1 dye at 150 and 130 minutes in RBA-1 and ρ0 astrocytes, respectively. In contrast to RBA-1, 30 minutes after 20 mM H2O2 exposure, ρ0 astrocytes formed marked plasma membrane blebs, lost the ability to retain Mito-R, and showed condensation of nuclei. Importantly, H2O2-induced ROS and accompanied mCa2+ elevation in control showed higher levels than ρ0 at early time point but vice versa at late time point. Our findings underscore dual phase of RC-defective cells harboring less mitochondrial stress due to low RC activity during short-term oxidative stress but augmented mROS-mediated mCa2+ stress during severe oxidative insult.

  2. Chloride transport in a glioma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Wolpaw, E.W.

    1984-01-01

    Maintenance of the extracellular environment is a major function of central nervous system astroglia. The transport of Cl/sup -/ across the cell membrane may be an integral part of this function, since Cl/sup -/ transport has been implicated in homeostasis of cell volume, pH, and extracellular K/sup +/ concentration. The work presented here investigated Cl/sup -/ transport in the glioma cell line LRM55. Results indicate that LRM55 cells are a good model for astroglia and that these cells contain three Cl/sup -/ transporters; a Cl/sup -//HCO/sub 3//sup -/ exchanger, a K/sup +//Cl/sup -/ cotransporter, and a Cl/sup -//SO/sub 4//sup 2 -/ exchanger. Ion transport studies measured the fluxes of Cl/sup -/ (as /sup 36/Cl/sup -/), K/sup +/ (as /sup 86/Rb/sup +/), and SO/sub 4//sup 2 -/ (as /sup 35/SO/sub 4//sup 2 -/). Cl/sup -/ flux was trans-simulated by Cl/sup -/ or HCO/sub 3//sup -/ and was inhibited by SITS or furosemide. External K/sup +/ stimulated Cl/sup -/ influx and external Cl/sup -/ stimulated Rb/sup +/ influx. Furosemide, but not SITS, inhibited the K/sup +//Cl/sup -/ cotransporter. High K/sup +/ medium increased cell volume and Cl/sup -/ content. Steady-state Cl/sup -/ concentration was at least twice that predicted from passive equilibration according to the Nernst equation. SO/sub 4//sup 2 -/ flux was trans-stimulated by SO/sub 4//sup 2 -/ or by Cl/sup -/. Cl/sup -/ was a competitive inhibitor of SO/sub 4//sup 2 -/ influx, but SO/sub 4//sup 2 -/ had no detectable effect on Cl/sup -/ influx or efflux. SO/sub 4//sup 2 -/ flux was inhibited by SITS or furosemide.

  3. Involvement of the Notch pathway in terminal astrocytic differentiation: role of PKA

    Directory of Open Access Journals (Sweden)

    Carla Angulo‑Rojo

    2013-12-01

    Full Text Available The Notch pathway is a highly conserved signaling system essential for modulating neurogenesis and promoting astrogenesis. Similarly, the cAMP signaling cascade can promote astrocytic commitment in several cell culture models, such as the C6 glioma cell line. These cells have the capacity to differentiate into oligodendrocytes or astrocytes, characteristics that allow their use as a glial progenitor model. In this context, we explore here the plausible involvement of cAMP in Notch-dependent signal transactions. The exposure of C6 cells to a non-hydrolysable cAMP analogue resulted in a sustained augmentation of Notch activity, as detected by nuclear translocation of its intracellular domain portion (NICD and transcriptional activity. The cAMP effect is mediated through the activation of the γ-secretase complex, responsible for Notch cleavage and is sensitive to inhibitors of the cAMP-dependent protein kinase, PKA. As expected, Notch cleavage and nuclear translocation resulted in the up-regulation of the mRNA levels of one of its target genes, the transcription factor Hair and enhancer of split 5. Moreover, the glutamate uptake activity, as well as the expression of astrocytic markers such as glial fibrillary acidic protein, S100β protein and GLAST was also enhanced in cAMP-exposed cells. Our results clearly suggest that during the process of C6 astrocytic differentiation, cAMP activates the PKA/γ-secretase/NICD/RBPJκ pathway and Notch1 expression, leading to transcriptional activation of the genes responsible for glial progenitor cell fate decision.

  4. Transplantation of specific human astrocytes promotes functional recovery after spinal cord injury.

    Directory of Open Access Journals (Sweden)

    Stephen J A Davies

    Full Text Available Repairing trauma to the central nervous system by replacement of glial support cells is an increasingly attractive therapeutic strategy. We have focused on the less-studied replacement of astrocytes, the major support cell in the central nervous system, by generating astrocytes from embryonic human glial precursor cells using two different astrocyte differentiation inducing factors. The resulting astrocytes differed in expression of multiple proteins thought to either promote or inhibit central nervous system homeostasis and regeneration. When transplanted into acute transection injuries of the adult rat spinal cord, astrocytes generated by exposing human glial precursor cells to bone morphogenetic protein promoted significant recovery of volitional foot placement, axonal growth and notably robust increases in neuronal survival in multiple spinal cord laminae. In marked contrast, human glial precursor cells and astrocytes generated from these cells by exposure to ciliary neurotrophic factor both failed to promote significant behavioral recovery or similarly robust neuronal survival and support of axon growth at sites of injury. Our studies thus demonstrate functional differences between human astrocyte populations and suggest that pre-differentiation of precursor cells into a specific astrocyte subtype is required to optimize astrocyte replacement therapies. To our knowledge, this study is the first to show functional differences in ability to promote repair of the injured adult central nervous system between two distinct subtypes of human astrocytes derived from a common fetal glial precursor population. These findings are consistent with our previous studies of transplanting specific subtypes of rodent glial precursor derived astrocytes into sites of spinal cord injury, and indicate a remarkable conservation from rat to human of functional differences between astrocyte subtypes. In addition, our studies provide a specific population of human

  5. Transplantation of specific human astrocytes promotes functional recovery after spinal cord injury.

    Science.gov (United States)

    Davies, Stephen J A; Shih, Chung-Hsuan; Noble, Mark; Mayer-Proschel, Margot; Davies, Jeannette E; Proschel, Christoph

    2011-03-02

    Repairing trauma to the central nervous system by replacement of glial support cells is an increasingly attractive therapeutic strategy. We have focused on the less-studied replacement of astrocytes, the major support cell in the central nervous system, by generating astrocytes from embryonic human glial precursor cells using two different astrocyte differentiation inducing factors. The resulting astrocytes differed in expression of multiple proteins thought to either promote or inhibit central nervous system homeostasis and regeneration. When transplanted into acute transection injuries of the adult rat spinal cord, astrocytes generated by exposing human glial precursor cells to bone morphogenetic protein promoted significant recovery of volitional foot placement, axonal growth and notably robust increases in neuronal survival in multiple spinal cord laminae. In marked contrast, human glial precursor cells and astrocytes generated from these cells by exposure to ciliary neurotrophic factor both failed to promote significant behavioral recovery or similarly robust neuronal survival and support of axon growth at sites of injury. Our studies thus demonstrate functional differences between human astrocyte populations and suggest that pre-differentiation of precursor cells into a specific astrocyte subtype is required to optimize astrocyte replacement therapies. To our knowledge, this study is the first to show functional differences in ability to promote repair of the injured adult central nervous system between two distinct subtypes of human astrocytes derived from a common fetal glial precursor population. These findings are consistent with our previous studies of transplanting specific subtypes of rodent glial precursor derived astrocytes into sites of spinal cord injury, and indicate a remarkable conservation from rat to human of functional differences between astrocyte subtypes. In addition, our studies provide a specific population of human astrocytes that

  6. Expression of zonula occludens-1 (ZO-1) and the transcription factor ZO-1-associated nucleic acid-binding protein (ZONAB)-MsY3 in glial cells and colocalization at oligodendrocyte and astrocyte gap junctions in mouse brain.

    Science.gov (United States)

    Penes, Mihai C; Li, Xinbo; Nagy, James I

    2005-07-01

    The PDZ domain-containing protein zonula occludens-1 (ZO-1) interacts with several members of the connexin (Cx) family of gap junction-forming proteins and has been localized to gap junctions, including those containing Cx47 in oligodendrocytes. We now provide evidence for ZO-1 expression in astrocytes in vivo and association with astrocytic connexins by confocal immunofluorescence demonstration of ZO-1 colocalization with astrocytic Cx30 and Cx43, and by ZO-1 coimmunoprecipitation with Cx30 and Cx43. Evidence for direct interaction of Cx30 with ZO-1 was obtained by pull-down assays that indicated binding of Cx30 to the second of the three PDZ domains in ZO-1. Further, we investigated mouse Y-box transcription factor MsY3, the canine ortholog of which has been termed ZO-1-associated nucleic acid-binding protein (ZONAB) and previously reported to interact with ZO-1. By immunofluorescence using specific antimouse ZONAB antibody, ZONAB was found to be associated with oligodendrocytes throughout mouse brain and spinal cord, and to be colocalized with oligodendrocytic Cx47 and Cx32 as well as with astrocytic Cx43. Our results extend the CNS cell types that express the multifunctional protein ZO-1, demonstrate an additional connexin (Cx30) that directly interacts with ZO-1, and show for the first time the association of a transcription factor (ZONAB) with ZO-1 localized to oligodendrocyte and astrocyte gap junctions. Given previous observations that ZONAB and ZO-1 in combination regulate gene expression, our results suggest roles of glial gap junction-mediated anchoring of signalling molecules in a wide variety of glial homeostatic processes. PMID:16045494

  7. Minocycline is cytoprotective in human trabecular meshwork cells and optic nerve head astrocytes by increasing expression of XIAP, survivin, and Bcl-2

    Directory of Open Access Journals (Sweden)

    Marcus Kernt

    2010-06-01

    Full Text Available Marcus Kernt, Aljoscha S Neubauer, Kirsten H Eibl, Armin Wolf, Michael W Ulbig, Anselm Kampik, Cristoph HirneissDepartment of Ophthalmology, Ludwig-Maximilians-University, Munich, GermanyIntroduction: Primary open-angle glaucoma (POAG is one of the leading causes of blindness. Activation of optic nerve head astrocytes (ONHA and loss of trabecular meshwork cells (TMC are pathognomonic for this neurodegenerative disease. Oxidative stress and elevated levels of transforming growth factor beta (TGFβ play an important role in the pathogenesis of POAG. This study investigates the possible antiapoptotic and cytoprotective effects of minocycline on TMC and ONHA under oxidative stress and increased TGFβ levels.Methods: TMC and ONHA were treated with minocycline 1–150 μM. Possible toxic effects and IC50 were evaluated after 48 hours. Cell proliferation and viability were examined in order to assess the protective effects of minocycline on TMC and ONHA. Expression of Bcl-2, XIAP, and survivin, as well as their mRNA expression, were assessed by real time polymerase chain reaction (RT-PCR and Western Blot analysis 48 hours after treatment with minocycline alone and additional incubation with TGFβ-2 or oxidative stress.Results: Minocycline 1–75 μM showed no toxic effects on TMC and ONHA. Under conditions of oxidative stress, both TMC and ONHA showed an increase in viability and an ability to proliferate when treated with minocycline 20–40 μM. RT-PCR and Western blotting yielded an overexpression of Bcl-2, XIAP, and survivin when TMC or ONHA were treated with minocycline 20–40 μM under conditions of oxidative stress and when additionally incubated with TGFβ-2.Conclusion: Minocycline up to 75 μM does not have toxic effects on TMC and ONHA. Treatment with minocycline 20–40 μM led to increased viability and proliferation under oxidative stress and TGFβ-2, as well as overexpression of Bcl-2, XIAP, and survivin. This protective pathway may help

  8. Cocaine-Mediated Autophagy in Astrocytes Involves Sigma 1 Receptor, PI3K, mTOR, Atg5/7, Beclin-1 and Induces Type II Programed Cell Death.

    Science.gov (United States)

    Cao, Lu; Walker, Mary P; Vaidya, Naveen K; Fu, Mingui; Kumar, Santosh; Kumar, Anil

    2016-09-01

    Cocaine, a commonly used drug of abuse, has been shown to cause neuropathological dysfunction and damage in the human brain. However, the role of autophagy in this process is not defined. Autophagy, generally protective in nature, can also be destructive leading to autophagic cell death. This study was designed to investigate whether cocaine induces autophagy in the cells of CNS origin. We employed astrocyte, the most abundant cell in the CNS, to define the effects of cocaine on autophagy. We measured levels of the autophagic marker protein LC3II in SVGA astrocytes after exposure with cocaine. The results showed that cocaine caused an increase in LC3II level in a dose- and time-dependent manner, with the peak observed at 1 mM cocaine after 6-h exposure. This result was also confirmed by detecting LC3II in SVGA astrocytes using confocal microscopy and transmission electron microscopy. Next, we sought to explore the mechanism by which cocaine induces the autophagic response. We found that cocaine-induced autophagy was mediated by sigma 1 receptor, and autophagy signaling proteins p-mTOR, Atg5, Atg7, and p-Bcl-2/Beclin-1 were also involved, and this was confirmed by using selective inhibitors and small interfering RNAs (siRNAs). In addition, we found that chronic treatment with cocaine resulted in cell death, which is caspase-3 independent and can be ameliorated by autophagy inhibitor. Therefore, this study demonstrated that cocaine induces autophagy in astrocytes and is associated with autophagic cell death. PMID:26243186

  9. Astrocytes drive upregulation of the multidrug resistance transporter ABCB1 (P-Glycoprotein) in endothelial cells of the blood-brain barrier in mutant superoxide dismutase 1-linked amyotrophic lateral sclerosis.

    Science.gov (United States)

    Qosa, Hisham; Lichter, Jessica; Sarlo, Mark; Markandaiah, Shashirekha S; McAvoy, Kevin; Richard, Jean-Philippe; Jablonski, Michael R; Maragakis, Nicholas J; Pasinelli, Piera; Trotti, Davide

    2016-08-01

    The efficacy of drugs targeting the CNS is influenced by their limited brain access, which can lead to complete pharmacoresistance. Recently a tissue-specific and selective upregulation of the multidrug efflux transporter ABCB1 or P-glycoprotein (P-gp) in the spinal cord of both patients and the mutant SOD1-G93A mouse model of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease that prevalently kills motor neurons has been reported. Here, we extended the analysis of P-gp expression in the SOD1-G93A ALS mouse model and found that P-gp upregulation was restricted to endothelial cells of the capillaries, while P-gp expression was not detected in other cells of the spinal cord parenchyma such as astrocytes, oligodendrocytes, and neurons. Using both in vitro human and mouse models of the blood-brain barrier (BBB), we found that mutant SOD1 astrocytes were driving P-gp upregulation in endothelial cells. In addition, a significant increase in reactive oxygen species production, Nrf2 and NFκB activation in endothelial cells exposed to mutant SOD1 astrocytes in both human and murine BBB models were observed. Most interestingly, astrocytes expressing FUS-H517Q, a different familial ALS-linked mutated gene, also drove NFκB-dependent upregulation of P-gp. However, the pathway was not dependent on oxidative stress but rather involved TNF-α release. Overall, these findings indicated that nuclear translocation of NFκB was a converging mechanism used by endothelial cells of the BBB to upregulate P-gp expression in mutant SOD1-linked ALS and possibly other forms of familial ALS. GLIA 2016 GLIA 2016;64:1298-1313. PMID:27158936

  10. Different cell death pathway in neurons and astrocytes after focal cerebral ischemia%局灶脑缺血神经细胞和星形胶质细胞可能不同死亡途径探讨

    Institute of Scientific and Technical Information of China (English)

    付学军; 褚晓凡; 邹良玉; 亓传洁; 饶宜光

    2011-01-01

    immunohistochemistry experiment group and electron microscope experiment group were obtained at 3h,6h,12h,24h and 48h after embelization,respectively. HE and GFAP staining,TUNEL and GFAP-TUNEL double labeled immunohistochemistry analysis were applied. Routine electron microscopic technology was also used to evaluate morphologic changes as well. Results Astrocytes in the core region became more swollen as the time of ischemia went on. Ater 12h ischemia, nuclear membrane cracked and chromatin leakage happened in astrocytes without coagulability changes. In neurons, cell nucleus deformation and karyopycnosis along with apoptotic bodies were observed. The degrees of cell injury in the marginal region of iscbemia at different time point were not of concordance. TUNEL positive cells were seen in central and marginal regions 3h after ischemia, mainly in marginal region, and were more as ischemia went on while decreased in core area. After 24h isohemia, TUNEL positive cells could scarcely be seen in core region. GFAP positive cells were decreased in core region after 6h ischemia and scarcely seen after 12h ischemia. In marginal region after 24h ischemia a number of GFAP positive cells were seen which drawed an apparent dividing line contrasted to the central region. GFAP and TUNEL double labeling positive cells were not seen at every time point. Conclusion Continuous ischemia can cause neuron in both core and marginal area coagulation death. Apoptosis may be main death pathway for neuron.As for astrocytes,swellen death was in both areas. It maybe belong to the oncosis pathway.

  11. EXPRESSION OF Fas LIGAND IN HUMAN COLON CANCER CELL LINES

    Institute of Scientific and Technical Information of China (English)

    张建军; 丁尔迅; 王强; 陈学云; 付志仁

    2001-01-01

    To investigate the expression of Fas ligand in human colon carcinoma cell lines. Methods: A total of six human colon cancer cell lines were examined for the expression of Fas ligand mRNA and cell surface protein by using RT-PCR and flow cytometry respectively. Results: The results showed that Fas ligand mRNA was expressed in all of the six cancer cell lines and Fas ligand cell surface protein was expressed in part of them. Conclusion: These data suggest that Fas ligand was expressed, at least in part, in human colon cancer cell lines and might facilitate to escape from immune surveillance of the host.

  12. DNA profiling and characterization of animal cell lines.

    Science.gov (United States)

    Stacey, Glyn N; Byrne, Ed; Hawkins, J Ross

    2014-01-01

    The history of the culture of animal cell lines is littered with published and much unpublished experience with cell lines that have become switched, mislabelled, or cross-contaminated during laboratory handling. To deliver valid and good quality research and to avoid waste of time and resources on such rogue lines, it is vital to perform some kind of qualification for the provenance of cell lines used in research and particularly in the development of biomedical products. DNA profiling provides a valuable tool to compare different sources of the same cells and, where original material or tissue is available, to confirm the correct identity of a cell line. This chapter provides a review of some of the most useful techniques to test the identity of cells in the cell culture laboratory and gives methods which have been used in the authentication of cell lines. PMID:24297409

  13. Biological characteristics of cell lines of human dental alveolus

    Institute of Scientific and Technical Information of China (English)

    陈世璋; 黄靖香; 孙明学; 赵斌

    2003-01-01

    Objective To investigate the biological characteristics of cell lines of healthy and diseased human dental alveoli. Methods Primary cell lines from either healthy or diseased human dental alveoli were obtained. Two cell lines, H-258 and H-171 derived from healthy and diseased human tissues respectively, were selected for morphological study and research on their growth and aging, using cell counting, and histochemical and immunohistochemical staining. Results Primary cell lines were successfully established from innormal dental alveoli. After freezing and thawing for three times, cell growth was continued and no morphological alterations were observed. The doubling time was 53.4 hours and mean division index (MDI) was 4‰. Cells were kept normal after twenty generations with no obvious reduction of doubling time and MDI. Of twenty-six primary cell lines derived from healthy human dental alveoli, only three cell lines achieved generation. After freezing and thawing for twice, cultured cells were still alive at a decreased growth speed, with doubling time of 85.9 hours and MDI of 3‰. Both cell lines, H-171 and H-258, shared the characteristics of osteoblast. Conclusions Primary cell lines of diseased human dental alveoli show greater growth potential. All cell lines of dental alveoli share characteristics of osteoblast. The technique we developed may be put into practice for the treatment of abnormal dental alveoli.

  14. Purification and Characterization of Progenitor and Mature Human Astrocytes Reveals Transcriptional and Functional Differences with Mouse.

    Science.gov (United States)

    Zhang, Ye; Sloan, Steven A; Clarke, Laura E; Caneda, Christine; Plaza, Colton A; Blumenthal, Paul D; Vogel, Hannes; Steinberg, Gary K; Edwards, Michael S B; Li, Gordon; Duncan, John A; Cheshier, Samuel H; Shuer, Lawrence M; Chang, Edward F; Grant, Gerald A; Gephart, Melanie G Hayden; Barres, Ben A

    2016-01-01

    The functional and molecular similarities and distinctions between human and murine astrocytes are poorly understood. Here, we report the development of an immunopanning method to acutely purify astrocytes from fetal, juvenile, and adult human brains and to maintain these cells in serum-free cultures. We found that human astrocytes have abilities similar to those of murine astrocytes in promoting neuronal survival, inducing functional synapse formation, and engulfing synaptosomes. In contrast to existing observations in mice, we found that mature human astrocytes respond robustly to glutamate. Next, we performed RNA sequencing of healthy human astrocytes along with astrocytes from epileptic and tumor foci and compared these to human neurons, oligodendrocytes, microglia, and endothelial cells (available at http://www.brainrnaseq.org). With these profiles, we identified novel human-specific astrocyte genes and discovered a transcriptome-wide transformation between astrocyte precursor cells and mature post-mitotic astrocytes. These data represent some of the first cell-type-specific molecular profiles of the healthy and diseased human brain.

  15. The Refsum disease marker phytanic acid, a branched chain fatty acid, affects Ca2+ homeostasis and mitochondria, and reduces cell viability in rat hippocampal astrocytes.

    Science.gov (United States)

    Kahlert, Stefan; Schönfeld, Peter; Reiser, Georg

    2005-02-01

    The saturated branched chain fatty acid, phytanic acid, a degradation product of chlorophyll, accumulates in Refsum disease, an inherited peroxisomal disorder with neurological clinical features. To elucidate the pathogenic mechanism, we investigated the influence of phytanic acid on cellular physiology of rat hippocampal astrocytes. Phytanic acid (100 microM) induced an immediate transient increase in cytosolic Ca2+ concentration, followed by a plateau. The peak of this biphasic Ca2+ response was largely independent of extracellular Ca2+, indicating activation of cellular Ca2+ stores by phytanic acid. Phytanic acid depolarized mitochondria without causing in situ swelling of mitochondria. The slow decrease of mitochondrial potential is not consistent with fast and simultaneous opening of the mitochondrial permeability transition pore. However, phytanic acid induced substantial generation of reactive oxygen species. Phytanic acid caused astroglia cell death after a few hours of exposure. We suggest that the cytotoxic effect of phytanic acid seems to be due to a combined action on Ca2+ regulation, mitochondrial depolarization, and increased ROS generation in brain cells.

  16. Enterovirus 71 VP1 activates calmodulin-dependent protein kinase II and results in the rearrangement of vimentin in human astrocyte cells.

    Directory of Open Access Journals (Sweden)

    Cong Haolong

    Full Text Available Enterovirus 71 (EV71 is one of the main causative agents of foot, hand and mouth disease. Its infection usually causes severe central nervous system diseases and complications in infected infants and young children. In the present study, we demonstrated that EV71 infection caused the rearrangement of vimentin in human astrocytoma cells. The rearranged vimentin, together with various EV71 components, formed aggresomes-like structures in the perinuclear region. Electron microscopy and viral RNA labeling indicated that the aggresomes were virus replication sites since most of the EV71 particles and the newly synthesized viral RNA were concentrated here. Further analysis revealed that the vimentin in the virus factories was serine-82 phosphorylated. More importantly, EV71 VP1 protein is responsible for the activation of calmodulin-dependent protein kinase II (CaMK-II which phosphorylated the N-terminal domain of vimentin on serine 82. Phosphorylation of vimentin and the formation of aggresomes were required for the replication of EV71 since the latter was decreased markedly after phosphorylation was blocked by KN93, a CaMK-II inhibitor. Thus, as one of the consequences of CaMK-II activation, vimentin phosphorylation and rearrangement may support virus replication by playing a structural role for the formation of the replication factories. Collectively, this study identified the replication centers of EV71 in human astrocyte cells. This may help us understand the replication mechanism and pathogenesis of EV71 in human.

  17. Astrocytes Surviving Severe Stress Can Still Protect Neighboring Neurons from Proteotoxic Injury.

    Science.gov (United States)

    Gleixner, Amanda M; Posimo, Jessica M; Pant, Deepti B; Henderson, Matthew P; Leak, Rehana K

    2016-09-01

    Astrocytes are one of the major cell types to combat cellular stress and protect neighboring neurons from injury. In order to fulfill this important role, astrocytes must sense and respond to toxic stimuli, perhaps including stimuli that are severely stressful and kill some of the astrocytes. The present study demonstrates that primary astrocytes that managed to survive severe proteotoxic stress were protected against subsequent challenges. These findings suggest that the phenomenon of preconditioning or tolerance can be extended from mild to severe stress for this cell type. Astrocytic stress adaptation lasted at least 96 h, the longest interval tested. Heat shock protein 70 (Hsp70) was raised in stressed astrocytes, but inhibition of neither Hsp70 nor Hsp32 activity abolished their resistance against a second proteotoxic challenge. Only inhibition of glutathione synthesis abolished astrocytic stress adaptation, consistent with our previous report. Primary neurons were plated upon previously stressed astrocytes, and the cocultures were then exposed to another proteotoxic challenge. Severely stressed astrocytes were still able to protect neighboring neurons against this injury, and the protection was unexpectedly independent of glutathione synthesis. Stressed astrocytes were even able to protect neurons after simultaneous application of proteasome and Hsp70 inhibitors, which otherwise elicited synergistic, severe loss of neurons when applied together. Astrocyte-induced neuroprotection against proteotoxicity was not elicited with astrocyte-conditioned media, suggesting that physical cell-to-cell contacts may be essential. These findings suggest that astrocytes may adapt to severe stress so that they can continue to protect neighboring cell types from profound injury. PMID:26374549

  18. Cloning of aminopeptidase Npromoter and its activity in hematopoietic cell and different tumor cell lines

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Aminopeptidase N (APN) promoter region was cloned and sequenced from peripheral blood mononuclear cells. The recombinant reporter construct containing the promoter and luciferase gene, designated pXP1-APNLuc, was introduced into myeloblastic cell line, T lymphocyte cell line and various tumor cell lines. Luciferase assay showed that APN upstream promoter is myeloid-specific for high expression in myeloblastic cell line and much lower expres sion in T lymphocyte cell line. The promoter activity was relatively high in lung adenoma cell line compared with other tumor cell lines including hepatoma cell line, tong cancer cell line and esophageal cancer cell line in which the promoter activity significantly diminished or was almost undetectable. The characteristics of APN promoter may pro vide a new strategy for specific myeloprotection while tumor patients are being treated with chemotherapy and/or radio therapy.

  19. Expression and cellular function of vSNARE proteins in brain astrocytes.

    Science.gov (United States)

    Ropert, N; Jalil, A; Li, D

    2016-05-26

    Gray matter protoplasmic astrocytes, a major type of glial cell in the mammalian brain, extend thin processes ensheathing neuronal synaptic terminals. Albeit electrically silent, astrocytes respond to neuronal activity with Ca(2+) signals that trigger the release of gliotransmitters, such as glutamate, d-serine, and ATP, which modulate synaptic transmission. It has been suggested that the astrocytic processes, together with neuronal pre- and post-synaptic elements, constitute a tripartite synapse, and that astrocytes actively regulate information processing. Astrocytic vesicles expressing VAMP2 and VAMP3 vesicular SNARE (vSNARE) proteins have been suggested to be a key feature of the tripartite synapse and mediate gliotransmitter release through Ca(2+)-regulated exocytosis. However, the concept of exocytotic release of gliotransmitters by astrocytes has been challenged. Here we review studies investigating the expression profile of VAMP2 and VAMP3 vSNARE proteins in rodent astrocytes, and the functional implication of VAMP2/VAMP3 vesicles in astrocyte signaling. We also discuss our recent data suggesting that astrocytic VAMP3 vesicles regulate the trafficking of glutamate transporters at the plasma membrane and glutamate uptake. A better understanding of the functional consequences of the astrocytic vSNARE vesicles on glutamate signaling, neuronal excitability and plasticity, will require the development of new strategies to selectively interrogate the astrocytic vesicles trafficking in vivo. PMID:26518463

  20. Expression Profile of MiR-128 in the Astrocytoma Patients and Cell Lines.

    Science.gov (United States)

    Xu, Jingjing; Liu, Yuqiong; Guo, Si; Ma, Shengli; Xiao, Lin; Wei, Na; Xue, Rui

    2016-09-01

    Malignant astrocytomas are the most common primary brain tumors. The critical characterizes of astrocyomas are their aggressive and infiltrative in the brain, which leads to uncontrollable by conventional forms of therapy. MicroRNAs are small RNAs that had been found to regulate their targets by specific binding to the 3'-untranslated region (3'UTR) of mRNA. Recent advances in understanding the molecular biology of these tumors have revealed that microRNA (miRNA) disruption may play important roles in the pathogenesis of astrocytomas. And some of the miRNA alterations were found in the serum of astrocytoma patients. In this study, we studied the expression profile of miR-128, in the different stages of astrocytoma tissues and two human astrocytoma cell lines, A172 and T98G cells. We found that the levels of miR-128 are decreased in the A172 and T98G cells when compared to normal human astrocyte (NHA). Furthermore, the levels of miR-128 decreased gradually to the pathological stages of astrocytomas. We also identified that TROVE2 is a novel target of miR-128 by the luciferase reporter system. Furthermore, the expression levels of TROVE2 are dramatically increased with the pathological stages increasing. Finally, the levels of TROVE2 are negatively correlated with miR-128 in astrocytoma tissues. Our data provided novel evidence for the miR-128 and TROVE2 in the development of human astrocytomas. PMID:26307612

  1. Effects of huperzine A on secretion of nerve growth factor in cultured rat cortical astrocytes and neurite outgrowth in rat PC12 cells

    Institute of Scientific and Technical Information of China (English)

    Li-li TANG; Rui WANG; Xi-can TANG

    2005-01-01

    Aim: To study the effects of huperzine A (HupA) on neuritogenic activity and the expression of nerve growth factor (NGF). Methods: After being treated with 10 μmol/L HupA, neurite outgrowth of PC12 cells was observed and counted under phase-contrast microscopy. Mitogenic activity was assayed by [3H]thymidine incorporation. Cell cytotoxicity was evaluated by lactate dehydrogenase (LDH)release. AChE activity, mRNA and protein expression were measured by the Ellman's method, RT-PCR, and Western blot, respectively. NGF mRNA and protein levels were determined by RT-PCR and ELISA assays. Results: Treatment of PC 12 cells with 10 μmol/L HupA for 48 h markedly increased the number of neuritebearing cells, but caused no significant alteration in cell viability or other signs of cytotoxicity. In addition to inhibiting AChE activity, 10 μmol/L HupA also increased the mRNA and protein levels of this enzyme. In addition, following 2 h exposure of the astrocytes to 10 μmol/L HupA, there was a significant up-regulation of mRNA for NGF and P75 low-affinity NGF receptor. The protein level of NGF was also increased after 24 h treatment with HupA. Conclusion: Our findings demonstrate for the first time that HupA has a direct or indirect neurotrophic activity, which might be beneficial in treatment of neurodegenerative disorders such as Alzheimer disease.

  2. Lrp4 in astrocytes modulates glutamatergic transmission.

    Science.gov (United States)

    Sun, Xiang-Dong; Li, Lei; Liu, Fang; Huang, Zhi-Hui; Bean, Jonathan C; Jiao, Hui-Feng; Barik, Arnab; Kim, Seon-Myung; Wu, Haitao; Shen, Chengyong; Tian, Yun; Lin, Thiri W; Bates, Ryan; Sathyamurthy, Anupama; Chen, Yong-Jun; Yin, Dong-Min; Xiong, Lei; Lin, Hui-Ping; Hu, Jin-Xia; Li, Bao-Ming; Gao, Tian-Ming; Xiong, Wen-Cheng; Mei, Lin

    2016-08-01

    Neurotransmission requires precise control of neurotransmitter release from axon terminals. This process is regulated by glial cells; however, the underlying mechanisms are not fully understood. We found that glutamate release in the brain was impaired in mice lacking low-density lipoprotein receptor-related protein 4 (Lrp4), a protein that is critical for neuromuscular junction formation. Electrophysiological studies revealed compromised release probability in astrocyte-specific Lrp4 knockout mice. Lrp4 mutant astrocytes suppressed glutamatergic transmission by enhancing the release of ATP, whose level was elevated in the hippocampus of Lrp4 mutant mice. Consequently, the mutant mice were impaired in locomotor activity and spatial memory and were resistant to seizure induction. These impairments could be ameliorated by blocking the adenosine A1 receptor. The results reveal a critical role for Lrp4, in response to agrin, in modulating astrocytic ATP release and synaptic transmission. Our findings provide insight into the interaction between neurons and astrocytes for synaptic homeostasis and/or plasticity. PMID:27294513

  3. Endocytosis of human immunodeficiency virus 1 (HIV-1) in astrocytes: a fiery path to its destination

    OpenAIRE

    Chauhan, Ashok; Khandkar, Mehrab

    2014-01-01

    Despite successful suppression of peripheral HIV-1 infection by combination antiretroviral therapy, immune activation by residual virus in the brain leads to HIV-associated neurocognitive disorders (HAND). In the brain, several types of cells, including microglia, perivascular macrophage, and astrocytes have been reported to be infected by HIV-1. Astrocytes, the most abundant cells in the brain, maintain homeostasis. The general consensus on HIV-1 infection in astrocytes is that it produces u...

  4. Neurotoxic potential and cellular uptake of T-2 toxin in human astrocytes in primary culture.

    Science.gov (United States)

    Weidner, Maria; Lenczyk, Marlies; Schwerdt, Gerald; Gekle, Michael; Humpf, Hans-Ulrich

    2013-03-18

    The trichothecene mycotoxin T-2 toxin, which is produced by fungi of the Fusarium species, is a worldwide occurring contaminant of cereal based food and feed. The cytotoxic properties of T-2 toxin are already well described with apoptosis being a major mechanism of action in various cell lines as well as in primary cells of different origin. However, only few data on neurotoxic properties of T-2 toxin are reported so far, but in vivo studies showed different effects of T-2 toxin on behavior as well as on levels of brain amines in animals. To further investigate the cytotoxic properties of T-2 toxin on cells derived from brain tissue, normal human astrocytes in primary culture (NHA) were used in this study. Besides studies of cytotoxicity, apoptosis (caspase-3-activation, Annexin V) and necrosis (LDH-release), the cellular uptake and metabolism of T-2 toxin in NHA was analyzed and compared to the uptake in an established human cell line (HT-29). The results show that human astrocytes were highly sensitive to the cytotoxic properties of T-2 toxin, and apoptosis, induced at low concentrations, was identified for the first time as the mechanism of toxic action in NHA. Furthermore, a strong accumulation of T-2 toxin in NHA and HT-29 cells was detected, and T-2 toxin was subjected to metabolism leading to HT-2 toxin, a commonly found metabolite after T-2 toxin incubation in both cell types. This formation seems to occur within the cells since incubations of T-2 toxin with cell depleted culture medium did not lead to any degradation of the parent toxin. The results of this study emphasize the neurotoxic potential of T-2 toxin in human astrocytes at low concentrations after short incubation times. PMID:23363530

  5. Development and characterization of a new human hepatic cell line

    Science.gov (United States)

    Ramboer, Eva; De Craene, Bram; De Kock, Joey; Berx, Geert; Rogiers, Vera; Vanhaecke, Tamara; Vinken, Mathieu

    2015-01-01

    The increasing demand and hampered use of primary human hepatocytes for research purposes have urged scientists to search for alternative cell sources, such as immortalized hepatic cell lines. The aim of this study was to develop a human hepatic cell line using the combined overexpression of TERT and the cell cycle regulators cyclin D1 and mutant isoform CDK4R24C. Following transduction of adult human primary hepatocytes with the selected immortalization genes, cell growth was triggered and a cell line was established. When cultured under appropriate conditions, the cell line expressed several hepatocytic markers and liver-enriched transcription factors at the transcriptional and/or translational level, secreted liver-specific proteins and showed glycogen deposition. These results suggest that the immortalization strategy applied to primary human hepatocytes could generate a novel hepatic cell line that seems to retain some key hepatic characteristics. PMID:26869867

  6. Abundance of Flt3 and its ligand in astrocytic tumors

    Directory of Open Access Journals (Sweden)

    Eßbach C

    2013-05-01

    Full Text Available C Eßbach,1 N Andrae,1 D Pachow,1 J-P Warnke,2 A Wilisch-Neumann,1 E Kirches,1 C Mawrin11Department of Neuropathology, Otto-von-Guericke University, Magdeburg, 2Department of Neurosurgery, Paracelsus Hospital, Zwickau, GermanyBackground: Molecular targeted therapies for astrocytic tumors are the subject of growing research interest, due to the limited response of these tumors, especially glioblastoma multiforme, to conventional chemotherapeutic regimens. Several of these approaches exploit the inhibition of receptor tyrosine kinases. To date, it has not been elucidated if fms-like tyrosine kinase-3 (Flt3 and its natural ligand (Flt3L are expressed in astrocytic tumors, although some of the clinically intended small-molecule receptor tyrosine kinase inhibitors affect Flt3, while others do not. More importantly, the recent proof of principle for successful stimulation of the immune system against gliomas in preclinical models via local Flt3L application requires elucidation of this receptor tyrosine kinase pathway in these tumors in more detail. This therapy is based on recruitment of Flt3-positive dendritic cells, but may be corroborated by activity of this signaling pathway in glioma cells.Methods: Receptor and ligand expression was analyzed by real-time polymerase chain reaction in 31 astrocytic tumors (six diffuse and 11 anaplastic astrocytomas, 14 glioblastomas derived from patients of both genders and in glioblastoma cell lines. The two most common activating mutations of the Flt3 gene, ie, internal tandem duplication and D835 point mutation, were assessed by specific polymerase chain reaction.Results: A relatively high abundance of Flt3L mRNA (4%–6% of the reference, β2 microglobulin could be demonstrated in all tumor samples. Flt3 expression could generally be demonstrated by 40 specific polymerase chain reaction cycles and gel electrophoresis in 87% of the tumors, including all grades, although the small quantities of the receptor did

  7. MODERATE CYTOTOXICITY OF PROANTHOCYANIDINS TO HUMAN TUMOR-CELL LINES

    NARCIS (Netherlands)

    KOLODZIEJ, H; HABERLAND, C; WOERDENBAG, HJ; KONINGS, AWT

    1995-01-01

    In the present study the cytotoxicity of 16 proanthocyanidins was evaluated in GLC(4), a human small cell lung carcinoma cell line, and in COLO 320, a human colorectal cancer cell line, using the microculture tetrazolium (MTT) assay. With IC50 values ranging from 18 to >200 mu m following continuous

  8. HIV-1-infected and immune-activated macrophages induce astrocytic differentiation of human cortical neural progenitor cells via the STAT3 pathway.

    Directory of Open Access Journals (Sweden)

    Hui Peng

    Full Text Available Diminished adult neurogenesis is considered a potential mechanism in the pathogenesis of HIV-1-associated dementia (HAD. In HAD, HIV-1-infected and immune-activated brain mononuclear phagocytes (MP; perivascular macrophages and microglia drive central nervous system (CNS inflammation and may alter normal neurogenesis. We previously demonstrated HIV-1-infected and lipopolysaccharide (LPS activated monocyte-derived macrophages (MDM inhibit human neural progenitor cell (NPC neurogenesis, while enhancing astrogliogenesis through the secretion of the inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α, in vitro and in vivo. Here we further test the hypothesis that HIV-1-infected/activated MDM promote NPC astrogliogenesis via activation of the transcription factor signal transducer and activator of transcription 3 (STAT3, a critical factor for astrogliogenesis. Our results show that LPS-activated MDM-conditioned medium (LPS-MCM and HIV-infected/LPS-activated MDM-conditioned medium (LPS+HIV-MCM induced Janus kinase 1 (Jak1 and STAT3 activation. Induction of the Jak-STAT3 activation correlated with increased glia fibrillary acidic protein (GFAP expression, demonstrating an induction of astrogliogenesis. Moreover, STAT3-targeting siRNA (siSTAT3 decreased MCM-induced STAT3 activation and NPC astrogliogenesis. Furthermore, inflammatory cytokines (including IL-6, IL-1β and TNF-α produced by LPS-activated and/or HIV-1-infected MDM may contribute to MCM-induced STAT3 activation and astrocytic differentiation. These observations were confirmed in severe combined immunodeficient (SCID mice with HIV-1 encephalitis (HIVE. In HIVE mice, siRNA control (without target sequence, sicon pre-transfected NPCs injected with HIV-1-infected MDM showed more astrocytic differentiation and less neuronal differentiation of NPCs as compared to NPC injection alone. siSTAT3 abrogated HIV-1-infected MDM-induced astrogliogenesis of injected NPCs. Collectively, these

  9. Distinct differentiation characteristics of individual human embryonic stem cell lines

    Directory of Open Access Journals (Sweden)

    Knuutila Sakari

    2006-08-01

    Full Text Available Abstract Background Individual differences between human embryonic stem cell (hESC lines are poorly understood. Here, we describe the derivation of five hESC lines (called FES 21, 22, 29, 30 and 61 from frozen-thawed human embryos and compare their individual differentiation characteristic. Results The cell lines were cultured either on human or mouse feeder cells. The cells grew significantly faster and could be passaged enzymatically only on mouse feeders. However, this was found to lead to chromosomal instability after prolonged culture. All hESC lines expressed the established markers of pluripotent cells as well as several primordial germ cell (PGC marker genes in a uniform manner. However, the cell lines showed distinct features in their spontaneous differentiation patterns. The embryoid body (EB formation frequency of FES 30 cell line was significantly lower than that of other lines and cells within the EBs differentiated less readily. Likewise, teratomas derived from FES 30 cells were constantly cystic and showed only minor solid tissue formation with a monotonous differentiation pattern as compared with the other lines. Conclusion hESC lines may differ substantially in their differentiation properties although they appear similar in the undifferentiated state.

  10. Galunisertib inhibits glioma vasculogenic mimicry formation induced by astrocytes

    OpenAIRE

    Chao Zhang; Wenliang Chen; Xin Zhang; Bin Huang; Aanjing Chen; Ying He; Jian Wang; Xingang Li

    2016-01-01

    Gliomas are among the most lethal primary brain tumors found in humans. In high-grade gliomas, vasculogenic mimicry is often detected and has been correlated with prognosis, thus suggesting its potential as a therapeutic target. Vasculogenic mimicry mainly forms vascular-like channels independent of endothelial cells; however, little is known about the relationship between astrocytes and vasculogenic mimicry. In our study, we demonstrated that the presence of astrocytes promoted vasculogenic ...

  11. Astrocytes revisited: concise historic outlook on glutamate homeostasis and signaling

    OpenAIRE

    Parpura, Vladimir; VERKHRATSKY, ALEXEI

    2012-01-01

    Astroglia is a main type of brain neuroglia, which includes many cell sub-types that differ in their morphology and physiological properties and yet are united by the main function, which is the maintenance of brain homeostasis. Astrocytes employ a variety of mechanisms for communicating with neuronal networks. The communication mediated by neurotransmitter glutamate has received a particular attention. Glutamate is de novo synthesized exclusively in astrocytes; astroglia-derived glutamine is...

  12. Analysis of three marine fish cell lines by rapd assay.

    Science.gov (United States)

    Guo, H R; Zhang, S C; Tong, S L; Xiang, J H

    2001-01-01

    We tested the applicability of the random amplified polymorphic deoxyribonucleic acid (RAPD) analysis for identification of three marine fish cell lines FG, SPH, and RSBF, and as a possible tool to detect cross-contamination. Sixty commercial 10-mer RAPD primers were tested on the cell lines and on samples collected from individual fish. The results obtained showed that the cell lines could be identified to the correspondent species on the basis of identical patterns produced by 35-48% of the primers tested; the total mean similarity indices for cell lines versus correspondent species of individual fish ranged from 0.825 to 0.851, indicating the existence of genetic variation in these cell lines in relation to the species of their origin. Also, four primers, which gave a monomorphic band pattern within species/line, but different among the species/line, were obtained. These primers can be useful for identification of these cell lines and for characterization of the genetic variation of these cell lines in relation to the species of their origin. This supported the use of RAPD analysis as an effective tool in species identification and cross-contamination test among different cell lines. PMID:11573817

  13. POTENTIAL CELL LINE TOXICITY OF ENVIRONMENTAL NANOPARTICLES

    Directory of Open Access Journals (Sweden)

    Mohan Durga

    2012-01-01

    Full Text Available In India, the unprecedented growth rate and urbanization along with the rapid increase in motor vehicle activity and industrialization are contributing to high levels of urban air pollution. The population is mainly exposed to high air pollution concentrations, where motor vehicle emissions constitute the main source of fine and ultrafine particles. Motor exhaust emissions is a mixture of gases and Particulate Matter (PM. Diesel and petrol fuels in vehicles produce combustion-derived particles as a result of combustion. Vehicle exhaust particles are the main constituents of environmental nanoparticles. In the present investigation, environmental nanoparticles such as Diesel Exhaust Particles (DEP and Petrol Exhaust Particles (PEP were collected from on-road vehicles using a specially designed collection chamber. The surface morphology of the collected particles was analyzed through Transmission Electron Microscope (TEM, and the elemental mapping was performed through EDAX analysis. Results indicated the presence of nanometer-size particles in both the categories of vehicle exhaust. These small-size particles of respirable range can enter the respiratory tract of humans and get deposited in the lungs and cause various effects inside the human body. The aim of this study is to assess the cytotoxicity of the collected Diesel Exhaust Nanoparticles (DENPs and Petrol Exhaust Nanoparticles (PENPs. Cytotoxicity endpoint, such as IC50 (50% Inhibitory Concentration, was determined after a 24-h exposure. Results of this study indicated that all five cell lines were sensitive to these vehicle exhaust nanoparticles at varying levels.

  14. Spatiotemporal characteristics of calcium dynamics in astrocytes

    Science.gov (United States)

    Kang, Minchul; Othmer, Hans G.

    2009-09-01

    Although Cai2+ waves in networks of astrocytes in vivo are well documented, propagation in vivo is much more complex than in culture, and there is no consensus concerning the dominant roles of intercellular and extracellular messengers [inositol 1,4,5-trisphosphate (IP3) and adenosine-5'-triphosphate (ATP)] that mediate Cai2+ waves. Moreover, to date only simplified models that take very little account of the geometrical struture of the networks have been studied. Our aim in this paper is to develop a mathematical model based on realistic cellular morphology and network connectivity, and a computational framework for simulating the model, in order to address these issues. In the model, Cai2+ wave propagation through a network of astrocytes is driven by IP3 diffusion between cells and ATP transport in the extracellular space. Numerical simulations of the model show that different kinetic and geometric assumptions give rise to differences in Cai2+ wave propagation patterns, as characterized by the velocity, propagation distance, time delay in propagation from one cell to another, and the evolution of Ca2+ response patterns. The temporal Cai2+ response patterns in cells are different from one cell to another, and the Cai2+ response patterns evolve from one type to another as a Cai2+ wave propagates. In addition, the spatial patterns of Cai2+ wave propagation depend on whether IP3, ATP, or both are mediating messengers. Finally, two different geometries that reflect the in vivo and in vitro configuration of astrocytic networks also yield distinct intracellular and extracellular kinetic patterns. The simulation results as well as the linear stability analysis of the model lead to the conclusion that Cai2+ waves in astrocyte networks are probably mediated by both intercellular IP3 transport and nonregenerative (only the glutamate-stimulated cell releases ATP) or partially regenerative extracellular ATP signaling.

  15. Physiopathologic dynamics of vesicle traffic in astrocytes.

    Science.gov (United States)

    Potokar, Maja; Stenovec, Matjaž; Kreft, Marko; Gabrijel, Mateja; Zorec, Robert

    2011-02-01

    The view of how astrocytes, a type of glial cells, contribute to the functioning of the central nervous system (CNS) has changed greatly in the last decade. Although glial cells outnumber neurons in the mammalian brain, it was considered for over a century that they played a subservient role to neurons. This view changed. Functions thought to be exclusively present in neurons, i.e. excitability mediated release of chemical messengers, has also been demonstrated in astrocytes. In this process, following an increase in cytosolic calcium activity, membrane bound vesicles, storing chemical messengers (gliotransmitters), fuse with the plasma membrane, a process known as exocytosis, permitting the exit of vesicle cargo into the extracellular space. Vesicles are delivered to and are removed from the site of exocytosis by an amazingly complex set of processes that we have only started to learn about recently. In this paper we review vesicle traffic, which is subject to physiological regulation and may be changed under pathological conditions.

  16. Cellular radiosensitivity of small-cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Krarup, M; Poulsen, H S; Spang-Thomsen, M

    1997-01-01

    . The multitarget single hit model was applied to calculate the cellular radiosensitivity (D0), the capacity for sublethal damage repair (Dq), and the extrapolation number (n). Values for alpha and beta were determined from best-fit curves according to the linear-quadratic model and these values were applied...... to calculate the surviving fraction after 2-Gy irradiation (SF2). RESULTS: In our investigation, the extrapolation method proved to be inappropriate for the study of in vitro cellular radiosensitivity due to lack of reproducibility. The results obtained by the clonogenic assay showed that the cell lines...

  17. Astrocyte morphology, heterogeneity and density in the developing African Giant Rat (Cricetomys gambianus

    Directory of Open Access Journals (Sweden)

    James Olukayode Olopade

    2015-05-01

    Full Text Available Astrocyte morphologies and heterogeneity were described in male African giant rats (AGR (Cricetomys gambianus, Waterhouse across three age groups (5 neonates, 5 juveniles and 5 adults using Silver impregnation method and immunohistochemistry against glia fibrillary acidic protein (GFAP. Immunopositive cell signaling, cell size and population were least in neonates, followed by adults and juveniles respectively. In neonates, astrocyte processes were mostly detected within the glia limitans of the mid and hind brain; their cell bodies measuring 32±4.8 µm in diameter against 91±5.4µm and 75± 1.9µm in juveniles and adults respectively. Astrocyte heterogeneity in juvenile and adult groups revealed eight subtypes to include fibrous astrocytes chiefly in the corpus callosum and brain stem, protoplasmic astrocytes in the cortex and dentate gyrus (DG; radial glia were found along the olfactory bulb (OB and subventricular zone (SVZ; velate astrocytes were mainly found in the cerebellum and hippocampus; marginal astrocytes close to the pia mater; Bergmann glia in the molecular layer of the cerebellum; perivascular and periventricular astrocytes in the cortex and third ventricle respectively. Cell counts from twelve anatomical regions of the brain were significantly higher in juveniles than in adults (p≤0.01 using unpaired student t-test in the cerebral cortex, pia, corpus callosum, rostral migratory stream (RMS, DG and cerebellum. Highest astrocyte count was found in the DG, while the least count was in the brain stem and sub cortex. Astrocytes along the periventricular layer of the OB are believed to be part of the radial glia system that transport newly formed cells towards the hippocampus and play roles in neurogenesis migration and homeostasis in the AGR. Therefore, astrocyte heterogeneity was examined across age groups in the AGR to determine whether age influences astrocytes population in different regions of the AGR brain and discuss

  18. Redistribution of monocarboxylate transporter 2 on the surface of astrocytes in the human epileptogenic hippocampus

    DEFF Research Database (Denmark)

    Lauritzen, Fredrik; Heuser, Kjell; de Lanerolle, Nihal C;

    2012-01-01

    astrocyte endfeet, respectively, facilitate the transport of monocarboxylates and protons across cell membranes. Recently, we reported that the density of MCT1 protein is reduced on endothelial cells and increased on astrocyte plasma membranes in the hippocampal formation in patients with MTLE and in...... several animal models of the disorder. Because the perivascular astrocyte endfeet comprise an important part of the neurovascular unit, we now assessed the distribution of the MCT2 in hippocampal formations in TLE patients with (MTLE) or without hippocampal sclerosis (non-MTLE). Light microscopic...... perivascular astrocyte endfeet. Interestingly, the loss of MCT2 on astrocyte endfeet in MTLE (n = 3) was accompanied by an upregulation of the protein on astrocyte membranes facing synapses in the neuropil, when compared with non-MTLE (n = 3). We propose that the altered distribution of MCT1 and MCT2 in TLE...

  19. Astrocytes mediate synapse elimination through MEGF10 and MERTK pathways

    Science.gov (United States)

    Chung, Won-Suk; Clarke, Laura E.; Wang, Gordon X.; Stafford, Benjamin K.; Sher, Alexander; Chakraborty, Chandrani; Joung, Julia; Foo, Lynette C.; Thompson, Andrew; Chen, Chinfei; Smith, Stephen J.; Barres, Ben A.

    2013-12-01

    To achieve its precise neural connectivity, the developing mammalian nervous system undergoes extensive activity-dependent synapse remodelling. Recently, microglial cells have been shown to be responsible for a portion of synaptic pruning, but the remaining mechanisms remain unknown. Here we report a new role for astrocytes in actively engulfing central nervous system synapses. This process helps to mediate synapse elimination, requires the MEGF10 and MERTK phagocytic pathways, and is strongly dependent on neuronal activity. Developing mice deficient in both astrocyte pathways fail to refine their retinogeniculate connections normally and retain excess functional synapses. Finally, we show that in the adult mouse brain, astrocytes continuously engulf both excitatory and inhibitory synapses. These studies reveal a novel role for astrocytes in mediating synapse elimination in the developing and adult brain, identify MEGF10 and MERTK as critical proteins in the synapse remodelling underlying neural circuit refinement, and have important implications for understanding learning and memory as well as neurological disease processes.

  20. Glutamate metabolism in the brain focusing on astrocytes

    DEFF Research Database (Denmark)

    Schousboe, Arne; Scafidi, Susanna; Bak, Lasse Kristoffer;

    2014-01-01

    Metabolism of glutamate, the main excitatory neurotransmitter and precursor of GABA, is exceedingly complex and highly compartmentalized in brain. Maintenance of these neurotransmitter pools is strictly dependent on the de novo synthesis of glutamine in astrocytes which requires both the anaplero......Metabolism of glutamate, the main excitatory neurotransmitter and precursor of GABA, is exceedingly complex and highly compartmentalized in brain. Maintenance of these neurotransmitter pools is strictly dependent on the de novo synthesis of glutamine in astrocytes which requires both......, as well as in nitrogen trafficking and ammonia homeostasis in brain. The anatomical specialization of astrocytic endfeet enables these cells to rapidly and efficiently remove neurotransmitters from the synaptic cleft to maintain homeostasis, and to provide glutamine to replenish neurotransmitter pools...... summarizes the evidence that astrocytes are essential and dynamic partners in both glutamatergic and GABAergic neurotransmission in brain....

  1. DNA content analysis of insect cell lines by flow cytometry

    OpenAIRE

    Léry, Xavier; Charpentier, Guy; Belloncik, Serge

    1999-01-01

    The DNA content of insect cell lines (6 lepidoptera, 1 coleoptera and 1 diptera) was determined by flow cytometry. The DNA profiles of the 8 cell lines tested were different. They were characterized by the presence of several peaks (2 to 7) corresponding to different ploidy levels, by differences in the fluorescence intensity of each peak and by the proportion of cells in each peak. Two cell lines (Cf124 and BmN) were constituted of 2 distinct populations of cells. The DNA profiles of the cel...

  2. Taurine Biosynthesis by Neurons and Astrocytes*

    OpenAIRE

    Vitvitsky, Victor; Garg, Sanjay K.; Banerjee, Ruma

    2011-01-01

    The physiological roles of taurine, a product of cysteine degradation and one of the most abundant amino acids in the body, remain elusive. Taurine deficiency leads to heart dysfunction, brain development abnormalities, retinal degradation, and other pathologies. The taurine synthetic pathway is proposed to be incomplete in astrocytes and neurons, and metabolic cooperation between these cell types is reportedly needed to complete the pathway. In this study, we analyzed taurine synthesis capab...

  3. Unveiling astrocytic control of cerebral blood flow with optogenetics.

    Science.gov (United States)

    Masamoto, Kazuto; Unekawa, Miyuki; Watanabe, Tatsushi; Toriumi, Haruki; Takuwa, Hiroyuki; Kawaguchi, Hiroshi; Kanno, Iwao; Matsui, Ko; Tanaka, Kenji F; Tomita, Yutaka; Suzuki, Norihiro

    2015-06-16

    Cortical neural activities lead to changes in the cerebral blood flow (CBF), which involves astrocytic control of cerebrovascular tone. However, the manner in which astrocytic activity specifically leads to vasodilation or vasoconstriction is difficult to determine. Here, cortical astrocytes genetically expressing a light-sensitive cation channel, channelrhodopsin-2 (ChR2), were transcranially activated with a blue laser while the spatiotemporal changes in CBF were noninvasively monitored with laser speckle flowgraphy in the anesthetised mouse cortex. A brief photostimulation induced a fast transient increase in CBF. The average response onset time was 0.7 ± 0.7 sec at the activation foci, and this CBF increase spread widely from the irradiation spot with an apparent propagation speed of 0.8-1.1 mm/sec. The broad increase in the CBF could be due to a propagation of diffusible vasoactive signals derived from the stimulated astrocytes. Pharmacological manipulation showed that topical administration of a K(+) channel inhibitor (BaCl2; 0.1-0.5 mM) significantly reduced the photostimulation-induced CBF responses, which indicates that the ChR2-evoked astrocytic activity involves K(+) signalling to the vascular smooth muscle cells. These findings demonstrate a unique model for exploring the role of the astrocytes in gliovascular coupling using non-invasive, time-controlled, cell-type specific perturbations.

  4. Probing astrocytes with carbon nanotubes and assessing their effects on astrocytic structural and functional properties

    Science.gov (United States)

    Gottipati, Manoj K.

    Single-walled carbon nanotubes, chemically-functionalized with polyethylene glycol (SWCNT-PEG) have been shown to modulate the morphology and proliferation characteristics of astrocytes in culture, when applied to the cells as colloidal solutes or as films upon which the cells can attach and grow. These changes were associated with a change in the immunoreactivity of the astrocyte-specific protein, glial fibrillary acidic protein (GFAP); the solutes and films caused an increase and a decrease in GFAP levels, respectively. To assess if these morpho-functional changes induced by the SWCNT-PEG modalities are dependent on GFAP or if the changes in GFAP levels are independent events, I used astrocytes isolated from GFAP knockout mice and found that selected changes induced by the SWCNT-PEG modalities are mediated by GFAP, namely the changes in perimeter, shape and cell death for colloidal solutes and the rate of proliferation for films. Since the loss GFAP has been shown to hamper the trafficking of glutamate transporters to the surface of astrocytes, which plays a vital role in the uptake of extracellular glutamate and maintaining homeostasis in the brain and spinal cord, in a subsequent study, I assessed if the SWCNT-PEG solute causes any change in the glutamate uptake characteristics of astrocytes. Using a radioactive glutamate uptake assay and immunolabeling, I found that SWCNT-PEG solute causes an increase in the uptake of glutamate from the extracellular space along with an increase in the immunoreactivity of the glutamate transporter, L-glutamate L-aspartate transporter (GLAST), on their cell surface, a likely consequence of the increase in GFAP levels induced by the SWCNT-PEG solute. These results imply that SWCNT-PEG could potentially be used as a viable candidate in neural prosthesis applications to prevent glutamate excitotoxicity, a pathological process observed in brain and spinal cord injuries, and alleviate the death toll of neurons surrounding the injury

  5. Neurospheres induced from bone marrow stromal cells are multipotent for differentiation into neuron, astrocyte, and oligodendrocyte phenotypes

    International Nuclear Information System (INIS)

    Bone marrow stromal cells (MSCs) can be expanded rapidly in vitro and have the potential to be differentiated into neuronal, glial and endodermal cell types. However, induction for differentiation does not always have stable result. We present a new method for efficient induction and acquisition of neural progenitors, neuronal- and glial-like cells from MSCs. We demonstrate that rat MSCs can be induced to neurospheres and most cells are positive for nestin, which is an early marker of neuronal progenitors. In addition, we had success in proliferation of these neurospheres with undifferentiated characteristics and finally we could obtain large numbers of neuronal and glial phenotypes. Many of the cells expressed β-tubulin III when they were cultivated with our method. MSCs can become a valuable cell source as an autograft for clinical application involving regeneration of the central nervous system

  6. The Role of Intermittent Hypoxia on the Proliferative Inhibition of Rat Cerebellar Astrocytes.

    Directory of Open Access Journals (Sweden)

    Sheng-Chun Chiu

    Full Text Available Sleep apnea syndrome, characterized by intermittent hypoxia (IH, is linked with increased oxidative stress. This study investigates the mechanisms underlying IH and the effects of IH-induced oxidative stress on cerebellar astrocytes. Rat primary cerebellar astrocytes were kept in an incubator with an oscillating O2 concentration between 20% and 5% every 30 min for 1-4 days. Although the cell loss increased with the duration, the IH incubation didn't induce apoptosis or necrosis, but rather a G0/G1 cell cycle arrest of cerebellar astrocytes was noted. ROS accumulation was associated with cell loss during IH. PARP activation, resulting in p21 activation and cyclin D1 degradation was associated with cell cycle G0/G1 arrest of IH-treated cerebellar astrocytes. Our results suggest that IH induces cell loss by enhancing oxidative stress, PARP activation and cell cycle G0/G1 arrest in rat primary cerebellar astrocytes.

  7. Chloroquine mediated molecular tuning of astrocytes for enhanced permissiveness to HIV infection

    OpenAIRE

    Vijaykumar, Theophilus S.; Nath, Avindra; Chauhan, Ashok

    2008-01-01

    We report in this study that minimum productive HIV infection in astrocytes (a predominant cell type in brain and persists for the entire life) occurs through endocytosis. The lysosomotropic agent chloroquine enhanced permissiveness of astrocytes to HIV infection possibly by circumventing degradation of endosome-entrapped viral particles. In particular, chloroquine may promote establishment of a stable long term viral reservoir in astrocytes and may eventually facilitate early onset of neurol...

  8. Insights into Human Astrocyte Response to H5N1 Infection by Microarray Analysis

    OpenAIRE

    Xian Lin; Ruifang Wang; Jun Zhang; Xin Sun; Zhong Zou; Shengyu Wang; Meilin Jin

    2015-01-01

    Influenza virus infects not only the respiratory system but also the central nervous system (CNS), leading to influenza-associated encephalopathy and encephalitis. Astrocytes are essential for brain homeostasis and neuronal function. These cells can also be infected by influenza virus. However, genome-wide changes in response to influenza viral infection in astrocytes have not been defined. In this study, we performed gene profiling of human astrocytes in response to H5N1. Innate immune and p...

  9. Trichloroethylene toxicity in a human hepatoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Thevenin, E.; McMillian, J. [Medical Univ. of Charleston South Carolina, SC (United States)

    1994-12-31

    The experiments conducted in this study were designed to determine the usefullness of hepatocyte cultures and a human hepatoma cell line as model systems for assessing human susceptibility to hepatocellular carcinoma due to exposure to trichloroethylene. The results from these studies will then be analyzed to determine if human cell lines can be used to conduct future experiments of this nature.

  10. Derivation of the human embryonic stem cell line RCM1

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCM-1 was derived from a failed to fertilise egg undergoing parthenogenetic stimulation. The cell line shows normal pluripotency marker expression and differentiation to three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

  11. Derivation of the human embryonic stem cell line RCM1.

    Science.gov (United States)

    De Sousa, P A; Tye, B J; Sneddon, S; Bruce, K; Dand, P; Russell, G; Collins, D M; Greenshields, A; McDonald, K; Bradburn, H; Gardner, J; Downie, J M; Courtney, A; Brison, D R

    2016-03-01

    The human embryonic stem cell line RCM-1 was derived from a failed to fertilise egg undergoing parthenogenetic stimulation. The cell line shows normal pluripotency marker expression and differentiation to three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available. PMID:27346018

  12. Cancer and inflammation studies using zebrafish cell lines

    NARCIS (Netherlands)

    He, Shuning

    2010-01-01

    As the zebrafish, Danio rerio, has been increasingly used as an animal model for biomedical research, we aimed to establish zebrafish cell line models for inflammation and cancer studies in this thesis. Several zebrafish cell lines were characterized and their genetic and physiological properties we

  13. Regulation of neurotrophic factors and energy metabolism by antidepressants in astrocytes

    KAUST Repository

    Martin, Jean Luc

    2013-09-01

    There is growing evidence that astrocytes are involved in the neuropathology of major depression. In particular, decreases in glial cell density observed in the cerebral cortex of individuals with major depressive disorder are accompanied by a reduction of several astrocytic markers suggesting that astrocyte dysfunction may contribute to the pathophysiology of major depression. In rodents, glial loss in the prefrontal cortex is sufficient to induce depressive-like behaviors and antidepressant treatment prevents the stress-induced reduction of astrocyte number in the hippocampus. Collectively, these data support the existence of a link between astrocyte loss or dysfunction, depressive-like behavior and antidepressant treatment. Astrocytes are increasingly recognized to play important roles in neuronal development, neurotransmission, synaptic plasticity and maintenance of brain homeostasis. It is also well established that astrocytes provide trophic, structural, and metabolic support to neurons. In this article, we review evidence that antidepressants regulate energy metabolism and neurotrophic factor expression with particular emphasis on studies in astrocytes. These observations support a role for astrocytes as new targets for antidepressants. The contribution of changes in astrocyte glucose metabolism and neurotrophic factor expression to the therapeutic effects of antidepressants remains to be established. © 2013 Bentham Science Publishers.

  14. Calcium Imaging of Living Astrocytes in the Mouse Spinal Cord following Sensory Stimulation

    Directory of Open Access Journals (Sweden)

    Giovanni Cirillo

    2012-01-01

    Full Text Available Astrocytic Ca2+ dynamics have been extensively studied in ex vivo models; however, the recent development of two-photon microscopy and astrocyte-specific labeling has allowed the study of Ca2+ signaling in living central nervous system. Ca2+ waves in astrocytes have been described in cultured cells and slice preparations, but evidence for astrocytic activation during sensory activity is lacking. There are currently few methods to image living spinal cord: breathing and heart-beating artifacts have impeded the widespread application of this technique. We here imaged the living spinal cord by two-photon microscopy in C57BL6/J mice. Through pressurized injection, we specifically loaded spinal astrocytes using the red fluorescent dye sulforhodamine 101 (SR101 and imaged astrocytic Ca2+ levels with Oregon-Green BAPTA-1 (OGB. Then, we studied astrocytic Ca2+ levels at rest and after right electrical hind paw stimulation. Sensory stimulation significantly increased astrocytic Ca2+ levels within the superficial dorsal horn of the spinal cord compared to rest. In conclusion, in vivo morphofunctional imaging of living astrocytes in spinal cord revealed that astrocytes actively participate to sensory stimulation.

  15. Characterization of xenoantiserum produced against B cell acute lymphoblastic leukemia cell line

    Directory of Open Access Journals (Sweden)

    Akagi,Tadaatsu

    1982-10-01

    Full Text Available Antiserum was produced in white rabbit by intravenously injecting living cells of a B cell acute lymphoblastic leukemia (ALL line (BALL-1. The reactivity of the antiserum against various lymphoid cell lines was examined by membrane immunofluorescence after appropriate absorption. Serum absorbed with non-T, non-B (NALL-1 and T-ALL (TALL-1 cells recognized B cell antigens distinct from Ia-like antigens on both normal and neoplastic B cells. After further absorption with tonsillar cells or normal B cell line (KO-HL-3, it reacted only with BALL-1 cells and did not react with other leukemia/lymphoma and normal B cell lines. The serum absorbed with tonsillar cells reacted only with BALL-1 and some B cell lines. Thus we were able to obtain antisera with specificity to B cell antigen, B-ALL antigen, and B cell line antigen.

  16. Regulated expression of erythropoietin by two human hepatoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Goldberg, M.A.; Glass, G.A.; Cunningham, J.M.; Bunn, H.F.

    1987-11-01

    The development of a cell culture system that produces erythropoietin (Epo) in a regulated manner has been the focus of much effort. The authors have screened multiple renal and hepatic cell lines for either constitutive or regulated expression of Epo. Only the human hepatoma cell lines, Hep3B and HepG2, made significant amounts of Epo as measured both by radioimmunoassay and in vitro bioassay (as much as 330 milliunits per 10/sup 6/ cells in 24 hr). The constitutive production of Epo increased dramatically as a function of cell density in both cell lines. At cell densities < 3.3 x 10/sup 5/ cells per cm/sup 2/, there was little constitutive release of Epo in the medium. With Hep3B cells grown at low cell densities, a mean 18-fold increase in Epo expression was seen in response to hypoxia and a 6-fold increase was observed in response to incubation in medium containing 50 ..mu..M cobalt(II) chloride. At similar low cell densities, Epo production in HepG2 cells could be enhanced an average of about 3-fold by stimulation with either hypoxia or cobalt(II) chloride. Upon such stimulation, both cell lines demonstrated markedly elevated levels of Epo mRNA. Hence, both Hep3B and HepG2 cell lines provide an excellent in vitro system in which to study the physiological regulation of Epo expression.

  17. Human embryonic stem cell lines derived from the Chinese population

    Institute of Scientific and Technical Information of China (English)

    Zhen Fu FANG; Fan JIN; Hui GAI; Ying CHEN; Li WU; Ai Lian LIU; Bin CHEN; Hui Zhen SHENG

    2005-01-01

    Six human embryonic stem cell lines were established from surplus blastocysts. The cell lines expressed alkaline phosphatase and molecules typical of primate embryonic stem cells, including Oct-4, Nanog, TDGF1, Sox2, EBAF,Thy-1, FGF4, Rex-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. Five of the six lines formed embryoid bodies that expressed markers of a variety of cell types; four of them formed teratomas with tissue types representative of all three embryonic germ layers. These human embryonic stem cells are capable of producing clones of undifferentiated morphology, and one of them was propagated to become a subline. Human embryonic stem cell lines from the Chinese population should facilitate stem cell research and may be valuable in studies of population genetics and ecology.

  18. Cell type-specific dependency on the PI3K/Akt signaling pathway for the endogenous Epo and VEGF induction by baicalein in neurons versus astrocytes.

    Directory of Open Access Journals (Sweden)

    Yu-Yo Sun

    Full Text Available The neuroprotective effect of baicalein is generally attributed to inhibition of 12/15-lipoxygenase (12/15-LOX and suppression of oxidative stress, but recent studies showed that baicalein also activates hypoxia-inducible factor-α (HIF1α through inhibition of prolyl hydrolase 2 (PHD2 and activation of the phosphatidylinositide-3 kinase (PI3K/Akt signaling pathway. Yet, the significance and regulation of prosurvival cytokines erythropoietin (Epo and vascular endothelial growth factor (VEGF, two transcriptional targets of HIF1α, in baicalein-mediated neuroprotection in neurons and astrocytes remains unknown. Here we investigated the causal relationship between the PI3K/Akt signaling pathway and Epo/VEGF expression in baicalein-mediated neuroprotection in primary rat cortical neurons and astrocytes. Our results show that baicalein induced Epo and VEGF expression in a HIF1α- and PI3K/Akt-dependent manner in neurons. Baicalein also protected neurons against excitotoxicity in a PI3K- and Epo/VEGF-dependent manner without affecting neuronal excitability. In contrast, at least a 10-fold higher concentration of baicalein was needed to induce Epo/VEGF production and PI3K/Akt activity in astrocytes for protection of neurons. Moreover, only baicalein-induced astrocytic VEGF, but not Epo expression requires HIF1α, while PI3K/Akt signaling had little role in baicalein-induced astrocytic Epo/VEGF expression. These results suggest distinct mechanisms of baicalein-mediated Epo/VEGF production in neurons and astrocytes for neuroprotection, and provide new insights into the mechanisms and potential of baicalein in treating brain injury in vivo.

  19. A novel gliotic P2 receptor mediating cyclooxygenase-2 induction in rat and human astrocytes.

    Science.gov (United States)

    Brambilla, R; Ceruti, S; Malorni, W; Cattabeni, F; Abbracchio, M P

    2000-07-01

    In astrocytic cultures maintained in vitro, a brief challenge with the ATP analog alpha,beta methyleneATP (alpha,betameATP) results, 3 days later, in marked elongation of astrocytic processes, an event that resembles the astrocytic hypertrophy known to occur in vivo during reactive astrogliosis. alpha,beta meATP-induced effects were observed in primary astrocytes obtained from both rat striatum and cortex (a brain area highly involved in chronic neurodegenerative pathologies), as well as in human astrocytoma cells (ADF cells). Purine-induced gliosis could be reversed by the non-selective P2X/P2Y receptor antagonist pyridoxalphosphate-6-azophenyl-2', 4'-disulphonic acid (PPADS), but not by oxidized ATP (an antagonist of the P2X(7) receptor), in line with previous studies of our laboratory suggesting the involvement of a P2Y receptor subtype. Induction of reactive gliosis was preceded by increased expression of cyclooxygenase-2 (COX-2), an enzyme whose excessive activation has been implicated in both acute and chronic neurodegenerative diseases. The selective COX-2 inhibitor NS-398 prevented both purine-induced astrogliosis and the associated COX-2 induction, suggesting that inhibition of the transcription of the COX-2 gene may also contribute to the anti-inflammatory properties of this agent. Significant blockade of both alpha,beta meATP-mediated reactive gliosis and COX-2 induction was also observed with PPADS. These data suggest that COX-2 mediates P2Y receptor-induced reactive astrogliosis, and that antagonists selective for this receptor subtype may represent a novel class of anti-inflammatory agents of potential interest in acute and chronic neurological disorders characterized by an inflammatory component and reactive gliosis.

  20. Accumulation of silver nanoparticles by cultured primary brain astrocytes

    Science.gov (United States)

    Luther, Eva M.; Koehler, Yvonne; Diendorf, Joerg; Epple, Matthias; Dringen, Ralf

    2011-09-01

    Silver nanoparticles (AgNP) are components of various food industry products and are frequently used for medical equipment and materials. Although such particles enter the vertebrate brain, little is known on their biocompatibility for brain cells. To study the consequences of an AgNP exposure of brain cells we have treated astrocyte-rich primary cultures with polyvinylpyrrolidone (PVP)-coated AgNP. The incubation of cultured astrocytes with micromolar concentrations of AgNP for up to 24 h resulted in a time- and concentration-dependent accumulation of silver, but did not compromise the cell viability nor lower the cellular glutathione content. In contrast, the incubation of astrocytes for 4 h with identical amounts of silver as AgNO3 already severely compromised the cell viability and completely deprived the cells of glutathione. The accumulation of AgNP by astrocytes was proportional to the concentration of AgNP applied and significantly lowered by about 30% in the presence of the endocytosis inhibitors chloroquine or amiloride. Incubation at 4 °C reduced the accumulation of AgNP by 80% compared to the values obtained for cells that had been exposed to AgNP at 37 °C. These data demonstrate that viable cultured brain astrocytes efficiently accumulate PVP-coated AgNP in a temperature-dependent process that most likely involves endocytotic pathways.

  1. Accumulation of silver nanoparticles by cultured primary brain astrocytes

    International Nuclear Information System (INIS)

    Silver nanoparticles (AgNP) are components of various food industry products and are frequently used for medical equipment and materials. Although such particles enter the vertebrate brain, little is known on their biocompatibility for brain cells. To study the consequences of an AgNP exposure of brain cells we have treated astrocyte-rich primary cultures with polyvinylpyrrolidone (PVP)-coated AgNP. The incubation of cultured astrocytes with micromolar concentrations of AgNP for up to 24 h resulted in a time- and concentration-dependent accumulation of silver, but did not compromise the cell viability nor lower the cellular glutathione content. In contrast, the incubation of astrocytes for 4 h with identical amounts of silver as AgNO3 already severely compromised the cell viability and completely deprived the cells of glutathione. The accumulation of AgNP by astrocytes was proportional to the concentration of AgNP applied and significantly lowered by about 30% in the presence of the endocytosis inhibitors chloroquine or amiloride. Incubation at 4 0C reduced the accumulation of AgNP by 80% compared to the values obtained for cells that had been exposed to AgNP at 37 0C. These data demonstrate that viable cultured brain astrocytes efficiently accumulate PVP-coated AgNP in a temperature-dependent process that most likely involves endocytotic pathways.

  2. Accumulation of silver nanoparticles by cultured primary brain astrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Luther, Eva M; Koehler, Yvonne; Dringen, Ralf [Center for Biomolecular Interactions Bremen, University of Bremen, PO Box 330440, D-28334 Bremen (Germany); Diendorf, Joerg; Epple, Matthias, E-mail: ralf.dringen@uni-bremen.de [Inorganic Chemistry and Center for Nanointegration Duisburg-Essen, University of Duisburg-Essen, Universitaetsstrasse 5-7, D-45117 Essen (Germany)

    2011-09-16

    Silver nanoparticles (AgNP) are components of various food industry products and are frequently used for medical equipment and materials. Although such particles enter the vertebrate brain, little is known on their biocompatibility for brain cells. To study the consequences of an AgNP exposure of brain cells we have treated astrocyte-rich primary cultures with polyvinylpyrrolidone (PVP)-coated AgNP. The incubation of cultured astrocytes with micromolar concentrations of AgNP for up to 24 h resulted in a time- and concentration-dependent accumulation of silver, but did not compromise the cell viability nor lower the cellular glutathione content. In contrast, the incubation of astrocytes for 4 h with identical amounts of silver as AgNO{sub 3} already severely compromised the cell viability and completely deprived the cells of glutathione. The accumulation of AgNP by astrocytes was proportional to the concentration of AgNP applied and significantly lowered by about 30% in the presence of the endocytosis inhibitors chloroquine or amiloride. Incubation at 4 {sup 0}C reduced the accumulation of AgNP by 80% compared to the values obtained for cells that had been exposed to AgNP at 37 {sup 0}C. These data demonstrate that viable cultured brain astrocytes efficiently accumulate PVP-coated AgNP in a temperature-dependent process that most likely involves endocytotic pathways.

  3. The transcriptional diversity of 25 Drosophila cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Cherbas, L.; Willingham, A.; Zhang, D.; Yang, L.; Zou, Y.; Eads, B. D.; Carlson, J. W.; Landolin, J. M.; Kapranov, P.; Dumais, J.; Samsonova, A.; Choi, J. -H.; Roberts, J.; Davis, C. A.; Tang, H.; van Baren, M. J.; Ghosh, S.; Dobin, A.; Bell, K.; Lin, W.; Langton, L.; Duff, M. O.; Tenney, A. E.; Zaleski, C.; Brent, M. R.; Hoskins, R. A.; Kaufman, T. C.; Andrews, J.; Graveley, B. R.; Perrimon, N.; Celniker, S. E.; Gingeras, T. R.; Cherbas, P.

    2010-12-22

    Drosophila melanogaster cell lines are important resources for cell biologists. Here, we catalog the expression of exons, genes, and unannotated transcriptional signals for 25 lines. Unannotated transcription is substantial (typically 19% of euchromatic signal). Conservatively, we identify 1405 novel transcribed regions; 684 of these appear to be new exons of neighboring, often distant, genes. Sixty-four percent of genes are expressed detectably in at least one line, but only 21% are detected in all lines. Each cell line expresses, on average, 5885 genes, including a common set of 3109. Expression levels vary over several orders of magnitude. Major signaling pathways are well represented: most differentiation pathways are ‘‘off’’ and survival/growth pathways ‘‘on.’’ Roughly 50% of the genes expressed by each line are not part of the common set, and these show considerable individuality. Thirty-one percent are expressed at a higher level in at least one cell line than in any single developmental stage, suggesting that each line is enriched for genes characteristic of small sets of cells. Most remarkable is that imaginal discderived lines can generally be assigned, on the basis of expression, to small territories within developing discs. These mappings reveal unexpected stability of even fine-grained spatial determination. No two cell lines show identical transcription factor expression. We conclude that each line has retained features of an individual founder cell superimposed on a common ‘‘cell line‘‘ gene expression pattern. Wereport the transcriptional profiles of 25 Drosophila melanogaster cell lines, principally by whole-genome tiling microarray analysis of total RNA, carried out as part of the modENCODE project. The data produced in this study add to our knowledge of the cell lines and of the Drosophila transcriptome in several ways. We summarize the expression of previously annotated genes in each of the 25 lines with emphasis on what

  4. Tricyclic Antidepressant Amitriptyline-induced Glial Cell Line-derived Neurotrophic Factor Production Involves Pertussis Toxin-sensitive Gαi/o Activation in Astroglial Cells.

    Science.gov (United States)

    Hisaoka-Nakashima, Kazue; Miyano, Kanako; Matsumoto, Chie; Kajitani, Naoto; Abe, Hiromi; Okada-Tsuchioka, Mami; Yokoyama, Akinobu; Uezono, Yasuhito; Morioka, Norimitsu; Nakata, Yoshihiro; Takebayashi, Minoru

    2015-05-29

    Further elaborating the mechanism of antidepressants, beyond modulation of monoaminergic neurotransmission, this study sought to elucidate the mechanism of amitriptyline-induced production of glial cell line-derived neurotrophic factor (GDNF) in astroglial cells. Previous studies demonstrated that an amitriptyline-evoked matrix metalloproteinase (MMP)/FGF receptor (FGFR)/FGFR substrate 2α (FRS2α)/ERK cascade is crucial for GDNF production, but how amitriptyline triggers this cascade remains unknown. MMP is activated by intracellular mediators such as G proteins, and this study sought to clarify the involvement of G protein signaling in amitriptyline-evoked GDNF production in rat C6 astroglial cells (C6 cells), primary cultured rat astrocytes, and normal human astrocytes. Amitriptyline-evoked GDNF mRNA expression and release were inhibited by pertussis toxin (PTX), a Gα(i/o) inhibitor, but not by NF449, a Gα(s) inhibitor, or YM-254890, a Gαq inhibitor. The activation of the GDNF production cascade (FGFR/FRS2α/ERK) was also inhibited by PTX. Deletion of Gα(ο1) and Gα(i3) by RNAi demonstrated that these G proteins play important roles in amitriptyline signaling. G protein activation was directly analyzed by electrical impedance-based biosensors (CellKey(TM) assay), using a label-free (without use of fluorescent proteins/probes or radioisotopes) and real time approach. Amitriptyline increased impedance, indicating Gα(i/o) activation that was suppressed by PTX treatment. The impedance evoked by amitriptyline was not affected by inhibitors of the GDNF production cascade. Furthermore, FGF2 treatment did not elicit any effect on impedance, indicating that amitriptyline targets PTX-sensitive Gα(i/o) upstream of the MMP/FGFR/FRS2α/ERK cascade. These results suggest novel targeting for the development of antidepressants.

  5. Astrocytes as a source for Extracellular matrix molecules and cytokines

    Directory of Open Access Journals (Sweden)

    Stefan eWiese

    2012-06-01

    Full Text Available Research of the past 25 years has shown that astrocytes do more than participating and building up the blood brain barrier and detoxify the active synapse by reuptake of neurotransmitters and ions. Indeed, astrocytes express neurotransmitter receptors and, as a consequence, respond to stimuli. Deeper knowledge of the differentiation processes during development of the central nervous system (CNS might help explaining and even help treating neurological diseases like Alzheimer’s disease, Amyotrophic lateral sclerosis (ALS and psychiatric disorders in which astrocytes have been shown to play a role. Astrocytes and oligodendrocytes develop from a multipotent stem cell that prior to this has produced primarily neuronal precursor cells. This switch towards the more astroglial differentiation is regulated by a change in receptor composition on the cell surface and responsiveness of the respective trophic factors Fibroblast growth factor (FGF and Epidermal growth factor (EGF. The glial precursor cell is driven into the astroglial direction by signaling molecules like Ciliary neurotrophic factor (CNTF, Bone Morphogenetic Proteins (BMPs, and EGF. However, the early astrocytes influence their environment not only by releasing and responding to diverse soluble factors but also express a wide range of extracellular matrix (ECM molecules, in particular proteoglycans of the lectican family and tenascins. Lately these ECM molecules have been shown to participate in glial development. In this regard, especially the matrix protein Tenascin C (Tnc proved to be an important regulator of astrocyte precursor cell proliferation and migration during spinal cord development. On the other hand, ECM molecules expressed by reactive astrocytes are also known to act mostly in an inhibitory fashion under pathophysiological conditions. In this regard, we further summarize recent data concerning the role of chondroitin sulfate proteoglycans and Tnc under pathological

  6. Derivation of human embryonic stem cell line Genea022

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea022 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through CGH and STR analysis. Pluripotency of Genea022 was demonstrated with 84% of cells expressed Nanog, 98% Oct4, 55% Tra1–60 and 97% SSEA4, gave a Pluritest Pluripotency score of 42.95, Novelty of 1.23, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination.

  7. Derivation of Genea052 human embryonic stem cell line

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea052 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through CGH and STR analysis. Pluripotency of Genea052 was demonstrated with 85% of cells expressing Nanog, 87% Oct4, 60% Tra1-60 and 97% SSEA4, a PluriTest Pluripotency score of 27.21, Novelty score of 1.2 and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and any visible contamination.

  8. Derivation of Genea047 human embryonic stem cell line

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea047 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype and female allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea047 was demonstrated with 88% of cells expressing Nanog, 95% Oct4, 59% Tra1-60 and 99% SSEA4, a PluriTest Pluripotency score of 30.86, Novelty score of 1.23 and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and any visible contamination.

  9. Derivation of Genea015 human embryonic stem cell line

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea015 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male Allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea015 was demonstrated with 80% of cells expressing Nanog, 97% Oct4, 75% Tra1-60 and 98% SSEA4, a PluriTest Pluripotency score of 29.52, Novelty score of 1.3 and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination.

  10. Derivation of human embryonic stem cell line Genea023

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea023 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through CGH and STR analysis. Pluripotency of Genea023 was demonstrated with 85% of cells expressed Nanog, 98% Oct4, 55% Tra1-60 and 98% SSEA4, gave a Pluritest Pluripotency score of 42.76, Novelty of 1.23, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination.

  11. Derivation of Genea042 human embryonic stem cell line

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea042 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype and female allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea042 was demonstrated with 81% of cells expressing Nanog, 95% Oct4, 53% Tra1-60 and 97% SSEA4, a PluriTest Pluripotency score of 30.06, Novelty score of 1.24 and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination.

  12. Modified TEM cell design exposure system for in vitro exposure of cultured human astrocytes to 900 MHz GSM mobile phone type signals

    International Nuclear Information System (INIS)

    Full text: A key to the rigour of any experiment seeking to investigate possible effects on living systems of the electromagnetic energy (EME) from mobile phones is to ensure that the exposures used are accurately known and reflect the actual exposures. To achieve well controlled and characterised radiofrequency (RF) exposures is not trivial, and has been a concern in many previous studies. At St Vincent's Hospital Centre for Immunology (CFI), an in vitro study is being performed of possible gene expression changes in cultured human astrocytes exposed to GSM mobile phone type signals. In order to provide rigorous RF dosimetry for the study, Telstra Research Laboratories (TRL) has developed a modified transverse electromagnetic (TEM) cell exposure system. This paper will describe salient aspects of the design and development of the system used at CFI. In the experimental design proposed by CFI, live human astrocyte cells are exposed in standard FalconTM 25 cm3 plastic culture flasks while incubated in a CO2 atmosphere at 37 deg C. The cells typically exist as a very thin monolayer (microns) adhered to the bottom of the flask under a layer of several millimetres of nutrient media. This particular arrangement presents a number of challenges for the design of an appropriate RF exposure system. Many RF exposure systems rely on measurements of average absorption within the target material to determine the specific absorption rate (SAR) in the sample. The actual SAR at any given point in the exposed volume may differ markedly from this average value, and typically varies quadratically with height (h) within the sample, where h is taken to be in the direction of the incident electric (E) field. This variance may be tolerable where the cells are distributed in solution throughout the volume, but this is not the case in this instance. Alternatively, keeping the sample very thin can reduce the variance. However, this limits the efficiency of the system, so that high input

  13. Astrocyte-neuron co-culture on microchips based on the model of SOD mutation to mimic ALS.

    Science.gov (United States)

    Kunze, Anja; Lengacher, Sylvain; Dirren, Elisabeth; Aebischer, Patrick; Magistretti, Pierre J; Renaud, Philippe

    2013-07-24

    Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease. ALS is believed to be a non-cell autonomous condition, as other cell types, including astrocytes, have been implicated in disease pathogenesis. Hence, to facilitate the development of therapeutics against ALS, it is crucial to better understand the interactions between astrocytes and neural cells. Furthermore, cell culture assays are needed that mimic the complexity of cell to cell communication at the same time as they provide control over the different microenvironmental parameters. Here, we aim to validate a previously developed microfluidic system for an astrocyte-neuron cell culture platform, in which astrocytes have been genetically modified to overexpress either a human wild-type (WT) or a mutated form of the super oxide dismutase enzyme 1 (SOD1). Cortical neural cells were co-cultured with infected astrocytes and studied for up to two weeks. Using our microfluidic device that prevents direct cell to cell contact, we could evaluate neural cell response in the vicinity of astrocytes. We showed that neuronal cell density was reduced by about 45% when neurons were co-cultured with SOD-mutant astrocytes. Moreover, we demonstrated that SOD-WT overexpressing astrocytes reduced oxidative stress on cortical neurons that were in close metabolic contact. In contrast, cortical neurons in metabolic contact with SOD-mutant astrocytes lost their synapsin protein expression after severe glutamate treatment, an indication of the toxicity potentiating effect of the SOD-mutant enzyme.

  14. Spatio-temporal pattern of proliferation of immunoglobulin G-containing astrocytes in the injured mouse cerebral hemisphere

    International Nuclear Information System (INIS)

    Following a mechanical injury of the cerebral hemisphere in adult mice, [3H]thymidine was injected at different time intervals to reveal proliferating cells. Brain section were double immunostained for immunoglobulin G (IgG) and glial fibrillary acidic protein (GFAP), and subjected to autoradiography. The procedure allowed to distinguish four cell types: (1) proliferating process-bearing cells immunopositive for IgG but expressing no GFAP (GFAP-/IgG+ PB cells) (2) proliferating and (3) non-proliferating astrocytes immunopositive both for GFAP and IgG (GFAP+/IgG+), (4) proliferating astrocytes expressing GFAP and containing no IgG (GFAP+/IgG-). Thereafter, the distribution of each cell type within the injury area was recorded at each stage of the experiment and displayed as contour line maps. During the whole posttraumatic period, areas of very strong proliferation of GFAP-/IgG+ PB-cells were located within deep of the cerebral cortex and in the white matter around the bottom part of the lesion. Few proliferating GFAP+/IgG+ astrocytes were mainly located in the same regions. However, changes in the distribution of proliferating astrocytes containing no IgG followed different spatio-temporal pattern. On the 4th day after injury areas of their proliferative activity surrounded both the bottom and superficial parts of the lesion. It seems that the immediate contact with the extravasated blood serum does not determine IgG cellular uptake. The uptake appears to depend on the cell type and its role in re-establishing the extracellular space in the injured brain tissue. (author). 15 refs, 7 figs

  15. Cytotoxinic Mechanism of Hydroxyapatite Nanoparticles on Human Hepatoma Cell Lines

    Institute of Scientific and Technical Information of China (English)

    CAO Xian-ying; QI Zhi-tao; DAI Hong-lian; YAN Yu-hua; LI Shi-pu

    2003-01-01

    Stable and single-dispersed HAP nanoparticles were synthesized with chemical method assisted by ultrasonic treatment.HAP nanoparticles were surveyed by AFM and Zataplus.The effect on the Bel-7402 human hepatoma cell lines treated with HAP nanoparticles was investigated by the MTT methods and observation of morphology,and the mechanism was studied in changes of cell cycle and ultrastructure.The result shows that inhibition of HAP nanoparticles on the Bel-7402 human hepatoma cell lines is obviously in vitro.HAP nanoparticles the entered cancer cytoplasm,and cell proliferation is stopped at G1 phase of cell cycle,thus,cancer cells die directly.

  16. Regulation of Kir4.1 expression in astrocytes and astrocytic tumors: a role for interleukin-1 β

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    Zurolo Emanuele

    2012-12-01

    Full Text Available Abstract Objective Decreased expression of inwardly rectifying potassium (Kir channels in astrocytes and glioma cells may contribute to impaired K+ buffering and increased propensity for seizures. Here, we evaluated the potential effect of inflammatory molecules, such as interleukin-1β (IL-1β on Kir4.1 mRNA and protein expression. Methods We investigated Kir4.1 (Kcnj10 and IL-1β mRNA expression in the temporal cortex in a rat model of temporal lobe epilepsy 24 h and 1 week after induction of status epilepticus (SE, using real-time PCR and western blot analysis. The U373 glioblastoma cell line and human fetal astrocytes were used to study the regulation of Kir4.1 expression in response to pro-inflammatory cytokines. Expression of Kir4.1 protein was also evaluated by means of immunohistochemistry in surgical specimens of patients with astrocytic tumors (n = 64, comparing the expression in tumor patients with (n = 38 and without epilepsy (n = 26. Results Twenty-four hours after onset of SE, Kir4.1 mRNA and protein were significantly down-regulated in temporal cortex of epileptic rats. This decrease in expression was followed by a return to control level at 1 week after SE. The transient downregulation of Kir4.1 corresponded to the time of prominent upregulation of IL-1β mRNA. Expression of Kir4.1 mRNA and protein in glial cells in culture was downregulated after exposure to IL-1β. Evaluation of Kir4.1 in tumor specimens showed a significantly lower Kir4.1 expression in the specimens of patients with epilepsy compared to patients without epilepsy. This paralleled the increased presence of activated microglial cells, as well as the increased expression of IL-1β and the cytoplasmic translocation of high mobility group box 1 (HMGB1. Conclusions Taken together, these findings indicate that alterations in expression of Kir4.1 occurring in epilepsy-associated lesions are possibly influenced by the local inflammatory environment and in

  17. Recombinant protein production from stable mammalian cell lines and pools.

    Science.gov (United States)

    Hacker, David L; Balasubramanian, Sowmya

    2016-06-01

    We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced.

  18. Establishment of human embryonic stem cell line from gamete donors

    Institute of Scientific and Technical Information of China (English)

    LI Tao; ZHOU Can-quan; MAI Qing-yun; ZHUANG Guang-lun

    2005-01-01

    Background Human embryonic stem (HES) cell derived from human blastocyst can be propagated indefinitely in the primitive undifferentiated state while remaining pluripotent. It has exciting potential in human developmental biology, drug discovery, and transplantation medicine. But there are insufficient HES cell lines for further study. Methods Three oocyte donors were studied, and 3 in vitro fertilization (IVF) cycles were carried out to get blastocysts for the establishment of HES cell line. Isolated from blastocysts immunosurgically, inner cell mass (ICM) was cultured and propagated on mouse embryonic fibroblasts (MEFs). Once established, morphology, cell surface markers, karyotype and differentiating ability of the cell line were thoroughly analyzed.Results Four ICMs from 7 blastocysts were cultured on MEFs. After culture, one cell line (cHES-1) was established and met the criteria for defining human pluripotent stem cells including a series of markers used to identify pluripotent stem cells, morphological similarity to primate embryonic stem cells and HES reported else where. Normal and stable karyotype maintained over 60 passages, and demonstrated ability to differentiate into a wide variety of cell types.Conclusions HES cell lines can be established from gamete donors at a relatively highly efficient rate. The establishment will exert a widespread impact on biomedical research.

  19. Susceptibility of various cell lines to Neospora caninum tachyzoites cultivation

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    Khordadmehr, M.,

    2014-05-01

    Full Text Available Neospora caninum is a coccidian protozoan parasite which is a major cause of bovine abortions and neonatal mortality in cattle, sheep, goat and horse. Occasionally, cultured cells are used for isolation and multiplication of the agent in vitro with several purposes. In this study the tachyzoite yields of N. caninum were compared in various cell cultures as the host cell lines. Among the cell cultures tested, two presented good susceptibility to the agent: cell lines Vero and MA-104. SW742 and TLI (in vitro suspension culture of lymphoid cells infected with Theileria lestoquardi showed moderate sensitivity. No viable tachyzoite were detected in the culture of MDCK and McCoy cell lines. These results demonstrate that MA-104 and SW742 cells present adequate susceptibility to N. caninum compared to Vero cells, which have been largely used to multiply the parasite in vitro. Moreover, these have easy manipulation, fast multiplication and relatively low nutritional requirements. In addition, the result of this study showed that TLI cell line as a suspension cell culture is susceptible to Nc-1 tachyzoites infection and could be used as an alternative host cell line for tachyzoites culture in vitro studies.

  20. Establishment of Germ-line Competent C57BL/6J Embryonic Stem Cell Lines

    Institute of Scientific and Technical Information of China (English)

    Gui-jun YAN; Zheng GU; Jian WANG; Jia-ke TSO

    2004-01-01

    Objective To establish C57BL/6J embryonic stem (ES) cell lines with potential germline contribution Methods ES cells were isolated from blastocyst inner cell mass of C57BL/6J mice, and cultured for 15 passages, and then injected into blastococels of lCR mice blastocysts to establish chimeric mice.Results Three ES cell lines (mC57ESl,mC57ES3, mC57ES7) derived from the inner cell mass of C57BL/6J mice blastocysts were established. They were characteristic of undifferentiated state, including normal XY karyotype, expression of a specific cell surface marker "stage-specific embryonic antigen-1" and alkaline phosphatase in continuous passage. When injected into immunodeficient mice, mC5 7ES1 cells consis tently differentiated into derivatives of all three embryonic germ layers. When mC57ES1cells were transferred into ICR mice blastocysts, 4 chimeric mice have been obtained.One male of them revealed successful germ-line transmission. Conclussion We have obtained C57BL/6J ES cell lines with a potential germ-line contribution, which can be used to generate transgenic and gene knock-out mice.

  1. Morphological assessment of neurite outgrowth in hippocampal neuron-astrocyte co-cultures.

    Science.gov (United States)

    Giordano, Gennaro; Costa, Lucio G

    2012-05-01

    Neurite outgrowth is a fundamental event in brain development, as well as in regeneration of damaged neurons. Astrocytes play a major role in neuritogenesis, by expressing and releasing factors that facilitate neurite outgrowth, such as extracellular matrix proteins, and factors that can inhibit neuritogenesis, such as the chondroitin sulfate proteoglycan neurocan. In this unit we describe a noncontact co-culture system of hippocampal neurons and cortical (or hippocampal) astrocytes for measurement of neurite outgrowth. Hippocampal pyramidal neurons are plated on glass coverslips, which are inverted onto an astrocyte feeder layer, allowing exposure of neurons to astrocyte-derived factors without direct contact between these two cell types. After co-culture, neurons are stained and photographed, and processes are assessed morphologically using Metamorph software. This method allows exposing astrocytes to various agents before co-culture in order to assess how these exposures may influence the ability of astrocytes to foster neurite outgrowth. PMID:22549268

  2. Astrocyte Depletion Impairs Redox Homeostasis and Triggers Neuronal Loss in the Adult CNS

    Directory of Open Access Journals (Sweden)

    Bettina Schreiner

    2015-09-01

    Full Text Available Although the importance of reactive astrocytes during CNS pathology is well established, the function of astroglia in adult CNS homeostasis is less well understood. With the use of conditional, astrocyte-restricted protein synthesis termination, we found that selective paralysis of GFAP+ astrocytes in vivo led to rapid neuronal cell loss and severe motor deficits. This occurred while structural astroglial support still persisted and in the absence of any major microvascular damage. Whereas loss of astrocyte function did lead to microglial activation, this had no impact on the neuronal loss and clinical decline. Neuronal injury was caused by oxidative stress resulting from the reduced redox scavenging capability of dysfunctional astrocytes and could be prevented by the in vivo treatment with scavengers of reactive oxygen and nitrogen species (ROS/RNS. Our results suggest that the subpopulation of GFAP+ astrocytes maintain neuronal health by controlling redox homeostasis in the adult CNS.

  3. Astrocytes as therapeutic targets of estrogenic compounds following brain injuries

    Directory of Open Access Journals (Sweden)

    George E. Barreto

    2015-03-01

    Full Text Available For decades, astrocytes have been considered to be non-excitable support cells that are relatively resistant to brain injury. This view has changed radically during the past twenty years. Multiple essential functions are performed by astrocytes in normal brain. Astrocytes are dynamically involved in synaptic transmission, metabolic and ionic homeostasis, and inflammatory maintenance of the blood brain barrier. Advances in our understanding of astrocytes include new observations about their structure, organization, and function. Astrocytes play an active and important role in the pathophysiology of brain damage. Brain injury impairs mitochondrial function and this is accompanied by increased oxidative stress, leading to prominent astrogliosis, which involves changes in gene expression and morphology, and therefore glial scar formation. Recent works have demonstrated a protective role of reactive astrocytes after brain injury. Nevertheless, others have pointed to an inhibitory role of astrocytes in axonal regeneration after injury. Reactive astrogliosis is a complex phenomenon that includes a mixture of positive and negative responses for neuronal survival and regeneration. Reactive astroglia maintains the integrity of the blood-brain barrier and the survival of the perilesional tissue, but may prevent axonal and damaged tissue regeneration. Neuroprotective strategies aiming at reducing gliosis and enhance brain plasticity are of potential interest for translational neuroscience research in brain injuries. In this context, neurosteroids have shown to be a promising strategy to protect brain against injury, as their effects may rely on reducing gliosis, brain inflammation and potentially modulating recovery from brain injury by engaging mechanisms of neural plasticity. In conclusion, in this work we will consider particularly the two-edged sword role of reactive astrocytes, which is an experimental paradigm helpful in discriminating destructive

  4. Metabolic aspects of Neuronal – Oligodendrocytic - Astrocytic (NOA interactions

    Directory of Open Access Journals (Sweden)

    Ana I Amaral

    2013-05-01

    Full Text Available Whereas astrocytes have been in the limelight on the metabolic glucose interaction scene for a while, oligodendrocytes are still waiting for a place. We would like to call oligodendrocyte interaction with astrocytes and neurons: NOA (neuron – oligodendrocyte – astrocyte interactions. One of the reasons to find out more about oligodendrocyte interaction with neurons and astrocytes is to detect markers of healthy oligodendrocyte metabolism, to be used in diagnosis and treatment assessment in diseases such as Perinatal hypoxic-ischemic encephalopathy and multiple sclerosis in which oligodendrocyte function is impaired, possibly due to glutamate toxicity. Glutamate receptors are expressed in oligodendrocytes and also vesicular glutamate release in the white matter has received considerable attention. It is also important to establish if the glial precursor cells recruited to damaged areas are developing oligodendrocyte characteristics or those of astrocytes. Thus, it is important to study astrocytes and oligodendrocytes separately to be able to differentiate between them. This is of particular importance in the white matter where the number of oligodendrocytes is considerable. The present review summarizes the not very extensive information published on glucose metabolism in oligodendrocytes in an attempt to stimulate research into this important field.

  5. [Novel function of astrocytes revealed by optogenetics].

    Science.gov (United States)

    Beppu, Kaoru; Matsui, Ko

    2014-12-01

    Astrocytes respond to neuronal activity. However, whether astrocytic activity has any significance in brain function is unknown. Signaling pathway leading from astrocytes to neurons would be required for astrocytes to participate in neuronal functions and, here, we investigated the presence of such pathway. Optogenetics was used to manipulate astrocytic activity. A light-sensitive protein, channelrhodopsin-2 (ChR2), was selectively expressed in astrocytes. Photostimulation of these astrocytes induced glutamate release which modulated neuronal activity and animal behavior. Such glutamate release was triggered by intracellular acidification produced by ChR2 photoactivation. Astrocytic acidification occurs upon brain ischemia, and we found that another optogenetic tool, archaerhodopsin (ArchT), could counter the acidification and suppress astrocytic glutamate release. Controlling of astrocytic pH may become a therapeutic strategy upon ischemia.

  6. Generation and characterization of human insulin-releasing cell lines

    OpenAIRE

    Joffé Elisa; Machado Marcel CC; Buchanan Cecilia; Terra Letícia F; Stigliano Iván; Krogh Karin; Peters Maria G; Labriola Leticia; Puricelli Lydia; Sogayar Mari C

    2009-01-01

    Abstract Background The in vitro culture of insulinomas provides an attractive tool to study cell proliferation and insulin synthesis and secretion. However, only a few human beta cell lines have been described, with long-term passage resulting in loss of insulin secretion. Therefore, we set out to establish and characterize human insulin-releasing cell lines. Results We generated ex-vivo primary cultures from two independent human insulinomas and from a human nesidioblastosis, all of which w...

  7. Generation and characterization of human insulin-releasing cell lines

    Directory of Open Access Journals (Sweden)

    Joffé Elisa

    2009-06-01

    Full Text Available Abstract Background The in vitro culture of insulinomas provides an attractive tool to study cell proliferation and insulin synthesis and secretion. However, only a few human beta cell lines have been described, with long-term passage resulting in loss of insulin secretion. Therefore, we set out to establish and characterize human insulin-releasing cell lines. Results We generated ex-vivo primary cultures from two independent human insulinomas and from a human nesidioblastosis, all of which were cultured up to passage number 20. All cell lines secreted human insulin and C-peptide. These cell lines expressed neuroendocrine and islets markers, confirming the expression profile found in the biopsies. Although all beta cell lineages survived an anchorage independent culture, none of them were able to invade an extracellular matrix substrate. Conclusion We have established three human insulin-releasing cell lines which maintain antigenic characteristics and insulin secretion profiles of the original tumors. These cell lines represent valuable tools for the study of molecular events underlying beta cell function and dysfunction.

  8. Generation and characterization of human insulin-releasing cell lines

    Science.gov (United States)

    Labriola, Leticia; Peters, Maria G; Krogh, Karin; Stigliano, Iván; Terra, Letícia F; Buchanan, Cecilia; Machado, Marcel CC; Joffé, Elisa Bal de Kier; Puricelli, Lydia; Sogayar, Mari C

    2009-01-01

    Background The in vitro culture of insulinomas provides an attractive tool to study cell proliferation and insulin synthesis and secretion. However, only a few human beta cell lines have been described, with long-term passage resulting in loss of insulin secretion. Therefore, we set out to establish and characterize human insulin-releasing cell lines. Results We generated ex-vivo primary cultures from two independent human insulinomas and from a human nesidioblastosis, all of which were cultured up to passage number 20. All cell lines secreted human insulin and C-peptide. These cell lines expressed neuroendocrine and islets markers, confirming the expression profile found in the biopsies. Although all beta cell lineages survived an anchorage independent culture, none of them were able to invade an extracellular matrix substrate. Conclusion We have established three human insulin-releasing cell lines which maintain antigenic characteristics and insulin secretion profiles of the original tumors. These cell lines represent valuable tools for the study of molecular events underlying beta cell function and dysfunction. PMID:19545371

  9. Active Sulforhodamine 101 Uptake into Hippocampal Astrocytes

    OpenAIRE

    Christian Schnell; Yohannes Hagos; Swen Hülsmann

    2012-01-01

    Sulforhodamine 101 (SR101) is widely used as a marker of astrocytes. In this study we investigated labeling of astrocytes by SR101 in acute slices from the ventrolateral medulla and the hippocampus of transgenic mice expressing EGFP under the control of the astrocyte-specific human GFAP promoter. While SR101 efficiently and specifically labeled EGFP-expressing astrocytes in hippocampus, we found that the same staining procedure failed to label astrocytes efficiently in the ventrol...

  10. Downregulation of the 18-kDa Translocator Protein: Effects on the Ammonia-Induced Mitochondrial Permeability Transition andCell Swelling in Cultured Astrocytes

    Science.gov (United States)

    Hepatic encephalopathy (HE) is a major neurological complication in patients with severe liver disease. While the pathogenesis of HE is unclear, an elevated CNS ammonia level is believed to be a major etiological factor, and astrocytes appear to be the primary target of its toxicity. A notable featu...

  11. Markers of murine embryonic and neural stem cells, neurons and astrocytes: reference points for developmental neurotoxicity testing

    Science.gov (United States)

    Developmental neurotoxicity (DNT) is a significant concern for environmental chemicals, as well as for food and drug constituents. The sensitivity of animal-based DNT models is unclear, and they are expensive and time consuming. Murine embryonic stem cells (mESC) recapitulate sev...

  12. Simultaneous neuron- and astrocyte-specific fluorescent marking.

    Science.gov (United States)

    Schulze, Wiebke; Hayata-Takano, Atsuko; Kamo, Toshihiko; Nakazawa, Takanobu; Nagayasu, Kazuki; Kasai, Atsushi; Seiriki, Kaoru; Shintani, Norihito; Ago, Yukio; Farfan, Camille; Hashimoto, Ryota; Baba, Akemichi; Hashimoto, Hitoshi

    2015-03-27

    Systematic and simultaneous analysis of multiple cell types in the brain is becoming important, but such tools have not yet been adequately developed. Here, we aimed to generate a method for the specific fluorescent labeling of neurons and astrocytes, two major cell types in the brain, and we have developed lentiviral vectors to express the red fluorescent protein tdTomato in neurons and the enhanced green fluorescent protein (EGFP) in astrocytes. Importantly, both fluorescent proteins are fused to histone 2B protein (H2B) to confer nuclear localization to distinguish between single cells. We also constructed several expression constructs, including a tandem alignment of the neuron- and astrocyte-expression cassettes for simultaneous labeling. Introducing these vectors and constructs in vitro and in vivo resulted in cell type-specific and nuclear-localized fluorescence signals enabling easy detection and distinguishability of neurons and astrocytes. This tool is expected to be utilized for the simultaneous analysis of changes in neurons and astrocytes in healthy and diseased brains.

  13. Investigation of radiosensitivity gene signatures in cancer cell lines.

    Directory of Open Access Journals (Sweden)

    John S Hall

    Full Text Available Intrinsic radiosensitivity is an important factor underlying radiotherapy response, but there is no method for its routine assessment in human tumours. Gene signatures are currently being derived and some were previously generated by expression profiling the NCI-60 cell line panel. It was hypothesised that focusing on more homogeneous tumour types would be a better approach. Two cell line cohorts were used derived from cervix [n = 16] and head and neck [n = 11] cancers. Radiosensitivity was measured as surviving fraction following irradiation with 2 Gy (SF2 by clonogenic assay. Differential gene expression between radiosensitive and radioresistant cell lines (SF2 median was investigated using Affymetrix GeneChip Exon 1.0ST (cervix or U133A Plus2 (head and neck arrays. There were differences within cell line cohorts relating to tissue of origin reflected by expression of the stratified epithelial marker p63. Of 138 genes identified as being associated with SF2, only 2 (1.4% were congruent between the cervix and head and neck carcinoma cell lines (MGST1 and TFPI, and these did not partition the published NCI-60 cell lines based on SF2. There was variable success in applying three published radiosensitivity signatures to our cohorts. One gene signature, originally trained on the NCI-60 cell lines, did partially separate sensitive and resistant cell lines in all three cell line datasets. The findings do not confirm our hypothesis but suggest that a common transcriptional signature can reflect the radiosensitivity of tumours of heterogeneous origins.

  14. Development of a cell line from Echinococcus granulosus germinal layer.

    Science.gov (United States)

    Albani, Clara María; Cumino, Andrea Carina; Elissondo, María Celina; Denegri, Guillermo María

    2013-10-01

    In vitro culture of parasitic helminths provides an important tool to study cell regeneration and physiology, as well as for molecular biology and genetic engineering studies. In the present study, we established in vitro propagation of cells from Echinococcus granulosus germinal cyst layer. E. granulosus germinal cells grew beyond 100 passages and showed no signs of reduced proliferation capacity. Microscopic analysis revealed that cells grew both attached to the substrate and in suspension, forming three-dimensional structures like mammalian stem cell aggregates. Examination of the chromosome number of attached germinal cells showed a high degree of heteroploidy, suggesting the occurrence of transformation during culture. Monolayer cells survived cryopreservation and were able to proliferate after thawing. Based on the characteristics displayed by E. granulosus germinal cells, we establish a cell line from the E. granulosus germinal layer. Furthermore, we propose that this cell line could be useful for drug screening and for obtaining parasite material.

  15. Voluntary Exercise Induces Astrocytic Structural Plasticity in the Globus Pallidus.

    Science.gov (United States)

    Tatsumi, Kouko; Okuda, Hiroaki; Morita-Takemura, Shoko; Tanaka, Tatsuhide; Isonishi, Ayami; Shinjo, Takeaki; Terada, Yuki; Wanaka, Akio

    2016-01-01

    Changes in astrocyte morphology are primarily attributed to the fine processes where intimate connections with neurons form the tripartite synapse and participate in neurotransmission. Recent evidence has shown that neurotransmission induces dynamic synaptic remodeling, suggesting that astrocytic fine processes may adapt their morphologies to the activity in their environment. To illustrate such a neuron-glia relationship in morphological detail, we employed a double transgenic Olig2(CreER/WT); ROSA26-GAP43-EGFP mice, in which Olig2-lineage cells can be visualized and traced with membrane-targeted GFP. Although Olig2-lineage cells in the adult brain usually become mature oligodendrocytes or oligodendrocyte precursor cells with NG2-proteoglycan expression, we found a population of Olig2-lineage astrocytes with bushy morphology in several brain regions. The globus pallidus (GP) preferentially contains Olig2-lineage astrocytes. Since the GP exerts pivotal motor functions in the indirect pathway of the basal ganglionic circuit, we subjected the double transgenic mice to voluntary wheel running to activate the GP and examined morphological changes of Olig2-lineage astrocytes at both the light and electron microscopic levels. The double transgenic mice were divided into three groups: control group mice were kept in a cage with a locked running wheel for 3 weeks, Runner group were allowed free access to a running wheel for 3 weeks, and the Runner-Rest group took a sedentary 3-week rest after a 3-week running period. GFP immunofluorescence analysis and immunoelectron microscopy revealed that astrocytic fine processes elaborated complex arborization in the Runner mice, and reverted to simple morphology comparable to that of the Control group in the Runner-Rest group. Our results indicated that the fine processes of the Olig2-lineage astrocytes underwent plastic changes that correlated with overall running activities, suggesting that they actively participate in motor

  16. Effect of type-2 astrocytes on the viability of dorsal root ganglion neurons and length of neuronal processes

    Institute of Scientific and Technical Information of China (English)

    Chunling Fan; Hui Wang; Dan Chen; Xiaoxin Cheng; Kun Xiong; Xuegang Luo; Qilin Cao

    2014-01-01

    The role of type-2 astrocytes in the repair of central nervous system injury remains poorly un-derstood. In this study, using a relatively simple culture condition in vitro, type-2 astrocytes, differentiated from oligodendrocyte precursor cells by induction with bone morphogenetic pro-tein-4, were co-cultured with dorsal root ganglion neurons. We examined the effects of type-2 astrocytes differentiated from oligodendrocyte precursor cells on the survival and growth of dorsal root ganglion neurons. Results demonstrated that the number of dorsal root ganglion neurons was higher following co-culture of oligodendrocyte precursor cells and type-2 astrocytes than when cultured alone, but lower than that of neurons co-cultured with type-1 astrocytes. The length of the longest process and the length of all processes of a single neuron were shortest in neurons cultured alone, followed by neurons co-cultured with type-2 astrocytes, then neurons co-cultured with oligodendrocyte precursor cells, and longest in neurons co-cultured with type-1 astrocytes. These results indicate that co-culture with type-2 astrocytes can increase neuronal survival rate and process length. However, compared with type-1 astrocytes and oligodendrocyte precursor cells, the promotion effects of type-2 astrocytes on the growth of dorsal root ganglion neurons were weaker.

  17. Differential heat shock response of primary human cell cultures and established cell lines

    DEFF Research Database (Denmark)

    Richter, W W; Issinger, O G

    1986-01-01

    degrees C treatment, whereas in immortalized cell lines usually 90% of the cells were found in suspension. Enhanced expression of the major heat shock protein (hsp 70) was found in all heat-treated cells. In contrast to the primary cell cultures, established and transformed cell lines synthesized a...

  18. Establishment of Jurkat tet-on cell line

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Tet-control system is developed to tightly control target gene expression in mammalian cells by using the regulatory elements of tetracycline-repressor of the transposor Tn10 from E.Coli.We have transfected reverse tetracycline-controlled transactivator gene (rtTA) into genome of Jurkat cells and established two Jurkat tet-on cell lines.Induction of luciferase reporter activity with doxycycline,a tetracycline derivative,is dose-dependent with a peak value of 32-fold increment.Establishment of Jurkat tet-on cell lines greatly facilitates quantitative studies on target gene functions in the cells.

  19. Antiproliferative effect of isopentenylated coumarins on several cancer cell lines.

    Science.gov (United States)

    Kawaii, S; Tomono, Y; Ogawa, K; Sugiura, M; Yano, M; Yoshizawa, Y; Ito, C; Furukawa, H

    2001-01-01

    33 coumarins, mainly the simple isopentenylated coumarins and derived pyrano- and furanocoumarins, were examined for their antiproliferative activity towards several cancer and normal human cell lines. The pyrano- and furanocoumarins showed strong activity against the cancer cell lines, whereas they had weak antiproliferative activity against the normal human cell lines. The decreasing rank order of potency was osthenone (10), clausarin (25), clausenidin (26), dentatin (24), nordentatin (23), imperatorin (29), seselin (27), xanthyletin (21), suberosin (17), phebalosin (8) and osthol (12). The structure-activity relationship established from the results revealed that the 1,1-dimethylallyl and isopentenyl groups have an important role for antiproliferative activity. PMID:11497276

  20. Primary cultures of astrocytes: Their value in understanding astrocytes in health and disease

    OpenAIRE

    Lange, Sofie C.; Bak, Lasse K.; Helle S. Waagepetersen; Schousboe, Arne; Norenberg, Michael D.

    2012-01-01

    During the past decades of astrocyte research it has become increasingly clear that astrocytes have taken a central position in all central nervous system activities. Much of our new understanding of astrocytes has been derived from studies conducted with primary cultures of astrocytes. Such cultures have been an invaluable tool for studying roles of astrocytes in physiological and pathological states. Many central astrocytic functions in metabolism, amino acid neurotransmission and calcium s...

  1. Microglia is activated by astrocytes in trimethyltin intoxication

    International Nuclear Information System (INIS)

    Microglia participates in most acute and chronic neuropathologies and its activation appears to involve interactions with neurons and other glial cells. Trimethyltin (TMT)-induced brain damage is a well-characterized model of neurodegeneration, in which microglial activation occurs before neuronal degeneration. The aim of this in vitro study was to investigate the role of astroglia in TMT-induced microgliosis by using nitric oxide (NO), inducible NO synthase (iNOS), and morphological changes as parameters for microglial activation. Our investigation discusses (a) whether microglial cells can be activated directly by TMT; (b) if astroglial cells are capable of triggering or modulating microglial activation; (c) how the morphology and survival of microglia and astrocytes are affected by TMT treatment; and (d) whether microglial-astroglial interactions depend on direct cell contact or on soluble factors. Our results show that microglia are more vulnerable to TMT than astrocytes are and cannot be activated directly by TMT with regard to the examined parameters. In bilayer coculture with viable astroglial cells, microglia produce NO in significant amounts at subcytotoxic concentrations of TMT (20 μmol/l). At these TMT concentrations, microglial cells in coculture convert into small round cells without cell processes, whereas flat, fibroblast-like astrocytes convert into thin process bearing stellate cells with a dense and compact cell body. We conclude that astrocytes trigger microglial activation after treatment with TMT, although the mechanisms of this interaction remain unknown

  2. Differential effects of bisphosphonates on breast cancer cell lines

    NARCIS (Netherlands)

    Verdijk, R.; Franke, H.R.; Wolbers, F.; Vermes, I.

    2007-01-01

    Bisphosphonates may induce direct anti-tumor effects in breast cancers cells in virtro. In this study, six bisphosphonates were administered to three breast caner cell lines. Cell proliferation was measured by quantification of th expressio of Cyclin D1 mRNA. Apoptosis was determined by flow cytome

  3. Molecular profiling reveals primary mesothelioma cell lines recapitulate human disease.

    Science.gov (United States)

    Chernova, T; Sun, X M; Powley, I R; Galavotti, S; Grosso, S; Murphy, F A; Miles, G J; Cresswell, L; Antonov, A V; Bennett, J; Nakas, A; Dinsdale, D; Cain, K; Bushell, M; Willis, A E; MacFarlane, M

    2016-07-01

    Malignant mesothelioma (MM) is an aggressive, fatal tumor strongly associated with asbestos exposure. There is an urgent need to improve MM patient outcomes and this requires functionally validated pre-clinical models. Mesothelioma-derived cell lines provide an essential and relatively robust tool and remain among the most widely used systems for candidate drug evaluation. Although a number of cell lines are commercially available, a detailed comparison of these commercial lines with freshly derived primary tumor cells to validate their suitability as pre-clinical models is lacking. To address this, patient-derived primary mesothelioma cell lines were established and characterized using complementary multidisciplinary approaches and bioinformatic analysis. Clinical markers of mesothelioma, transcriptional and metabolic profiles, as well as the status of p53 and the tumor suppressor genes CDKN2A and NF2, were examined in primary cell lines and in two widely used commercial lines. Expression of MM-associated markers, as well as the status of CDKN2A, NF2, the 'gatekeeper' in MM development, and their products demonstrated that primary cell lines are more representative of the tumor close to its native state and show a degree of molecular diversity, thus capturing the disease heterogeneity in a patient cohort. Molecular profiling revealed a significantly different transcriptome and marked metabolic shift towards a greater glycolytic phenotype in commercial compared with primary cell lines. Our results highlight that multiple, appropriately characterised, patient-derived tumor cell lines are required to enable concurrent evaluation of molecular profiles versus drug response. Furthermore, application of this approach to other difficult-to-treat tumors would generate improved cellular models for pre-clinical evaluation of novel targeted therapies. PMID:26891694

  4. Pituitary Adenylate cyclase-activating polypeptide orchestrates neuronal regulation of the astrocytic glutamate-releasing mechanism system xc (.).

    Science.gov (United States)

    Kong, Linghai; Albano, Rebecca; Madayag, Aric; Raddatz, Nicholas; Mantsch, John R; Choi, SuJean; Lobner, Doug; Baker, David A

    2016-05-01

    Glutamate signaling is achieved by an elaborate network involving neurons and astrocytes. Hence, it is critical to better understand how neurons and astrocytes interact to coordinate the cellular regulation of glutamate signaling. In these studies, we used rat cortical cell cultures to examine whether neurons or releasable neuronal factors were capable of regulating system xc (-) (Sxc), a glutamate-releasing mechanism that is expressed primarily by astrocytes and has been shown to regulate synaptic transmission. We found that astrocytes cultured with neurons or exposed to neuronal-conditioned media displayed significantly higher levels of Sxc activity. Next, we demonstrated that the pituitary adenylate cyclase-activating polypeptide (PACAP) may be a neuronal factor capable of regulating astrocytes. In support, we found that PACAP expression was restricted to neurons, and that PACAP receptors were expressed in astrocytes. Interestingly, blockade of PACAP receptors in cultures comprised of astrocytes and neurons significantly decreased Sxc activity to the level observed in purified astrocytes, whereas application of PACAP to purified astrocytes increased Sxc activity to the level observed in cultures comprised of neurons and astrocytes. Collectively, these data reveal that neurons coordinate the actions of glutamate-related mechanisms expressed by astrocytes, such as Sxc, a process that likely involves PACAP. A critical gap in modeling excitatory signaling is how distinct components of the glutamate system expressed by neurons and astrocytes are coordinated. In these studies, we found that system xc (-) (Sxc), a glutamate release mechanism expressed by astrocytes, is regulated by releasable neuronal factors including PACAP. This represents a novel form of neuron-astrocyte communication, and highlights the possibility that pathological changes involving astrocytic Sxc may stem from altered neuronal activity.

  5. Fractionated dose skews differentiation of Glial progenitor cells into immature oligodendrocytes and astrocytes, with lower mature oligodendrocytes formation, as compared to singe low dose of low and high LET radiation

    International Nuclear Information System (INIS)

    In the proposed study, the effect of fractionated, low dose versus single low dose of low LET X-rays and charged particles on induction of base excision repair enzyme Apurinic Endonuclease-1 (Ape1) are determined, which is known to inhibit cell differentiation, and found that at lower doses of 10,25 and 50 cGy there was a very significant induction of Apel which correlated to number of fractions, whereas at 100 cGy this induction was significantly lower. Also, there was a clear correlation between increase in fractions and higher immature OL and astrocyte formation

  6. Mercury specifically induces LINE-1 activity in a human neuroblastoma cell line.

    Science.gov (United States)

    Habibi, Laleh; Shokrgozar, Mohammad Ali; Tabrizi, Mina; Modarressi, Mohammad Hossein; Akrami, Seyed Mohammad

    2014-01-01

    L1 retro-elements comprise 17% of the human genome. Approximately 100 copies of these autonomous mobile elements are active in our DNA and can cause mutations, gene disruptions, and genomic instability. Therefore, human cells control the activities of L1 elements, in order to prevent their deleterious effects through different mechanisms. However, some toxic agents increase the retrotransposition activity of L1 elements in somatic cells. In order to identify specific effects of neurotoxic metals on L1 activity in neuronal cells, we studied the effects of mercury and cobalt on L1-retroelement activity by measuring levels of cellular transcription, protein expression, and genomic retrotransposition in a neuroblastoma cell line compared with the effects in three non-neuronal cell lines. Our results show that mercury increased the expression of L1 RNA, the activity of the L1 5'UTR, and L1 retrotransposition exclusively in the neuroblastoma cell line but not in non-neuronal cell lines. However, cobalt increased the expression of L1 RNA in neuroblastoma cells, HeLa cells, and wild-type human fibroblasts, and also increased the activity of the L1 5'UTR as well as the SV40 promoter in HeLa cells but not in neuroblastoma cells. Exposure to cobalt did not result in increased retrotransposition activity in HeLa cells or neuroblastoma cells. We conclude that non-toxic levels of the neurotoxic agent mercury could influence DNA by increasing L1 activities, specifically in neuronal cells, and may make these cells susceptible to neurodegeneration over time.

  7. Membrane lipidome of an epithelial cell line

    DEFF Research Database (Denmark)

    Sampaio, Julio L; Gerl, Mathias J; Klose, Christian;

    2011-01-01

    Tissue differentiation is an important process that involves major cellular membrane remodeling. We used Madin-Darby canine kidney cells as a model for epithelium formation and investigated the remodeling of the total cell membrane lipidome during the transition from a nonpolarized morphology...

  8. Pathway-specific differences between tumor cell lines and normal and tumor tissue cells

    Directory of Open Access Journals (Sweden)

    Tozeren Aydin

    2006-11-01

    Full Text Available Abstract Background Cell lines are used in experimental investigation of cancer but their capacity to represent tumor cells has yet to be quantified. The aim of the study was to identify significant alterations in pathway usage in cell lines in comparison with normal and tumor tissue. Methods This study utilized a pathway-specific enrichment analysis of publicly accessible microarray data and quantified the gene expression differences between cell lines, tumor, and normal tissue cells for six different tissue types. KEGG pathways that are significantly different between cell lines and tumors, cell lines and normal tissues and tumor and normal tissue were identified through enrichment tests on gene lists obtained using Significance Analysis of Microarrays (SAM. Results Cellular pathways that were significantly upregulated in cell lines compared to tumor cells and normal cells of the same tissue type included ATP synthesis, cell communication, cell cycle, oxidative phosphorylation, purine, pyrimidine and pyruvate metabolism, and proteasome. Results on metabolic pathways suggested an increase in the velocity nucleotide metabolism and RNA production. Pathways that were downregulated in cell lines compared to tumor and normal tissue included cell communication, cell adhesion molecules (CAMs, and ECM-receptor interaction. Only a fraction of the significantly altered genes in tumor-to-normal comparison had similar expressions in cancer cell lines and tumor cells. These genes were tissue-specific and were distributed sparsely among multiple pathways. Conclusion Significantly altered genes in tumors compared to normal tissue were largely tissue specific. Among these genes downregulation was a major trend. In contrast, cell lines contained large sets of significantly upregulated genes that were common to multiple tissue types. Pathway upregulation in cell lines was most pronounced over metabolic pathways including cell nucleotide metabolism and oxidative

  9. Medium-chain fatty acids inhibit mitochondrial metabolism in astrocytes promoting astrocyte-neuron lactate and ketone body shuttle systems.

    Science.gov (United States)

    Thevenet, Jonathan; De Marchi, Umberto; Domingo, Jaime Santo; Christinat, Nicolas; Bultot, Laurent; Lefebvre, Gregory; Sakamoto, Kei; Descombes, Patrick; Masoodi, Mojgan; Wiederkehr, Andreas

    2016-05-01

    Medium-chain triglycerides have been used as part of a ketogenic diet effective in reducing epileptic episodes. The health benefits of the derived medium-chain fatty acids (MCFAs) are thought to result from the stimulation of liver ketogenesis providing fuel for the brain. We tested whether MCFAs have direct effects on energy metabolism in induced pluripotent stem cell-derived human astrocytes and neurons. Using single-cell imaging, we observed an acute pronounced reduction of the mitochondrial electrical potential and a concomitant drop of the NAD(P)H signal in astrocytes, but not in neurons. Despite the observed effects on mitochondrial function, MCFAs did not lower intracellular ATP levels or activate the energy sensor AMP-activated protein kinase. ATP concentrations in astrocytes were unaltered, even when blocking the respiratory chain, suggesting compensation through accelerated glycolysis. The MCFA decanoic acid (300 μM) promoted glycolysis and augmented lactate formation by 49.6%. The shorter fatty acid octanoic acid (300 μM) did not affect glycolysis but increased the rates of astrocyte ketogenesis 2.17-fold compared with that of control cells. MCFAs may have brain health benefits through the modulation of astrocyte metabolism leading to activation of shuttle systems that provide fuel to neighboring neurons in the form of lactate and ketone bodies.-Thevenet, J., De Marchi, U., Santo Domingo, J., Christinat, N., Bultot, L., Lefebvre, G., Sakamoto, K., Descombes, P., Masoodi, M., Wiederkehr, A. Medium-chain fatty acids inhibit mitochondrial metabolism in astrocytes promoting astrocyte-neuron lactate and ketone body shuttle systems. PMID:26839375

  10. Global Conservation of Protein Status between Cell Lines and Xenografts

    Directory of Open Access Journals (Sweden)

    Julian Biau

    2016-08-01

    Full Text Available Common preclinical models for testing anticancer treatment include cultured human tumor cell lines in monolayer, and xenografts derived from these cell lines in immunodeficient mice. Our goal was to determine how similar the xenografts are compared with their original cell line and to determine whether it is possible to predict the stability of a xenograft model beforehand. We studied a selection of 89 protein markers of interest in 14 human cell cultures and respective subcutaneous xenografts using the reverse-phase protein array technology. We specifically focused on proteins and posttranslational modifications involved in DNA repair, PI3K pathway, apoptosis, tyrosine kinase signaling, stress, cell cycle, MAPK/ERK signaling, SAPK/JNK signaling, NFκB signaling, and adhesion/cytoskeleton. Using hierarchical clustering, most cell culture-xenograft pairs cluster together, suggesting a global conservation of protein signature. Particularly, Akt, NFkB, EGFR, and Vimentin showed very stable protein expression and phosphorylation levels highlighting that 4 of 10 pathways were highly correlated whatever the model. Other proteins were heterogeneously conserved depending on the cell line. Finally, cell line models with low Akt pathway activation and low levels of Vimentin gave rise to more reliable xenograft models. These results may be useful for the extrapolation of cell culture experiments to in vivo models in novel targeted drug discovery.

  11. Phenotypes and karyotypes of human malignant mesothelioma cell lines.

    Directory of Open Access Journals (Sweden)

    Vandana Relan

    Full Text Available BACKGROUND: Malignant mesothelioma is an aggressive tumour of serosal surfaces most commonly pleura. Characterised cell lines represent a valuable tool to study the biology of mesothelioma. The aim of this study was to develop and biologically characterise six malignant mesothelioma cell lines to evaluate their potential as models of human malignant mesothelioma. METHODS: Five lines were initiated from pleural biopsies, and one from pleural effusion of patients with histologically proven malignant mesothelioma. Mesothelial origin was assessed by standard morphology, Transmission Electron Microscopy (TEM and immunocytochemistry. Growth characteristics were assayed using population doubling times. Spectral karyotyping was performed to assess chromosomal abnormalities. Authentication of donor specific derivation was undertaken by DNA fingerprinting using a panel of SNPs. RESULTS: Most of cell lines exhibited spindle cell shape, with some retaining stellate shapes. At passage 2 to 6 all lines stained positively for calretinin and cytokeratin 19, and demonstrated capacity for anchorage-independent growth. At passage 4 to 16, doubling times ranged from 30-72 hours, and on spectral karyotyping all lines exhibited numerical chromosomal abnormalities ranging from 41 to 113. Monosomy of chromosomes 8, 14, 22 or 17 was observed in three lines. One line displayed four different karyotypes at passage 8, but only one karyotype at passage 42, and another displayed polyploidy at passage 40 which was not present at early passages. At passages 5-17, TEM showed characteristic features of mesothelioma ultrastructure in all lines including microvilli and tight intercellular junctions. CONCLUSION: These six cell lines exhibit varying cell morphology, a range of doubling times, and show diverse passage-dependent structural chromosomal changes observed in malignant tumours. However they retain characteristic immunocytochemical protein expression profiles of

  12. Strategies for selecting recombinant CHO cell lines for cGMP manufacturing: improving the efficiency of cell line generation.

    Science.gov (United States)

    Porter, Alison J; Racher, Andrew J; Preziosi, Richard; Dickson, Alan J

    2010-01-01

    Transfectants with a wide range of cellular phenotypes are obtained during the process of cell line generation. For the successful manufacture of a therapeutic protein, a means is required to identify a cell line with desirable growth and productivity characteristics from this phenotypically wide-ranging transfectant population. This identification process is on the critical path for first-in-human studies. We have stringently examined a typical selection strategy used to isolate cell lines suitable for cGMP manufacturing. One-hundred and seventy-five transfectants were evaluated as they progressed through the different assessment stages of the selection strategy. High producing cell lines, suitable for cGMP manufacturing, were identified. However, our analyses showed that the frequency of isolation of the highest producing cell lines was low and that ranking positions were not consistent between each assessment stage, suggesting that there is potential to improve upon the strategy. Attempts to increase the frequency of isolation of the 10 highest producing cell lines, by in silico analysis of alternative selection strategies, were unsuccessful. We identified alternative strategies with similar predictive capabilities to the typical selection strategy. One alternate strategy required fewer cell lines to be progressed at the assessment stages but the stochastic nature of the models means that cell line numbers are likely to change between programs. In summary, our studies illuminate the potential for improvement to this and future selection strategies, based around use of assessments that are more informative or that reduce variance, paving the way to improved efficiency of generation of manufacturing cell lines. PMID:20623584

  13. Channel-Mediated Lactate Release by K+-Stimulated Astrocytes

    KAUST Repository

    Sotelo-Hitschfeld, T.

    2015-03-11

    Excitatory synaptic transmission is accompanied by a local surge in interstitial lactate that occurs despite adequate oxygen availability, a puzzling phenomenon termed aerobic glycolysis. In addition to its role as an energy substrate, recent studies have shown that lactate modulates neuronal excitability acting through various targets, including NMDA receptors and G-protein-coupled receptors specific for lactate, but little is known about the cellular and molecular mechanisms responsible for the increase in interstitial lactate. Using a panel of genetically encoded fluorescence nanosensors for energy metabolites, we show here that mouse astrocytes in culture, in cortical slices, and in vivo maintain a steady-state reservoir of lactate. The reservoir was released to the extracellular space immediately after exposure of astrocytes to a physiological rise in extracellular K+ or cell depolarization. Cell-attached patch-clamp analysis of cultured astrocytes revealed a 37 pS lactate-permeable ion channel activated by cell depolarization. The channel was modulated by lactate itself, resulting in a positive feedback loop for lactate release. A rapid fall in intracellular lactate levels was also observed in cortical astrocytes of anesthetized mice in response to local field stimulation. The existence of an astrocytic lactate reservoir and its quick mobilization via an ion channel in response to a neuronal cue provides fresh support to lactate roles in neuronal fueling and in gliotransmission.

  14. MORPHOMETRIC SUBTYPING FOR A PANEL OF BREAST CANCER CELL LINES

    Energy Technology Data Exchange (ETDEWEB)

    Han, Ju; Chang, Hang; Fontenay, Gerald; Wang, Nicholas J.; Gray, Joe W.; Parvin, Bahram

    2009-05-08

    A panel of cell lines of diverse molecular background offers an improved model system for high-content screening, comparative analysis, and cell systems biology. A computational pipeline has been developed to collect images from cell-based assays, segment individual cells and colonies, represent segmented objects in a multidimensional space, and cluster them for identifying distinct subpopulations. While each segmentation strategy can vary for different imaging assays, representation and subpopulation analysis share a common thread. Application of this pipeline to a library of 41 breast cancer cell lines is demonstrated. These cell lines are grown in 2D and imaged through immunofluorescence microscopy. Subpopulations in this panel are identified and shown to correlate with previous subtyping literature that was derived from transcript data.

  15. Biobanking human embryonic stem cell lines

    DEFF Research Database (Denmark)

    Holm, Søren

    2016-01-01

    Stem cell banks curating and distributing human embryonic stem cells have been established in a number of countries and by a number of private institutions. This paper identifies and critically discusses a number of arguments that are used to justify the importance of such banks in policy...... are curiously absent from the particular stem cell banking policy discourse. This to some extent artificially isolates this discourse from the broader discussions about the flows of reproductive materials and tissues in modern society, and such isolation may lead to the interests of important actors being...

  16. Hyperglycaemia and diabetes impair gap junctional communication among astrocytes

    Directory of Open Access Journals (Sweden)

    Gautam K Gandhi

    2010-03-01

    Full Text Available Sensory and cognitive impairments have been documented in diabetic humans and animals, but the pathophysiology of diabetes in the central nervous system is poorly understood. Because a high glucose level disrupts gap junctional communication in various cell types and astrocytes are extensively coupled by gap junctions to form large syncytia, the influence of experimental diabetes on gap junction channel-mediated dye transfer was assessed in astrocytes in tissue culture and in brain slices from diabetic rats. Astrocytes grown in 15–25 mmol/l glucose had a slow-onset, poorly reversible decrement in gap junctional communication compared with those grown in 5.5 mmol/l glucose. Astrocytes in brain slices from adult STZ (streptozotocin-treated rats at 20–24 weeks after the onset of diabetes also exhibited reduced dye transfer. In cultured astrocytes grown in high glucose, increased oxidative stress preceded the decrement in dye transfer by several days, and gap junctional impairment was prevented, but not rescued, after its manifestation by compounds that can block or reduce oxidative stress. In sharp contrast with these findings, chaperone molecules known to facilitate protein folding could prevent and rescue gap junctional impairment, even in the presence of elevated glucose level and oxidative stress. Immunostaining of Cx (connexin 43 and 30, but not Cx26, was altered by growth in high glucose. Disruption of astrocytic trafficking of metabolites and signalling molecules may alter interactions among astrocytes, neurons and endothelial cells and contribute to changes in brain function in diabetes. Involvement of the microvasculature may contribute to diabetic complications in the brain, the cardiovascular system and other organs.

  17. Effects of aspartame metabolites on astrocytes and neurons.

    Science.gov (United States)

    Rycerz, Karol; Jaworska-Adamu, Jadwiga Elżbieta

    2013-01-01

    Aspartame, a widespread sweetener used in many food products, is considered as a highly hazardous compound. Aspartame was discovered in 1965 and raises a lot of controversy up to date. Astrocytes are glial cells, the presence and functions of which are closely connected with the central nervous system (CNS). The aim of this article is to demonstrate the direct and indirect role of astrocytes participating in the harmful effects of aspartame metabolites on neurons. The artificial sweetener is broken down into phenylalanine (50%), aspartic acid (40%) and methanol (10%) during metabolism in the body. The excess of phenylalanine blocks the transport of important amino acids to the brain contributing to reduced levels of dopamine and serotonin. Astrocytes directly affect the transport of this amino acid and also indirectly by modulation of carriers in the endothelium. Aspartic acid at high concentrations is a toxin that causes hyperexcitability of neurons and is also a precursor of other excitatory amino acid - glutamates. Their excess in quantity and lack of astrocytic uptake induces excitotoxicity and leads to the degeneration of astrocytes and neurons. The methanol metabolites cause CNS depression, vision disorders and other symptoms leading ultimately to metabolic acidosis and coma. Astrocytes do not play a significant role in methanol poisoning due to a permanent consumption of large amounts of aspartame. Despite intense speculations about the carcinogenicity of aspartame, the latest studies show that its metabolite - diketopiperazine - is cancirogenic in the CNS. It contributes to the formation of tumors in the CNS such as gliomas, medulloblastomas and meningiomas. Glial cells are the main source of tumors, which can be caused inter alia by the sweetener in the brain. On the one hand the action of astrocytes during aspartame poisoning may be advantageous for neuro-protection while on the other it may intensify the destruction of neurons. The role of the glia in

  18. Synapses lacking astrocyte appear in the amygdala during consolidation of Pavlovian threat conditioning.

    Science.gov (United States)

    Ostroff, Linnaea E; Manzur, Mustfa K; Cain, Christopher K; Ledoux, Joseph E

    2014-06-15

    There is growing evidence that astrocytes, long held to merely provide metabolic support in the adult brain, participate in both synaptic plasticity and learning and memory. Astrocytic processes are sometimes present at the synaptic cleft, suggesting that they might act directly at individual synapses. Associative learning induces synaptic plasticity and morphological changes at synapses in the lateral amygdala (LA). To determine whether astrocytic contacts are involved in these changes, we examined LA synapses after either threat conditioning (also called fear conditioning) or conditioned inhibition in adult rats by using serial section transmission electron microscopy (ssTEM) reconstructions. There was a transient increase in the density of synapses with no astrocytic contact after threat conditioning, especially on enlarged spines containing both polyribosomes and a spine apparatus. In contrast, synapses with astrocytic contacts were smaller after conditioned inhibition. This suggests that during memory consolidation astrocytic processes are absent if synapses are enlarging but present if they are shrinking. We measured the perimeter of each synapse and its degree of astrocyte coverage, and found that only about 20-30% of each synapse was ensheathed. The amount of synapse perimeter surrounded by astrocyte did not scale with synapse size, giving large synapses a disproportionately long astrocyte-free perimeter and resulting in a net increase in astrocyte-free perimeter after threat conditioning. Thus astrocytic processes do not mechanically isolate LA synapses, but may instead interact through local signaling, possibly via cell-surface receptors. Our results suggest that contact with astrocytic processes opposes synapse growth during memory consolidation.

  19. Cold storage and cryopreservation of tick cell lines

    Directory of Open Access Journals (Sweden)

    Lallinger Gertrud

    2010-04-01

    Full Text Available Abstract Background Tick cell lines are now available from fifteen ixodid and argasid species of medical and veterinary importance. However, some tick cell lines can be difficult to cryopreserve, and improved protocols for short- and long-term low temperature storage will greatly enhance their use as tools in tick and tick-borne pathogen research. In the present study, different protocols were evaluated for cold storage and cryopreservation of tick cell lines derived from Rhipicephalus (Boophilus decoloratus, Rhipicephalus (Boophilus microplus, Ixodes ricinus and Ixodes scapularis. For short-term cold storage, cells were kept under refrigeration at 6°C for 15, 30 and 45 days. For cryopreservation in liquid nitrogen, use of a sucrose-phosphate-glutamate freezing buffer (SPG as cryoprotectant was compared with dimethylsulfoxide (DMSO supplemented with sucrose. Cell viability was determined by the trypan blue exclusion test and cell morphology was evaluated in Giemsa-stained cytocentrifuge smears. Results Cold storage at 6°C for up to 30 days was successful in preserving R. (B. microplus, R. (B. decoloratus, I. ricinus and I. scapularis cell lines; lines from the latter three species could be easily re-cultivated after 45 days under refrigeration. While cell lines from all four tick species cryopreserved with 6% DMSO were successfully resuscitated, the R. (B. decoloratus cells did not survive freezing in SPG and of the other three species, only the R. (B. microplus cells resumed growth during the observation period. Conclusions This constitutes the first report on successful short-term refrigeration of cells derived from R. (B. decoloratus, R. (B. microplus, and I. ricinus, and use of SPG as an alternative to DMSO for cryopreservation, thus making an important contribution to more reliable and convenient tick cell culture maintenance.

  20. NT2 derived neuronal and astrocytic network signalling.

    Directory of Open Access Journals (Sweden)

    Eric J Hill

    Full Text Available A major focus of stem cell research is the generation of neurons that may then be implanted to treat neurodegenerative diseases. However, a picture is emerging where astrocytes are partners to neurons in sustaining and modulating brain function. We therefore investigated the functional properties of NT2 derived astrocytes and neurons using electrophysiological and calcium imaging approaches. NT2 neurons (NT2Ns expressed sodium dependent action potentials, as well as responses to depolarisation and the neurotransmitter glutamate. NT2Ns exhibited spontaneous and coordinated calcium elevations in clusters and in extended processes, indicating local and long distance signalling. Tetrodotoxin sensitive network activity could also be evoked by electrical stimulation. Similarly, NT2 astrocytes (NT2As exhibited morphology and functional properties consistent with this glial cell type. NT2As responded to neuronal activity and to exogenously applied neurotransmitters with calcium elevations, and in contrast to neurons, also exhibited spontaneous rhythmic calcium oscillations. NT2As also generated propagating calcium waves that were gap junction and purinergic signalling dependent. Our results show that NT2 derived astrocytes exhibit appropriate functionality and that NT2N networks interact with NT2A networks in co-culture. These findings underline the utility of such cultures to investigate human brain cell type signalling under controlled conditions. Furthermore, since stem cell derived neuron function and survival is of great importance therapeutically, our findings suggest that the presence of complementary astrocytes may be valuable in supporting stem cell derived neuronal networks. Indeed, this also supports the intriguing possibility of selective therapeutic replacement of astrocytes in diseases where these cells are either lost or lose functionality.

  1. Morphine Protects Spinal Cord Astrocytes from Glutamate-Induced Apoptosis via Reducing Endoplasmic Reticulum Stress

    Directory of Open Access Journals (Sweden)

    Chao Zhang

    2016-10-01

    Full Text Available Glutamate is not only a neurotransmitter but also an important neurotoxin in central nervous system (CNS. Chronic elevation of glutamate induces both neuronal and glial cell apoptosis. However, its effect on astrocytes is complex and still remains unclear. In this study, we investigated whether morphine, a common opioid ligand, could affect glutamate-induced apoptosis in astrocytes. Primary cultured astrocytes were incubated with glutamate in the presence/absence of morphine. It was found that morphine could reduce glutamate-induced apoptosis of astrocytes. Furthermore, glutamate activated Ca2+ release, thereby inducing endoplasmic reticulum (ER stress in astrocytes, while morphine attenuated this deleterious effect. Using siRNA to reduce the expression of κ-opioid receptor, morphine could not effectively inhibit glutamate-stimulated Ca2+ release in astrocytes, the protective effect of morphine on glutamate-injured astrocytes was also suppressed. These results suggested that morphine could protect astrocytes from glutamate-induced apoptosis via reducing Ca2+ overload and ER stress pathways. In conclusion, this study indicated that excitotoxicity participated in the glutamate mediated apoptosis in astrocytes, while morphine attenuated this deleterious effect via regulating Ca2+ release and ER stress.

  2. Trophic and tropic effects of striatal astrocytes on cografted mesencephalic dopamine neurons and their axons.

    Science.gov (United States)

    Pierret, P; Quenneville, N; Vandaele, S; Abbaszadeh, R; Lanctôt, C; Crine, P; Doucet, G

    1998-01-01

    Astrocytes from the ventral mesencephalon and from the striatum respectively promote the dendritic and axonal arborization of dopamine (DA) neurons in vitro. To test this response in vivo, astrocytes in primary cultures from the neonatal cerebral cortex, ventral mesencephalon, or striatum were coimplanted with fetal ventral mesencephalic tissue into the intact or DA-denervated striatum of adult rats and these cografts examined after 3-6 months by tyrosine hydroxylase (TH) immunohistochemistry (intact recipients) or after 5-6 months by in vitro [3H]DA-uptake autoradiography (DA-denervated recipients). In contrast with single ventral mesencephalic grafts, all types of cograft displayed a rather uniform distribution of TH-immunoreactive perikarya. The average size of TH-immunoreactive cell bodies was not significantly different in cografts containing cortical or mesencephalic astrocytes and in single ventral mesencephalic grafts, but it was significantly larger in cografts containing striatal astrocytes. Nevertheless, the number of [3H]DA-labeled terminals in the DA-lesioned host striatum was clearly smaller with cografts of striatal astrocytes than with single mesencephalic grafts or with cografts containing cortical astrocytes. On the other hand, cografts of striatal astrocytes contained much higher numbers of [3H]DA-labeled terminals than the other types of graft or cograft. Thus, while cografted astrocytes in general influence the distribution of DA neurons within the graft, astrocytes from the neonatal striatum have a trophic effect on DA perikarya and a tropic effect on DA axons, keeping the latter within the graft.

  3. Magnetic resonance spectroscopy in tumor cell lines research

    International Nuclear Information System (INIS)

    MRS can be used non-invasively to study the several trace metabolites and energy metabolism in vivo. By quantitatively analyzing the compounds changes we could detect abnormal metabolism in tumor and its surrounding tissue, and estimate tumor infiltration in vivo and vitro. In recent years, MRS has been applied in cell line research and is becoming a promising method. In this article we summarized the applications of MRS in cell lines in studying diagnosis, treatment, and tumor mechanisms. (authors)

  4. Effects of cultured astrocytes from rat cerebral cortex onthe neurite development of PC12 cells%星形神经胶质细胞对PC12神经元突起生长发育的影响

    Institute of Scientific and Technical Information of China (English)

    莫永炎; 邵紫韫; 陈瑗; 周玫; 张宝

    2004-01-01

    背景:星形细胞对神经元有提供营养、支持及调节突触活性作用,但它对神经元发育的影响还尚不清楚.目的:探讨体外培养的Sprague Dawley大鼠大脑皮质星形细胞对PC12神经元突起生长发育的作用.设计:完全随机设计,对照实验研究.方法:以培养的星形细胞与PC12神经元按不同细胞数目比例(50:1~1:1)共同培养,并用其制备的条件培养液培养PG12细胞.主要观察指标:用快速灵敏的MTT'比色法测定PC12神经元的细胞活力,用光学相差显微镜观察PC12细胞形态学变化.结果:①星形细胞条件培养液可增强PG12细胞活力(MTT测定的A值由0.255±0.012提高到0.510±0.036,P<0.001),却不能促使PC12神经元突起的生出.②当将星形细胞与PC12细胞按30:1~1:1的比例共同培养时,既可提高PC12细胞折光性和光晕又可促使其突起的生长;但按50:1~40:1的比例共同培养时,只观察到提高PC12细胞折光性和光晕,而无促使其突起生长发育的作用.结论:PC12神经元细胞活力的提高与星形细胞分泌到条件培养液中的可溶性因子有关,而PC12神经元突起生长发育可能是和与星形细胞的直接接触以及二者的细胞数目比有关.%BACKGROUND: Although astrocytes are kown to provide structural andtrophic support to neurons and modulate synaptic activity, their role is farfrom being completely understood.OBJECTIVE: To investigate effects of cultured astrocytes fromSprague-Dawley rat cerebral cortex on the neurite development of PC12 cellsderived from rat pheochromocytoma.DESIGN: Completely randomized controlled trial.METHODS: PC12 cells were co-cultured with astrocyte according to dif-ferent astrocytes/neurons ratio(50: 1 -1: 1), or cultured with serum-freeconditioned medium of astrocytes (ACM).MAIN OUTCOME MEASURES: The vitality of PC12 cells was measuredby sensitive MTT method and their morphologic features were observed byOlympus light microscope.RFSULTS: When PC

  5. Inhibition of Protease-Resistant Prion Protein Formation in a Transformed Deer Cell Line Infected with Chronic Wasting Disease‡

    Science.gov (United States)

    Raymond, Gregory J.; Olsen, Emily A.; Lee, Kil Sun; Raymond, Lynne D.; Bryant, P. Kruger; Baron, Gerald S.; Caughey, Winslow S.; Kocisko, David A.; McHolland, Linda E.; Favara, Cynthia; Langeveld, Jan P. M.; van Zijderveld, Fred G.; Mayer, Richard T.; Miller, Michael W.; Williams, Elizabeth S.; Caughey, Byron

    2006-01-01

    Chronic wasting disease (CWD) is an emerging transmissible spongiform encephalopathy (prion disease) of North American cervids, i.e., mule deer, white-tailed deer, and elk (wapiti). To facilitate in vitro studies of CWD, we have developed a transformed deer cell line that is persistently infected with CWD. Primary cultures derived from uninfected mule deer brain tissue were transformed by transfection with a plasmid containing the simian virus 40 genome. A transformed cell line (MDB) was exposed to microsomes prepared from the brainstem of a CWD-affected mule deer. CWD-associated, protease-resistant prion protein (PrPCWD) was used as an indicator of CWD infection. Although no PrPCWD was detected in any of these cultures after two passes, dilution cloning of cells yielded one PrPCWD-positive clone out of 51. This clone, designated MDBCWD, has maintained stable PrPCWD production through 32 serial passes thus far. A second round of dilution cloning yielded 20 PrPCWD-positive subclones out of 30, one of which was designated MDBCWD2. The MDBCWD2 cell line was positive for fibronectin and negative for microtubule-associated protein 2 (a neuronal marker) and glial fibrillary acidic protein (an activated astrocyte marker), consistent with derivation from brain fibroblasts (e.g., meningeal fibroblasts). Two inhibitors of rodent scrapie protease-resistant PrP accumulation, pentosan polysulfate and a porphyrin compound, indium (III) meso-tetra(4-sulfonatophenyl)porphine chloride, potently blocked PrPCWD accumulation in MDBCWD cells. This demonstrates the utility of these cells in a rapid in vitro screening assay for PrPCWD inhibitors and suggests that these compounds have potential to be active against CWD in vivo. PMID:16378962

  6. Reliable in vitro studies require appropriate ovarian cancer cell lines.

    Science.gov (United States)

    Jacob, Francis; Nixdorf, Sheri; Hacker, Neville F; Heinzelmann-Schwarz, Viola A

    2014-01-01

    Ovarian cancer is the fifth most common cause of cancer death in women and the leading cause of death from gynaecological malignancies. Of the 75% women diagnosed with locally advanced or disseminated disease, only 30% will survive five years following treatment. This poor prognosis is due to the following reasons: limited understanding of the tumor origin, unclear initiating events and early developmental stages of ovarian cancer, lack of reliable ovarian cancer-specific biomarkers, and drug resistance in advanced cases. In the past, in vitro studies using cell line models have been an invaluable tool for basic, discovery-driven cancer research. However, numerous issues including misidentification and cross-contamination of cell lines have hindered research efforts. In this study we examined all ovarian cancer cell lines available from cell banks. Hereby, we identified inconsistencies in the reporting, difficulties in the identification of cell origin or clinical data of the donor patients, restricted ethnic and histological type representation, and a lack of tubal and peritoneal cancer cell lines. We recommend that all cell lines should be distributed via official cell banks only with strict guidelines regarding the minimal available information required to improve the quality of ovarian cancer research in future. PMID:24936210

  7. Neuroimmunological Implications of AQP4 in Astrocytes

    Science.gov (United States)

    Ikeshima-Kataoka, Hiroko

    2016-01-01

    The brain has high-order functions and is composed of several kinds of cells, such as neurons and glial cells. It is becoming clear that many kinds of neurodegenerative diseases are more-or-less influenced by astrocytes, which are a type of glial cell. Aquaporin-4 (AQP4), a membrane-bound protein that regulates water permeability is a member of the aquaporin family of water channel proteins that is expressed in the endfeet of astrocytes in the central nervous system (CNS). Recently, AQP4 has been shown to function, not only as a water channel protein, but also as an adhesion molecule that is involved in cell migration and neuroexcitation, synaptic plasticity, and learning/memory through mechanisms involved in long-term potentiation or long-term depression. The most extensively examined role of AQP4 is its ability to act as a neuroimmunological inducer. Previously, we showed that AQP4 plays an important role in neuroimmunological functions in injured mouse brain in concert with the proinflammatory inducer osteopontin (OPN). The aim of this review is to summarize the functional implication of AQP4, focusing especially on its neuroimmunological roles. This review is a good opportunity to compile recent knowledge and could contribute to the therapeutic treatment of autoimmune diseases through strategies targeting AQP4. Finally, the author would like to hypothesize on AQP4’s role in interaction between reactive astrocytes and reactive microglial cells, which might occur in neurodegenerative diseases. Furthermore, a therapeutic strategy for AQP4-related neurodegenerative diseases is proposed. PMID:27517922

  8. Neuroimmunological Implications of AQP4 in Astrocytes.

    Science.gov (United States)

    Ikeshima-Kataoka, Hiroko

    2016-01-01

    The brain has high-order functions and is composed of several kinds of cells, such as neurons and glial cells. It is becoming clear that many kinds of neurodegenerative diseases are more-or-less influenced by astrocytes, which are a type of glial cell. Aquaporin-4 (AQP4), a membrane-bound protein that regulates water permeability is a member of the aquaporin family of water channel proteins that is expressed in the endfeet of astrocytes in the central nervous system (CNS). Recently, AQP4 has been shown to function, not only as a water channel protein, but also as an adhesion molecule that is involved in cell migration and neuroexcitation, synaptic plasticity, and learning/memory through mechanisms involved in long-term potentiation or long-term depression. The most extensively examined role of AQP4 is its ability to act as a neuroimmunological inducer. Previously, we showed that AQP4 plays an important role in neuroimmunological functions in injured mouse brain in concert with the proinflammatory inducer osteopontin (OPN). The aim of this review is to summarize the functional implication of AQP4, focusing especially on its neuroimmunological roles. This review is a good opportunity to compile recent knowledge and could contribute to the therapeutic treatment of autoimmune diseases through strategies targeting AQP4. Finally, the author would like to hypothesize on AQP4's role in interaction between reactive astrocytes and reactive microglial cells, which might occur in neurodegenerative diseases. Furthermore, a therapeutic strategy for AQP4-related neurodegenerative diseases is proposed. PMID:27517922

  9. In vitro Rb-1 gene transfer to retinoblastoma cell lines

    International Nuclear Information System (INIS)

    After transfection of Rb-vector to packaging cell line (CRIP) by Ca-P precipitation method, we could select nineteen colonies of G-418 resistant clone by ring cloning. Each colony was transduced to NIH3T3 cells to select the one which produces high titer virus. After NIH3T3 cells transduction, we could get 28 colony counts for the high, 127 for the middle, and 6 for the low viral titer. With the supernatant of the high viral titer colony (CRIPRb 2-5). We transduct retinoblastoma cell lines. 5 figs, 11 refs. (Author)

  10. Expressional patterns of chaperones in ten human tumor cell lines

    Directory of Open Access Journals (Sweden)

    Slavc Irene

    2004-12-01

    Full Text Available Abstract Background Chaperones (CH play an important role in tumor biology but no systematic work on expressional patterns has been reported so far. The aim of the study was therefore to present an analytical method for the concomitant determination of several CH in human tumor cell lines, to generate expressional patterns in the individual cell lines and to search for tumor and non-tumor cell line specific CH expression. Human tumor cell lines of neuroblastoma, colorectal and adenocarcinoma of the ovary, osteosarcoma, rhabdomyosarcoma, malignant melanoma, lung, cervical and breast cancer, promyelocytic leukaemia were homogenised, proteins were separated on two-dimensional gel electrophoresis with in-gel digestion of proteins and MALDI-TOF/TOF analysis was carried out for the identification of CH. Results A series of CH was identified including the main CH groups as HSP90/HATPas_C, HSP70, Cpn60_TCP1, DnaJ, Thioredoxin, TPR, Pro_isomerase, HSP20, ERP29_C, KE2, Prefoldin, DUF704, BAG, GrpE and DcpS. Conclusions The ten individual tumor cell lines showed different expression patterns, which are important for the design of CH studies in tumor cell lines. The results can serve as a reference map and form the basis of a concomitant determination of CH by a protein chemical rather than an immunochemical method, independent of antibody availability or specificity.

  11. Intercellular synchronization of diffusively coupled astrocytes

    CERN Document Server

    Alam, Md Jahoor; Devi, Gurumayum Reenaroy; Singh, Heisnam Dinachandra; Singh, R K Brojen; Sharma, B Indrajit

    2010-01-01

    We examine the synchrony of the dynamics of localized [Ca^{2+}]_i oscillations in internal pool of astrocytes via diffusing coupling of a network of such cells in a certain topology where cytosolic Ca^{2+} and inositol 1,4,5-triphosphate (IP3) are coupling molecules; and possible long range interaction among the cells. Our numerical results claim that the cells exhibit fairly well coordinated behaviour through this coupling mechanism. It is also seen in the results that as the number of coupling molecular species is increased, the rate of synchrony is also increased correspondingly. Apart from the topology of the cells taken, as the number of coupled cells around any one of the cells in the system is increased, the cell process information faster.

  12. Effect of type-2 astrocytes on the viability of dorsal root ganglion neurons and length of neuronal processes

    OpenAIRE

    Fan, Chunling; Wang, Hui; Chen, Dan; Cheng, Xiaoxin; Xiong, Kun; Luo, Xuegang; Cao, Qilin

    2014-01-01

    The role of type-2 astrocytes in the repair of central nervous system injury remains poorly understood. In this study, using a relatively simple culture condition in vitro, type-2 astrocytes, differentiated from oligodendrocyte precursor cells by induction with bone morphogenetic protein-4, were co-cultured with dorsal root ganglion neurons. We examined the effects of type-2 astrocytes differentiated from oligodendrocyte precursor cells on the survival and growth of dorsal root ganglion neuro...

  13. Neurohypophysial Receptor Gene Expression by Thymic T Cell Subsets and Thymic T Cell Lymphoma Cell Lines

    Directory of Open Access Journals (Sweden)

    I. Hansenne

    2004-01-01

    transcribed in thymic epithelium, while immature T lymphocytes express functional neurohypophysial receptors. Neurohypophysial receptors belong to the G protein-linked seven-transmembrane receptor superfamily and are encoded by four distinct genes, OTR, V1R, V2R and V3R. The objective of this study was to identify the nature of neurohypophysial receptor in thymic T cell subsets purified by immunomagnetic selection, as well as in murine thymic lymphoma cell lines RL12-NP and BW5147. OTR is transcribed in all thymic T cell subsets and T cell lines, while V3R transcription is restricted to CD4+ CD8+ and CD8+ thymic cells. Neither V1R nor V2R transcripts are detected in any kind of T cells. The OTR protein was identified by immunocytochemistry on thymocytes freshly isolated from C57BL/6 mice. In murine fetal thymic organ cultures, a specific OTR antagonist does not modify the percentage of T cell subsets, but increases late T cell apoptosis further evidencing the involvement of OT/OTR signaling in the control of T cell proliferation and survival. According to these data, OTR and V3R are differentially expressed during T cell ontogeny. Moreover, the restriction of OTR transcription to T cell lines derived from thymic lymphomas may be important in the context of T cell leukemia pathogenesis and treatment.

  14. Galunisertib inhibits glioma vasculogenic mimicry formation induced by astrocytes.

    Science.gov (United States)

    Zhang, Chao; Chen, Wenliang; Zhang, Xin; Huang, Bin; Chen, Aanjing; He, Ying; Wang, Jian; Li, Xingang

    2016-01-01

    Gliomas are among the most lethal primary brain tumors found in humans. In high-grade gliomas, vasculogenic mimicry is often detected and has been correlated with prognosis, thus suggesting its potential as a therapeutic target. Vasculogenic mimicry mainly forms vascular-like channels independent of endothelial cells; however, little is known about the relationship between astrocytes and vasculogenic mimicry. In our study, we demonstrated that the presence of astrocytes promoted vasculogenic mimicry. With suspension microarray technology and in vitro tube formation assays, we identified that astrocytes relied on TGF-β1 to enhance vasculogenic mimicry. We also found that vasculogenic mimicry was inhibited by galunisertib, a promising TGF-β1 inhibitor currently being studied in an ongoing trial in glioma patients. The inhibition was partially attributed to a decrease in autophagy after galunisertib treatment. Moreover, we observed a decrease in VE-cadherin and smooth muscle actin-α expression, as well as down-regulation of Akt and Flk phosphorylation in galunisertib-treated glioma cells. By comparing tumor weight and volume in a xenograft model, we acquired promising results to support our theory. This study expands our understanding of the role of astrocytes in gliomas and demonstrates that galunisertib inhibits glioma vasculogenic mimicry induced by astrocytes. PMID:26976322

  15. Integrated Brain Circuits: Astrocytic Networks Modulate Neuronal Activity and Behavior

    Science.gov (United States)

    Halassa, Michael M.; Haydon, Philip G.

    2011-01-01

    The past decade has seen an explosion of research on roles of neuron-astrocyte interactions in the control of brain function. We highlight recent studies performed on the tripartite synapse, the structure consisting of pre- and postsynaptic elements of the synapse and an associated astrocytic process. Astrocytes respond to neuronal activity and neuro-transmitters, through the activation of metabotropic receptors, and can release the gliotransmitters ATP, D-serine, and glutamate, which act on neurons. Astrocyte-derived ATP modulates synaptic transmission, either directly or through its metabolic product adenosine. D-serine modulates NMDA receptor function, whereas glia-derived glutamate can play important roles in relapse following withdrawal from drugs of abuse. Cell type–specific molecular genetics has allowed a new level of examination of the function of astrocytes in brain function and has revealed an important role of these glial cells that is mediated by adenosine accumulation in the control of sleep and in cognitive impairments that follow sleep deprivation. PMID:20148679

  16. The analgesic effect on neuropathic pain of retrogradely transported botulinum neurotoxin A involves Schwann cells and astrocytes.

    Directory of Open Access Journals (Sweden)

    Sara Marinelli

    Full Text Available In recent years a growing debate is about whether botulinum neurotoxins are retrogradely transported from the site of injection. Immunodetection of cleaved SNAP-25 (cl-SNAP-25, the protein of the SNARE complex targeted by botulinum neurotoxin serotype A (BoNT/A, could represent an excellent approach to investigate the mechanism of action on the nociceptive pathways at peripheral and/or central level. After peripheral administration of BoNT/A, we analyzed the expression of cl-SNAP-25, from the hindpaw's nerve endings to the spinal cord, together with the behavioral effects on neuropathic pain. We used the chronic constriction injury of the sciatic nerve in CD1 mice as animal model of neuropathic pain. We evaluated immunostaining of cl-SNAP-25 in the peripheral nerve endings, along the sciatic nerve, in dorsal root ganglia and in spinal dorsal horns after intraplantar injection of saline or BoNT/A, alone or colocalized with either glial fibrillar acidic protein, GFAP, or complement receptor 3/cluster of differentiation 11b, CD11b, or neuronal nuclei, NeuN, depending on the area investigated. Immunofluorescence analysis shows the presence of the cl-SNAP-25 in all tissues examined, from the peripheral endings to the spinal cord, suggesting a retrograde transport of BoNT/A. Moreover, we performed in vitro experiments to ascertain if BoNT/A was able to interact with the proliferative state of Schwann cells (SC. We found that BoNT/A modulates the proliferation of SC and inhibits the acetylcholine release from SC, evidencing a new biological effect of the toxin and further supporting the retrograde transport of the toxin along the nerve and its ability to influence regenerative processes. The present results strongly sustain a combinatorial action at peripheral and central neural levels and encourage the use of BoNT/A for the pathological pain conditions difficult to treat in clinical practice and dramatically impairing patients' quality of life.

  17. The Analgesic Effect on Neuropathic Pain of Retrogradely Transported botulinum Neurotoxin A Involves Schwann Cells and Astrocytes

    Science.gov (United States)

    Ricordy, Ruggero; Uggenti, Carolina; Tata, Ada Maria; Luvisetto, Siro; Pavone, Flaminia

    2012-01-01

    In recent years a growing debate is about whether botulinum neurotoxins are retrogradely transported from the site of injection. Immunodetection of cleaved SNAP-25 (cl-SNAP-25), the protein of the SNARE complex targeted by botulinum neurotoxin serotype A (BoNT/A), could represent an excellent approach to investigate the mechanism of action on the nociceptive pathways at peripheral and/or central level. After peripheral administration of BoNT/A, we analyzed the expression of cl-SNAP-25, from the hindpaw’s nerve endings to the spinal cord, together with the behavioral effects on neuropathic pain. We used the chronic constriction injury of the sciatic nerve in CD1 mice as animal model of neuropathic pain. We evaluated immunostaining of cl-SNAP-25 in the peripheral nerve endings, along the sciatic nerve, in dorsal root ganglia and in spinal dorsal horns after intraplantar injection of saline or BoNT/A, alone or colocalized with either glial fibrillar acidic protein, GFAP, or complement receptor 3/cluster of differentiation 11b, CD11b, or neuronal nuclei, NeuN, depending on the area investigated. Immunofluorescence analysis shows the presence of the cl-SNAP-25 in all tissues examined, from the peripheral endings to the spinal cord, suggesting a retrograde transport of BoNT/A. Moreover, we performed in vitro experiments to ascertain if BoNT/A was able to interact with the proliferative state of Schwann cells (SC). We found that BoNT/A modulates the proliferation of SC and inhibits the acetylcholine release from SC, evidencing a new biological effect of the toxin and further supporting the retrograde transport of the toxin along the nerve and its ability to influence regenerative processes. The present results strongly sustain a combinatorial action at peripheral and central neural levels and encourage the use of BoNT/A for the pathological pain conditions difficult to treat in clinical practice and dramatically impairing patients’ quality of life. PMID:23110146

  18. Transcription profiles of non-immortalized breast cancer cell lines

    International Nuclear Information System (INIS)

    Searches for differentially expressed genes in tumours have made extensive use of array technology. Most samples have been obtained from tumour biopsies or from established tumour-derived cell lines. Here we compare cultures of non-immortalized breast cancer cells, normal non-immortalized breast cells and immortalized normal and breast cancer cells to identify which elements of a defined set of well-known cancer-related genes are differentially expressed. Cultures of cells from pleural effusions or ascitic fluids from breast cancer patients (MSSMs) were used in addition to commercially-available normal breast epithelial cells (HMECs), established breast cancer cell lines (T-est) and established normal breast cells (N-est). The Atlas Human Cancer 1.2 cDNA expression array was employed. The data obtained were analysed using widely-available statistical and clustering software and further validated through real-time PCR. According to Significance Analysis of Microarray (SAM) and AtlasImage software, 48 genes differed at least 2-fold in adjusted intensities between HMECs and MSSMs (p < 0.01). Some of these genes have already been directly linked with breast cancer, metastasis and malignant progression, whilst others encode receptors linked to signal transduction pathways or are otherwise related to cell proliferation. Fifty genes showed at least a 2.5-fold difference between MSSMs and T-est cells according to AtlasImage, 2-fold according to SAM. Most of these classified as genes related to metabolism and cell communication. The expression profiles of 1176 genes were determined in finite life-span cultures of metastatic breast cancer cells and of normal breast cells. Significant differences were detected between the finite life-span breast cancer cell cultures and the established breast cancer cell lines. These data suggest caution in extrapolating information from established lines for application to clinical cancer research

  19. Physiological properties of astroglial cell lines derived from mice with high (SAMP8 and low (SAMR1, ICR levels of endogenous retrovirus

    Directory of Open Access Journals (Sweden)

    Choi Eun-Kyoung

    2008-11-01

    Full Text Available Abstract Previous studies have reported that various inbred SAM mouse strains differ markedly with regard to a variety of parameters, such as capacity for learning and memory, life spans and brain histopathology. A potential cause of differences seen in these strains may be based on the fact that some strains have a high concentration of infectious murine leukemia virus (MuLV in the brain, whereas other strains have little or no virus. To elucidate the effect of a higher titer of endogenous retrovirus in astroglial cells of the brain, we established astroglial cell lines from SAMR1 and SAMP8 mice, which are, respectively, resistant and prone to deficit in learning and memory and shortened life span. MuLV-negative astroglial cell lines established from ICR mice served as controls. Comparison of these cell lines showed differences in: 1 levels of the capsid antigen CAgag in both cell lysates and culture media, 2 expression of genomic retroelements, 3 the number of virus particles, 4 titer of infectious virus, 5 morphology, 6 replication rate of cells in culture and final cell concentrations, 7 expression pattern of proinflammatory cytokine genes. The results show that the expression of MuLV is much higher in SAMP8 than SAMR1 astrocyte cultures and that there are physiological differences in astroglia from the 2 strains. These results raise the possibility that the distinct physiological differences between SAMP8 and SAMR1 are a function of activation of endogenous retrovirus.

  20. The effects of trypsin on rat brain astrocyte activation.

    Directory of Open Access Journals (Sweden)

    Masoud Fereidoni

    2013-12-01

    Full Text Available Astrocytes are cells within the central nervous system which are activated in a wide spectrum of infections, and autoimmune and neurodegenerative diseases. In pathologic states, they produce inflammatory cytokines, chemokines, and nitric oxide (NO, and sometimes they induce apoptosis. Their protease-activated receptors (PARs can be activated by proteases, e.g. thrombin and trypsin, which are important in brain inflammation. The current study aimed to investigate the effects of different concentrations of trypsin (1 to 100U/ml on cultured astrocytes.In the present study, two-day rat infants' brains were isolated and homogenized after meninges removal, then cultivated in DMEM + 10% FBS medium. 10 days later, astrocytes were harvested and recultivated for more purification (up to 95%, using Immunocytochemistry method, in order to be employed for tests. They were affected by different concentrations of trypsin (1, 5, 10, 15, 20, 40, 60, 80, and 100 U/ml. To reveal the inflammation progress, NO concentrations (the Griess test were assessed after 24 and 48 hours.The results showed that trypsin concentration up to 20 U/ml caused a significant increase in NO, in a dose-dependent manner, on cultured astrocytes (P < 0.001. Trypsin 20 U/ml increased NO production fivefold the control group (P < 0.001. At higher concentrations than 20 U/ml, NO production diminished (P < 0.001. At 100 U/ml, NO production was less than the control group (P < 0.001.Inflammatory effects of trypsin 5-20 U/ml are probably due to the stimulation of astrocytes' PAR-2 receptors and the increasing of the activation of NF-κB, PKC, MAPKs. Stimulation of astrocytes' PAR-2 receptors causes an increase in iNOS activation which in turn leads to NO production. However, higher trypsin concentration possibly made astrocyte apoptosis; therefore, NO production diminished. These assumptions need to be further investigated.

  1. The inhibitory neurotransmitter GABA evokes long-lasting Ca(2+) oscillations in cortical astrocytes.

    Science.gov (United States)

    Mariotti, Letizia; Losi, Gabriele; Sessolo, Michele; Marcon, Iacopo; Carmignoto, Giorgio

    2016-03-01

    Studies over the last decade provided evidence that in a dynamic interaction with neurons glial cell astrocytes contribut to fundamental phenomena in the brain. Most of the knowledge on this derives, however, from studies monitoring the astrocyte Ca(2+) response to glutamate. Whether astrocytes can similarly respond to other neurotransmitters, including the inhibitory neurotransmitter GABA, is relatively unexplored. By using confocal and two photon laser-scanning microscopy the astrocyte response to GABA in the mouse somatosensory and temporal cortex was studied. In slices from developing (P15-20) and adult (P30-60) mice, it was found that in a subpopulation of astrocytes GABA evoked somatic Ca(2+) oscillations. This response was mediated by GABAB receptors and involved both Gi/o protein and inositol 1,4,5-trisphosphate (IP3 ) signalling pathways. In vivo experiments from young adult mice, revealed that also cortical astrocytes in the living brain exibit GABAB receptor-mediated Ca(2+) elevations. At all astrocytic processes tested, local GABA or Baclofen brief applications induced long-lasting Ca(2+) oscillations, suggesting that all astrocytes have the potential to respond to GABA. Finally, in patch-clamp recordings it was found that Ca(2+) oscillations induced by Baclofen evoked astrocytic glutamate release and slow inward currents (SICs) in pyramidal cells from wild type but not IP3 R2(-/-) mice, in which astrocytic GABAB receptor-mediated Ca(2+) elevations are impaired. These data suggest that cortical astrocytes in the mouse brain can sense the activity of GABAergic interneurons and through their specific recruitment contribut to the distinct role played on the cortical network by the different subsets of GABAergic interneurons. PMID:26496414

  2. Effect of 8-bromo-cAMP and dexamethasone on glutamate metabolism in rat astrocytes

    International Nuclear Information System (INIS)

    Glutamine synthetase (GS) activity in cultured rat astrocytes was measured in extracts and compared to the intracellular rate of glutamine synthesis by intact control astrocytes or astrocytes exposed to 1 mM 8-bromo-cAMP (8Br-cAMP) + 1 microM dexamethasone (DEX) for 4 days. GS activity in extracts of astrocytes treated with 8Br-cAMP + DEX was 7.5 times greater than the activity in extracts of control astrocytes. In contrast, the intracellular rate of glutamine synthesis by intact cells increased only 2-fold, suggesting that additional intracellular effectors regulate the expression of GS activity inside the intact cell. The rate of glutamine synthesis by astrocytes was 4.3 times greater in MEM than in HEPES buffered Hank's salts. Synthesis of glutamine by intact astrocytes cultured in MEM was independent of the external glutamine or ammonia concentrations but was increased by higher extracellular glutamate concentrations. In studies with intact astrocytes 80% of the original [U-14C]glutamate was recovered in the medium as radioactive glutamine, 2-3% as aspartate, and 7% as glutamate after 2 hours for both control and treated astrocytes. The results suggest: (1) astrocytes are highly efficient in the conversion of glutamate to glutamine; (2) induction of GS activity increases the rate of glutamate conversion to glutamine by astrocytes and the rate of glutamine release into the medium; (3) endogenous intracellular regulators of GS activity control the flux of glutamate through this enzymatic reaction; and (4) the composition of the medium alters the rate of glutamine synthesis from external glutamate

  3. Cell Line Modeling to Study Biomarker Panel in Prostate Cancer

    Science.gov (United States)

    NickKholgh, Bita; Fang, Xiaolan; Winters, Shira M.; Raina, Anvi; Pandya, Komal S.; Gyabaah, Kenneth; Fino, Nora; Balaji, K.C.

    2016-01-01

    BACKGROUND African–American men with prostate cancer (PCa) present with higher-grade and -stage tumors compared to Caucasians. While the disparity may result from multiple factors, a biological basis is often strongly suspected. Currently, few well-characterized experimental model systems are available to study the biological basis of racial disparity in PCa. We report a validated in vitro cell line model system that could be used for the purpose. METHODS We assembled a PCa cell line model that included currently available African–American PCa cell lines and LNCaP (androgen-dependent) and C4-2 (castration-resistant) Caucasian PCa cells. The utility of the cell lines in studying the biological basis of variance in a malignant phenotype was explored using a multiplex biomarker panel consisting of proteins that have been proven to play a role in the progression of PCa. The panel expression was evaluated by Western blot and RT-PCR in cell lines and validated in human PCa tissues by RT-PCR. As proof-of-principle to demonstrate the utility of our model in functional studies, we performed MTS viability assays and molecular studies. RESULTS The dysregulation of the multiplex biomarker panel in primary African–American cell line (E006AA) was similar to metastatic Caucasian cell lines, which would suggest that the cell line model could be used to study an inherent aggressive phenotype in African–American men with PCa. We had previously demonstrated that Protein kinase D1 (PKD1) is a novel kinase that is down regulated in advanced prostate cancer. We established the functional relevance by over expressing PKD1, which resulted in decreased proliferation and epithelial mesenchymal transition (EMT) in PCa cells. Moreover, we established the feasibility of studying the expression of the multiplex biomarker panel in archived human PCa tissue from African–Americans and Caucasians as a prelude to future translational studies. CONCLUSION We have characterized a novel in

  4. Peroxynitrite decomposition catalyst prevents apoptotic cell death in a human astrocytoma cell line incubated with supernatants of HIV-infected macrophages

    Directory of Open Access Journals (Sweden)

    Perno Carlo

    2002-09-01

    Full Text Available Abstract Background Oxidative stress has shown to contribute in the mechanisms underlying apoptotic cell death occuring in AIDS-dementia complex. Here we investigated the role of peroxynitrite in apoptosis occurring in astroglial cells incubated with supernatants of HIV-infected human primary macrophages (M/M. Results Flow cytometric analysis (FACS of human cultured astrocytes shortly incubated with HIV-1-infected M/M supernatants showed apoptotic cell death, an effect accompanied by pronounced staining for nitrotyrosine (footprint of peroxynitrite and by abnormal formation of malondialdehyde (MDA. Pretreatment of astrocytes with the peroxynitrite decomposition catalyst FeTMPS antagonized HIV-related astrocytic apoptosis, MDA formation and nitrotyrosine staining. Conclusions Taken together, our results suggest that inibition of peroxynitrite leads to protection against peroxidative stress accompanying HIV-related apoptosis of astrocytes. Overall results support the role of peroxynitrite in HIV-related programmed death of astrocytes and suggest the use of peroxynitrite decomposition catalyst to counteract HIV-1-related neurological disorders.

  5. Solid Oxide Fuel Cell Systems PVL Line

    Energy Technology Data Exchange (ETDEWEB)

    Susan Shearer - Stark State College; Gregory Rush - Rolls-Royce Fuel Cell Systems

    2012-05-01

    In July 2010, Stark State College (SSC), received Grant DE-EE0003229 from the U.S. Department of Energy (DOE), Golden Field Office, for the development of the electrical and control systems, and mechanical commissioning of a unique 20kW scale high-pressure, high temperature, natural gas fueled Stack Block Test System (SBTS). SSC worked closely with subcontractor, Rolls-Royce Fuel Cell Systems (US) Inc. (RRFCS) over a 13 month period to successfully complete the project activities. This system will be utilized by RRFCS for pre-commercial technology development and training of SSC student interns. In the longer term, when RRFCS is producing commercial products, SSC will utilize the equipment for workforce training. In addition to DOE Hydrogen, Fuel Cells, and Infrastructure Technologies program funding, RRFCS internal funds, funds from the state of Ohio, and funding from the DOE Solid State Energy Conversion Alliance (SECA) program have been utilized to design, develop and commission this equipment. Construction of the SBTS (mechanical components) was performed under a Grant from the State of Ohio through Ohio's Third Frontier program (Grant TECH 08-053). This Ohio program supported development of a system that uses natural gas as a fuel. Funding was provided under the Department of Energy (DOE) Solid-state Energy Conversion Alliance (SECA) program for modifications required to test on coal synthesis gas. The subject DOE program provided funding for the electrical build, control system development and mechanical commissioning. Performance testing, which includes electrical commissioning, was subsequently performed under the DOE SECA program. Rolls-Royce Fuel Cell Systems is developing a megawatt-scale solid oxide fuel cell (SOFC) stationary power generation system. This system, based on RRFCS proprietary technology, is fueled with natural gas, and operates at elevated pressure. A critical success factor for development of the full scale system is the capability

  6. Co-culture of astrocytes with neurons from injured brain A time-dependent dichotomy

    Institute of Scientific and Technical Information of China (English)

    Xiaojing Xu; Min Wang; Jing Liu; Jingya Lv; Yanan Hu; Huanxiang Zhang

    2011-01-01

    As supportive cells for neuronal growth and development, much effort has been devoted to the role of astrocytes in the normal state. However, the effect of the astrocytes after injury remains elusive. In the present study, neurons isolated from the subventricular zone of injured neonatal rat brains were co-cultured with astrocytes. After 6 days, these astrocytes showed a mature neuron-like appearance and the number of survivingneurons, primary dendrites and total branches was significantly higher than those at 3 days. The neurons began to shrink at 9 days after co-culture with shorter and thinner processes and the number of primary dendrites and total branches was significantly reduced. These experimental findings indicate that astrocytes in the injured brain promote the development of neurons in the early stages of co-culture while these cells reversely inhibit neuronal growth and development at the later states.

  7. Steroid hormone secretion in inflammatory breast cancer cell lines.

    Science.gov (United States)

    Illera, Juan Carlos; Caceres, Sara; Peña, Laura; de Andres, Paloma J; Monsalve, Beatriz; Illera, Maria J; Woodward, Wendy A; Reuben, James M; Silvan, Gema

    2015-12-01

    Inflammatory breast carcinoma (IBC) is a special type of breast cancer with a poor survival rate. Though several IBC cell lines have been established, recently a first IMC cell line was established. The aims of this study were: (1) to validate a highly sensitive, reliable, accurate and direct amplified enzyme immunoassay (EIA) to measure several cell-secreted steroid hormones: progesterone (P4), androstenedione (A4), testosterone (T), 17β-estradiol (E2) and estrone sulfate (SO4E1) in the culture medium. (2) To assess whether hormone production profile by IPC-366 cells validates the IMC model for human IBC. We validated a non-competitive amplified EIA for inflammatory breast cancer cell lines based on the results of accuracy, precision, sensitivity and parallelism. The low detection limits of the technique were: P4=13.2 pg/well, A4=2.3 pg/well, T=11.4 pg/well, E2=1.9 pg/well and SO4E1=4.5 pg/well. Intra- and inter-assay coefficient of variation percentages were 90%. In all hormones studied SUM149 have higher levels (1.4 times, but not significant) than IPC-366, and the correlation index between SUM149 and IPC-366 concentrations were >97%. We can coclude that cells of both cell lines, IPC-366 and SUM149, are capable to produce steroid hormone in culture media. The presented EIA methodology is very valuable for the detection of steroid production in culture media and could be used in hormone regulation studies and therapeutic agents in cell lines of inflammatory and non-inflammatory mammary carcinoma or other cancer cell lines in preclinical studies. PMID:26495931

  8. MOLECULAR AND CYTOGENETIC ANALYSIS OF LUNG TUMOR CELL LINES

    Science.gov (United States)

    We have measured the levels of amplification of oncogenes and tumor marker genes or other genes of interest in nine human lung tumor cell lines in comparison to normal human bronchial epithelial cells or normal blood lymphocytes to test the hypothesis that aberrant amplification ...

  9. Transportation characteristics of nolatrexed in three tumor cell lines

    Institute of Scientific and Technical Information of China (English)

    LI Yi-lei; ZHAO Ai-guo; WU Shu-guang

    2002-01-01

    Objective:To investigate the association of the transportation characteristics of nolatrexed in tumor cells with its drug sensitivity. Methods: The sensitivity of 3 tumor cell lines, C6, SRS82 and LoVo, to nolatrexed were determined by growth inhibition study. After exposure to 20 μmol/L nolatrexed at different time intervals ranging from 0 to 30 min, or to nolatrexed at different concentrations ranging from 0 to 40μmol/L for 10 min, the intracellular drug concentration was measured using high-performance liquid chromatography. Results: C6 was the most sensitive cell line among the three, with sensitivity 6. 8-fold and 13.8-fold those of SRS-82 and LoVo cells respectively. Transportation of nolatrexed in the 3 cell lines were qualitatively similar, which rapidly achieved steady-state within 5 min, and linear relationship between the intracellular and extracellular drug concentration was observed. The intracellular steady-state level achieved in C6 was significantly higher than those in the other two cell lines, the latter having comparable levels. Conclusion: Nolatrexed enters the cell very quickly and different transport capacities are involved in the generation of varied sensitivity to nolatrexed in tumor cells.

  10. Frequency and distribution of Notch mutations in tumor cell lines

    International Nuclear Information System (INIS)

    Deregulated Notch signaling is linked to a variety of tumors and it is therefore important to learn more about the frequency and distribution of Notch mutations in a tumor context. In this report, we use data from the recently developed Cancer Cell Line Encyclopedia to assess the frequency and distribution of Notch mutations in a large panel of cancer cell lines in silico. Our results show that the mutation frequency of Notch receptor and ligand genes is at par with that for established oncogenes and higher than for a set of house-keeping genes. Mutations were found across all four Notch receptor genes, but with notable differences between protein domains, mutations were for example more prevalent in the regions encoding the LNR and PEST domains in the Notch intracellular domain. Furthermore, an in silico estimation of functional impact showed that deleterious mutations cluster to the ligand-binding and the intracellular domains of NOTCH1. For most cell line groups, the mutation frequency of Notch genes is higher than in associated primary tumors. Our results shed new light on the spectrum of Notch mutations after in vitro culturing of tumor cells. The higher mutation frequency in tumor cell lines indicates that Notch mutations are associated with a growth advantage in vitro, and thus may be considered to be driver mutations in a tumor cell line context. The online version of this article (doi:10.1186/s12885-015-1278-x) contains supplementary material, which is available to authorized users

  11. Continuous production of erythropoietin by an established human renal carcinoma cell line: development of the cell line

    Energy Technology Data Exchange (ETDEWEB)

    Sherwood, J.B.; Shouval, D.

    1986-01-01

    Establishment of a stable, transformed human renal carcinoma cell line that produces erythropoietin in vitro and has maintained this function continuously since 1981 and for > 150 passages in monolayer culture was accomplished by transplantation of human renal clear cell carcinoma tissue from a patient with erythrocytosis into an immunosuppressed athymic mouse. In addition to its immunocrossreactivity with native human urinary erythropoietin, the tumor erythropoietin demonstrates biological activity in the in vitro mouse erythroid colony-forming unit assay and in tumor-bearing nude mice. The cloned renal carcinoma cell line has an abnormal human karyotype and has ultrastructural features characteristic of human renal clear cell carcinoma. This cell line provides a reproducible model system for the production of an erythropoietin-like material and for the study of its synthesis and secretion.

  12. Derivation of human embryonic stem cell line Genea019

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea019 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype, female Allele pattern and unaffected Htt CAG repeat length, compared to HD affected sibling Genea020. Pluripotency of Genea019 was demonstrated with 75% of cells expressing Nanog, 89% Oct4, 48% Tra1-60 and 85% SSEA4, a Pluritest Pluripotency score of 22.97, Novelty score of 1.42, tri-lineage teratoma formation and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination.

  13. EFFECTS OF CULTURED ASTROCYTES FROM RAT CEREBRAL CORTEX ON THE DEVELOPMENT OF PC12 CELLS%星形神经胶质细胞对PC12细胞生长发育的影响

    Institute of Scientific and Technical Information of China (English)

    莫永炎; 陈瑗; 周玫; 张宝

    2000-01-01

    在神经系统的生长发育过程中,星形胶质细胞对神经元生长发育的作用是一项重要的研究课题。本文以体外培养的SD大鼠大脑皮质星形胶质细胞与PC12神经元按不同细胞数目比例(50:1~1:1)共同培养,并用其制备的条件培养液培养PC12细胞,用快速灵敏的MTT比色法测定PC12神经元的细胞活力,用光学相差显微镜观察PC12细胞形态学变化。结果显示,星形胶质细胞条件培养液可增强PC12细胞活力(MTT测定的 OD值由0.255±0.012提高到0.510±0.036,P<0.001,且细胞折光性较对照组强),却不能促使PC12神经元突起的生出。将星形胶质细胞与PC12细胞按30:1~1:1的比例共同培养时,既可提高PC12细胞折光性和光晕又可促使其突起的生长;如按50:1~40:1的比例共同培养时,只观察到提高PC12细胞折光性和光晕,而无促使其突起生长发育的作用。本文结果提示,PC12神经元细胞活力的提高与星形胶质细胞分泌到条件培养液中的可溶性因子有关,而PC12神经元突起生长发育可能是和与星形胶质细胞的直接接触以及二者的细胞数且比有关。%To investigate effects of cultured astrocytes from Sprague Dawley rat cerebral cortex on the development of PC12 cellsderived from rat pheochromocytoma, PC12 cells were cocultured with astrocyte according to different astrocytes/neurons ratio(50:1~1:1) , or with serum-free conditioned medium of astrocytes(ACM). The vitality of PC12 cells was measured by sensi-tive MTT method and their morphologic features were observed by Olympus light microscope. The results showed: (1) WhenPC12 cells were cultured with ACM, compared with the control group, the vitality of PC12 cells was increased significantly (0.255+0. 012 vs 0. 510±0. 036, P<0. 001) and the morphological changes were not obvious in the experimental group. (2) WhenPC12 cells were cocultured with astrocyte in the ratio of 30: 1

  14. Establishment, immortalisation and characterisation of pteropid bat cell lines.

    Directory of Open Access Journals (Sweden)

    Gary Crameri

    Full Text Available BACKGROUND: Bats are the suspected natural reservoir hosts for a number of new and emerging zoonotic viruses including Nipah virus, Hendra virus, severe acute respiratory syndrome coronavirus and Ebola virus. Since the discovery of SARS-like coronaviruses in Chinese horseshoe bats, attempts to isolate a SL-CoV from bats have failed and attempts to isolate other bat-borne viruses in various mammalian cell lines have been similarly unsuccessful. New stable bat cell lines are needed to help with these investigations and as tools to assist in the study of bat immunology and virus-host interactions. METHODOLOGY/FINDINGS: Black flying foxes (Pteropus alecto were captured from the wild and transported live to the laboratory for primary cell culture preparation using a variety of different methods and culture media. Primary cells were successfully cultured from 20 different organs. Cell immortalisation can occur spontaneously, however we used a retroviral system to immortalise cells via the transfer and stable production of the Simian virus 40 Large T antigen and the human telomerase reverse transcriptase protein. Initial infection experiments with both cloned and uncloned cell lines using Hendra and Nipah viruses demonstrated varying degrees of infection efficiency between the different cell lines, although it was possible to infect cells in all tissue types. CONCLUSIONS/SIGNIFICANCE: The approaches developed and optimised in this study should be applicable to bats of other species. We are in the process of generating further cell lines from a number of different bat species using the methodology established in this study.

  15. Monoclonal antibodies against the human leukemia cell line K 562.

    Science.gov (United States)

    Böttger, V; Hering, S; Jantscheff, P; Micheel, B

    1985-01-01

    Three monoclonal antibodies raised against K 562, a cell line originally established from a patient with chronic myeloid leukemia (CML) in terminal blast crisis, were selected according to their distinct reaction pattern. Whereas two antibodies (ZIK-C1-A/C5 and ZIK-C1-A/H5 also designated C and H) recognized antigens, present on K 562 cells and other immature and mature hematopoietic cells (cell lines and normal blood and bone marrow cells), antibody ZIK-C1-A/D9 also designated Y showed an exclusive binding to K 562 cells. The results obtained (here and in the following paper) indicate, that antibody ZIK-C1-A/D9 defines an early differentiation antigen of hematopoiesis or a leukemia-associated antigen.

  16. IFN-γ signaling to astrocytes protects from autoimmune mediated neurological disability.

    Directory of Open Access Journals (Sweden)

    Claudia Hindinger

    Full Text Available Demyelination and axonal degeneration are determinants of progressive neurological disability in patients with multiple sclerosis (MS. Cells resident within the central nervous system (CNS are active participants in development, progression and subsequent control of autoimmune disease; however, their individual contributions are not well understood. Astrocytes, the most abundant CNS cell type, are highly sensitive to environmental cues and are implicated in both detrimental and protective outcomes during autoimmune demyelination. Experimental autoimmune encephalomyelitis (EAE was induced in transgenic mice expressing signaling defective dominant-negative interferon gamma (IFN-γ receptors on astrocytes to determine the influence of inflammation on astrocyte activity. Inhibition of IFN-γ signaling to astrocytes did not influence disease incidence, onset, initial progression of symptoms, blood brain barrier (BBB integrity or the composition of the acute CNS inflammatory response. Nevertheless, increased demyelination at peak acute disease in the absence of IFN-γ signaling to astrocytes correlated with sustained clinical symptoms. Following peak disease, diminished clinical remission, increased mortality and sustained astrocyte activation within the gray matter demonstrate a critical role of IFN-γ signaling to astrocytes in neuroprotection. Diminished disease remission was associated with escalating demyelination, axonal degeneration and sustained inflammation. The CNS infiltrating leukocyte composition was not altered; however, decreased IL-10 and IL-27 correlated with sustained disease. These data indicate that astrocytes play a critical role in limiting CNS autoimmune disease dependent upon a neuroprotective signaling pathway mediated by engagement of IFN-γ receptors.

  17. Proteomic analysis of astrocytic secretion that regulates neurogenesis using quantitative amine-specific isobaric tagging

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Hu; Zhou, Wenhao [Children' s Hospital of Fudan University, 399 Wanyuan Road, Shanghai 201102 (China); Wei, Liming; Zhong, Fan [Institutes of Biomedical Sciences, Fudan University, 138 Yixueyuan Roda, Shanghai 200032 (China); Yang, Yi, E-mail: yyang@shmu.edu.cn [Children' s Hospital of Fudan University, 399 Wanyuan Road, Shanghai 201102 (China)

    2010-01-08

    Astrocytes are essential components of neurogenic niches that affect neurogenesis through membrane association and/or the release of soluble factors. To identify factors released from astrocytes that could regulate neural stem cell differentiation and proliferation, we used mild oxygen-glucose deprivation (OGD) to inhibit the secretory capacity of astrocytes. Using the Transwell co-culture system, we found that OGD-treated astrocytes could not promote neural stem cell differentiation and proliferation. Next, isobaric tagging for the relative and absolute quantitation (iTRAQ) proteomics techniques was performed to identify the proteins in the supernatants of astrocytes (with or without OGD). Through a multi-step analysis and gene ontology classification, 130 extracellular proteins were identified, most of which were involved in neuronal development, the inflammatory response, extracellular matrix composition and supportive functions. Of these proteins, 44 had never been reported to be produced by astrocytes. Using ProteinPilot software analysis, we found that 60 extracellular proteins were significantly altered (27 upregulated and 33 downregulated) in the supernatant of OGD-treated astrocytes. Among these proteins, 7 have been reported to be able to regulate neurogenesis, while others may have the potential to regulate neurogenesis. This study profiles the major proteins released by astrocytes, which play important roles in the modulation of neurogenesis.

  18. Characteristics of calcium signaling in astrocytes induced by photostimulation with femtosecond laser

    Science.gov (United States)

    Zhao, Yuan; Zhang, Yuan; Zhou, Wei; Liu, Xiuli; Zeng, Shaoqun; Luo, Qingming

    2010-05-01

    Astrocytes have been identified to actively contribute to brain functions through Ca2+ signaling, serving as a bridge to communicate with neurons and other brain cells. However, conventional stimulation techniques are hard to apply to delicate investigations on astrocytes. Our group previously reported photostimulation with a femtosecond laser to evoke astrocytic calcium (Ca2+) waves, providing a noninvasive and efficient approach with highly precise targeting. In this work, detailed characteristics of astrocytic Ca2+ signaling induced by photostimulation are presented. In a purified astrocytic culture, after the illumination of a femtosecond laser onto one cell, a Ca2+ wave throughout the network with reduced speed is induced, and intracellular Ca2+ oscillations are observed. The intercellular propagation is pharmacologically confirmed to be mainly mediated by ATP through P2Y receptors. Different patterns of Ca2+ elevations with increased amplitude in the stimulated astrocyte are discovered by varying the femtosecond laser power, which is correspondingly followed by broader intercellular waves. These indicate that the strength of photogenerated Ca2+ signaling in astrocytes has a positive relationship with the stimulating laser power. Therefore, distinct Ca2+ signaling is feasibly available for specific studies on astrocytes by employing precisely controlled photostimulation.

  19. The histone deacetylase inhibitor suberoylanilide hydroxamic acid attenuates human astrocyte neurotoxicity induced by interferon-γ

    Directory of Open Access Journals (Sweden)

    Hashioka Sadayuki

    2012-05-01

    Full Text Available Abstract Backgrounds Increasing evidence shows that the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA possesses potent anti-inflammatory and immunomodulatory properties. It is tempting to evaluate the potential of SAHA as a therapeutic agent in various neuroinflammatory and neurodegenerative disorders. Methods We examined the effects of SAHA on interferon (IFN-γ-induced neurotoxicity of human astrocytes and on IFN-γ-induced phosphorylation of signal transducer and activator of transcription (STAT 3 in human astrocytes. We also studied the effects of SAHA on the astrocytic production of two representative IFN-γ-inducible inflammatory molecules, namely IFN-γ-inducible T cell α chemoattractant (I-TAC and intercellular adhesion molecule-1 (ICAM-1. Results SAHA significantly attenuated the toxicity of astrocytes activated by IFN-γ towards SH-SY5Y human neuronal cells. In the IFN-γ-activated astrocytes, SAHA reduced the STAT3 phosphorylation. SAHA also inhibited the IFN-γ-induced astrocytic production of I-TAC, but not ICAM-1. These results indicate that SAHA suppresses IFN-γ-induced neurotoxicity of human astrocytes through inhibition of the STAT3 signaling pathway. Conclusion Due to its anti-neurotoxic and anti-inflammatory properties, SAHA appears to have the therapeutic or preventive potential for a wide range of neuroinflammatory disorders associated with activated astrocytes.

  20. Gain-of-function defects of astrocytic Kir4.1 channels in children with autism spectrum disorders and epilepsy

    Science.gov (United States)

    Sicca, Federico; Ambrosini, Elena; Marchese, Maria; Sforna, Luigi; Servettini, Ilenio; Valvo, Giulia; Brignone, Maria Stefania; Lanciotti, Angela; Moro, Francesca; Grottesi, Alessandro; Catacuzzeno, Luigi; Baldini, Sara; Hasan, Sonia; D’Adamo, Maria Cristina; Franciolini, Fabio; Molinari, Paola; Santorelli, Filippo M.; Pessia, Mauro

    2016-01-01

    Dysfunction of the inwardly-rectifying potassium channels Kir4.1 (KCNJ10) represents a pathogenic mechanism contributing to Autism-Epilepsy comorbidity. To define the role of Kir4.1 variants in the disorder, we sequenced KCNJ10 in a sample of affected individuals, and performed genotype-phenotype correlations. The effects of mutations on channel activity, protein trafficking, and astrocyte function were investigated in Xenopus laevis oocytes, and in human astrocytoma cell lines. An in vivo model of the disorder was also explored through generation of kcnj10a morphant zebrafish overexpressing the mutated human KCNJ10. We detected germline heterozygous KCNJ10 variants in 19/175 affected children. Epileptic spasms with dysregulated sensory processing represented the main disease phenotype. When investigated on astrocyte-like cells, the p.R18Q mutation exerted a gain-of-function effect by enhancing Kir4.1 membrane expression and current density. Similarly, the p.R348H variant led to gain of channel function through hindrance of pH-dependent current inhibition. The frequent polymorphism p.R271C seemed, instead, to have no obvious functional effects. Our results confirm that variants in KCNJ10 deserve attention in autism-epilepsy, and provide insight into the molecular mechanisms of autism and seizures. Similar to neurons, astrocyte dysfunction may result in abnormal synaptic transmission and electrical discharge, and should be regarded as a possible pharmacological target in autism-epilepsy. PMID:27677466

  1. Comparative Metabolic Flux Profiling of Melanoma Cell Lines

    Science.gov (United States)

    Scott, David A.; Richardson, Adam D.; Filipp, Fabian V.; Knutzen, Christine A.; Chiang, Gary G.; Ronai, Ze'ev A.; Osterman, Andrei L.; Smith, Jeffrey W.

    2011-01-01

    Metabolic rewiring is an established hallmark of cancer, but the details of this rewiring at a systems level are not well characterized. Here we acquire this insight in a melanoma cell line panel by tracking metabolic flux using isotopically labeled nutrients. Metabolic profiling and flux balance analysis were used to compare normal melanocytes to melanoma cell lines in both normoxic and hypoxic conditions. All melanoma cells exhibited the Warburg phenomenon; they used more glucose and produced more lactate than melanocytes. Other changes were observed in melanoma cells that are not described by the Warburg phenomenon. Hypoxic conditions increased fermentation of glucose to lactate in both melanocytes and melanoma cells (the Pasteur effect). However, metabolism was not strictly glycolytic, as the tricarboxylic acid (TCA) cycle was functional in all melanoma lines, even under hypoxia. Furthermore, glutamine was also a key nutrient providing a substantial anaplerotic contribution to the TCA cycle. In the WM35 melanoma line glutamine was metabolized in the “reverse” (reductive) direction in the TCA cycle, particularly under hypoxia. This reverse flux allowed the melanoma cells to synthesize fatty acids from glutamine while glucose was primarily converted to lactate. Altogether, this study, which is the first comprehensive comparative analysis of metabolism in melanoma cells, provides a foundation for targeting metabolism for therapeutic benefit in melanoma. PMID:21998308

  2. Astrocytes in oligodendrocyte lineage development and white matter pathology

    Directory of Open Access Journals (Sweden)

    Jiasi eLi

    2016-05-01

    Full Text Available White matter is primarily composed of myelin and myelinated axons. Structural and functional completeness of myelin is critical for the reliable and efficient transmission of information. White matter injury has been associated with the development of many demyelinating diseases. Despite a variety of scientific advances aimed at promoting re-myelination, their benefit has proven at best to be marginal. Research suggests that the failure of the re-myelination process may be the result of an unfavorable microenvironment. Astrocytes, are the most ample and diverse type of glial cells in central nervous system which display multiple functions for the cells of the oligodendrocytes lineage. As such, much attention has recently been drawn to astrocyte function in terms of white matter myelin repair. They are different in white matter from those in grey matter in specific regards to development, morphology, location, protein expression and other supportive functions. During the process of demyelination and re-myelination, the functions of astrocytes are dynamic in that they are able to change functions in accordance to different time points, triggers or reactive pathways resulting in vastly different biologic effects. They have pivotal effects on oligodendrocytes and other cell types in the oligodendrocyte lineage by serving as an energy supplier, a participant of immunological and inflammatory functions, a source of trophic factors and iron and a sustainer of homeostasis. Astrocytic impairment has been shown to be directly linked to the development of neuromyelities optica. In addition, astroctyes have also been implicated in other white matter conditions such as psychiatric disorders and neurodegenerative diseases such as Alzheimer’s disease, multiple sclerosis and amyotrophic lateral sclerosis. Inhibiting specifically detrimental signaling pathways in astrocytes while preserving their beneficial functions may be a promising approach for

  3. Investigation on the suitable pressure for the preservation of astrocyte

    Energy Technology Data Exchange (ETDEWEB)

    Sotome, S; Shimizu, A [Department of Environmental Engineering for Symbiosis, Soka University, 1-326 Tangi-cho, Hachioji, Tokyo 192-8577 (Japan); Nakajima, K [Department of Bioinformatics, Soka University, 1-326 Tangi-cho, Hachioji, Tokyo 192-8577 (Japan); Yoshimura, Y, E-mail: sotome_shinichi@yahoo.co.j [Department of Applied Chemistry, National Defence Academy, 1-10-20 Hashirimizu, Yokosuka, Kanagawa 239-8686 (Japan)

    2010-03-01

    The effects of pressure on the survival rate of astrocytes in growth medium (DMEM) were investigated at room temperature and at 4{sup 0}C, in an effort to establish the best conditions for the preservation. Survival rate at 4{sup 0}C was found to be higher than that at room temperature. The survival rate of astrocytes preserved for 4 days at 4{sup 0}C increased with increasing pressure up to 1.6 MPa, but decreased with increasing pressure above 1.6 MPa. At 10 MPa, all astrocytes died. The survival rate of cultured astrocytes decreased significantly following pressurization for 2 hours and the subsequent preservation for 2 days at atmospheric pressure. Therefore, it is necessary to maintain pressure when preserving astrocytes. These results indicate that the cells can be stored at 4{sup 0}C under pressurization without freezing and without adding cryoprotective agents. Moreover, it may be possible to use this procedure as a new preservation method when cryopreservation is impractical.

  4. Investigation on the suitable pressure for the preservation of astrocyte

    Science.gov (United States)

    Sotome, S.; Nakajima, K.; Yoshimura, Y.; Shimizu, A.

    2010-03-01

    The effects of pressure on the survival rate of astrocytes in growth medium (DMEM) were investigated at room temperature and at 4°C, in an effort to establish the best conditions for the preservation. Survival rate at 4°C was found to be higher than that at room temperature. The survival rate of astrocytes preserved for 4 days at 4°C increased with increasing pressure up to 1.6 MPa, but decreased with increasing pressure above 1.6 MPa. At 10 MPa, all astrocytes died. The survival rate of cultured astrocytes decreased significantly following pressurization for 2 hours and the subsequent preservation for 2 days at atmospheric pressure. Therefore, it is necessary to maintain pressure when preserving astrocytes. These results indicate that the cells can be stored at 4°C under pressurization without freezing and without adding cryoprotective agents. Moreover, it may be possible to use this procedure as a new preservation method when cryopreservation is impractical.

  5. Skin Biopsy and Patient-Specific Stem Cell Lines

    Science.gov (United States)

    Li, Yao; Nguyen, Huy V.; Tsang, Stephen H.

    2016-01-01

    The generation of patient-specific induced pluripotent stem (iPS) cells permits the development of next-generation patient-specific systems biology models reflecting personalized genomics profiles to better understand pathophysiology. In this chapter, we describe how to create a patient-specific iPS cell line. There are three major steps: (1) performing a skin biopsy procedure on the patient; (2) extracting human fibroblast cells from the skin biopsy tissue; and (3) reprogramming patient-specific fibroblast cells into the pluripotent stem cell stage. PMID:26141312

  6. Human cell lines: A promising alternative for recombinant FIX production.

    Science.gov (United States)

    de Sousa Bomfim, Aline; Cristina Corrêa de Freitas, Marcela; Picanço-Castro, Virgínia; de Abreu Soares Neto, Mário; Swiech, Kamilla; Tadeu Covas, Dimas; Maria de Sousa Russo, Elisa

    2016-05-01

    Factor IX (FIX) is a vitamin K-dependent protein, and it has become a valuable pharmaceutical in the Hemophilia B treatment. We evaluated the potential of recombinant human FIX (rhFIX) expression in 293T and SK-Hep-1 human cell lines. SK-Hep-1-FIX cells produced higher levels of biologically active protein. The growth profile of 293T-FIX cells was not influenced by lentiviral integration number into the cellular genome. SK-Hep-1-FIX cells showed a significantly lower growth rate than SK-Hep-1 cells. γ-carboxylation process is significant to FIX biological activity, thus we performed a expression analysis of genes involved in this process. The 293T gene expression suggests that this cell line could efficiently carboxylate FIX, however only 28% of the total secreted protein is active. SK-Hep-1 cells did not express high amounts of VKORC1 and carboxylase, but this cell line secreted large amounts of active protein. Enrichment of culture medium with Ca(+2) and Mg(+2) ions did not affect positively rhFIX expression in SK-Hep-1 cells. In 293T cells, the addition of 0.5 mM Ca(+2) and 1 mM Mg(+2) resulted in higher rhFIX concentration. SK-Hep-1 cell line proved to be very effective in rhFIX production, and it can be used as a novel biotechnological platform for the production of recombinant proteins.

  7. Human cell lines: A promising alternative for recombinant FIX production.

    Science.gov (United States)

    de Sousa Bomfim, Aline; Cristina Corrêa de Freitas, Marcela; Picanço-Castro, Virgínia; de Abreu Soares Neto, Mário; Swiech, Kamilla; Tadeu Covas, Dimas; Maria de Sousa Russo, Elisa

    2016-05-01

    Factor IX (FIX) is a vitamin K-dependent protein, and it has become a valuable pharmaceutical in the Hemophilia B treatment. We evaluated the potential of recombinant human FIX (rhFIX) expression in 293T and SK-Hep-1 human cell lines. SK-Hep-1-FIX cells produced higher levels of biologically active protein. The growth profile of 293T-FIX cells was not influenced by lentiviral integration number into the cellular genome. SK-Hep-1-FIX cells showed a significantly lower growth rate than SK-Hep-1 cells. γ-carboxylation process is significant to FIX biological activity, thus we performed a expression analysis of genes involved in this process. The 293T gene expression suggests that this cell line could efficiently carboxylate FIX, however only 28% of the total secreted protein is active. SK-Hep-1 cells did not express high amounts of VKORC1 and carboxylase, but this cell line secreted large amounts of active protein. Enrichment of culture medium with Ca(+2) and Mg(+2) ions did not affect positively rhFIX expression in SK-Hep-1 cells. In 293T cells, the addition of 0.5 mM Ca(+2) and 1 mM Mg(+2) resulted in higher rhFIX concentration. SK-Hep-1 cell line proved to be very effective in rhFIX production, and it can be used as a novel biotechnological platform for the production of recombinant proteins. PMID:26802680

  8. Pre-conditioning induces the precocious differentiation of neonatal astrocytes to enhance their neuroprotective properties

    Directory of Open Access Journals (Sweden)

    Sandra J Hewett

    2011-07-01

    Full Text Available Hypoxic preconditioning reprogrammes the brain's response to subsequent H/I (hypoxia–ischaemia injury by enhancing neuroprotective mechanisms. Given that astrocytes normally support neuronal survival and function, the purpose of the present study was to test the hypothesis that a hypoxic preconditioning stimulus would activate an adaptive astrocytic response. We analysed several functional parameters 24 h after exposing rat pups to 3 h of systemic hypoxia (8% O2. Hypoxia increased neocortical astrocyte maturation as evidenced by the loss of GFAP (glial fibrillary acidic protein-positive cells with radial morphologies and the acquisition of multipolar GFAP-positive cells. Interestingly, many of these astrocytes had nuclear S100B. Accompanying their differentiation, there was increased expression of GFAP, GS (glutamine synthetase, EAAT-1 (excitatory amino acid transporter-1; also known as GLAST, MCT-1 (monocarboxylate transporter-1 and ceruloplasmin. A subsequent H/I insult did not result in any further astrocyte activation. Some responses were cell autonomous, as levels of GS and MCT-1 increased subsequent to hypoxia in cultured forebrain astrocytes. In contrast, the expression of GFAP, GLAST and ceruloplasmin remained unaltered. Additional experiments utilized astrocytes exposed to exogenous dbcAMP (dibutyryl-cAMP, which mimicked several aspects of the preconditioning response, to determine whether activated astrocytes could protect neurons from subsequent excitotoxic injury. dbcAMP treatment increased GS and glutamate transporter expression and function, and as hypothesized, protected neurons from glutamate excitotoxicity. Taken altogether, these results indicate that a preconditioning stimulus causes the precocious differentiation of astrocytes and increases the acquisition of multiple astrocytic functions that will contribute to the neuroprotection conferred by a sublethal preconditioning stress.

  9. Analysis of LINE-1 expression in human pluripotent cells.

    Science.gov (United States)

    Muñoz-Lopez, Martin; Garcia-Cañadas, Marta; Macia, Angela; Morell, Santiago; Garcia-Perez, Jose L

    2012-01-01

    Half of the human genome is composed of repeated DNA, and some types are mobile within our genome (transposons and retrotransposons). Despite their abundance, only a small fraction of them are currently active in our genome (Long Interspersed Element-1 (LINE-1), Alu, and SVA elements). LINE-1 or L1 elements are a family of active non-LTR retrotransposons, the ongoing mobilization of which still impacts our genome. As selfish DNA elements, L1 activity is more prominent in early human development, where new insertions would be transmitted to the progeny. Here, we describe the conventional methods aimed to determine the expression level of LINE-1 elements in pluripotent human cells.

  10. Comparison of repair capability of four human tumor cell lines

    International Nuclear Information System (INIS)

    A fast and reliable method for assessment of repair capacity of cells based on the restoration of transcription of the gene for the green fluorescent protein carried by a plasmid vector is presented. Repair capacity of the cells counted under fluorescent microscope 24 hours following transcription. This approach has been applied to compare the rates of repair of different types of DNA lesions in four human tumor cell lines. The analysis of the obtained results show that there are considerable differences in the repair capacity of the different cell lines towards the different types of lesions. It should be noted that the difference in repair capacity of the host cells are better evident when plasmids with lower rather that higher number of lesions per unit length of plasmid DNA are used. This is probably because the egfp genes with fewer lesions can be fully repaired while egfp genes with more lesions remain inactive even after some of the lesions have been repaired

  11. Establishment of cell suspension line of Populus tomentosa Carr

    Institute of Scientific and Technical Information of China (English)

    YAO Na; ZHANG Zhi-yi; AN Xin-min; YANG Kai

    2008-01-01

    Leaves of fine Populus tomentosa genotype TC152 were used as explants to establish cell suspension lines. The effects of plant growth regulators on callus induction and establishment of cell suspension lines were studied. The callus induction rate was the highest on a MS solid medium supplemented with 1.0 mg·L-1 2,4-D. A cell suspension line could be obtained by inoculating calli which were not subeultured into a MS liquid medium supplemented with 1.5 mg·L-1 2,4-D. The best subculture medium was MS+ 0.8 mg·L-1 2,4-D + 30 g·L-1 sucrose with a subculture cycle of seven days.

  12. Dipeptidyl peptidase IV in two human glioma cell lines

    Directory of Open Access Journals (Sweden)

    A Sedo

    2009-12-01

    Full Text Available There is growing evidence that dipeptidyl peptidase IV [DPP-IV, EC 3.4.14.5] takes part in the metabolism of biologically active peptides participating in the regulation of growth and transformation of glial cells. However, the knowledge on the DPP-IV expression in human glial and glioma cells is still very limited. In this study, using histochemical and biochemical techniques, the DPP-IV activity was demonstrated in two commercially available human glioma cell lines of different transformation degree, as represented by U373 astrocytoma (Grade III and U87 glioblastoma multiforme (Grade IV lines. Higher total activity of the enzyme, as well as its preferential localisation in the plasma membrane, was observed in U87 cells. Compared to U373 population, U87 cells were morphologically more pleiomorphic, they were cycling at lower rate and expressing less Glial Fibrillary Acidic Protein. The data revealed positive correlation between the degree of transformation of cells and activity of DPP-IV. Great difference in expression of this enzyme, together with the phenotypic differences of cells, makes these lines a suitable standard model for further 57 studies of function of this enzyme in human glioma cells.

  13. Haptoglobin increases with age in rat hippocampus and modulates Apolipoprotein E mediated cholesterol trafficking in neuroblastoma cell lines

    Directory of Open Access Journals (Sweden)

    Maria Stefania eSpagnuolo

    2014-08-01

    Full Text Available Alteration in cholesterol metabolism has been implicated in the pathogenesis of several neurodegenerative disorders. Apolipoprotein E (ApoE is the major component of brain lipoproteins supporting cholesterol transport. We previously reported that the acute-phase protein Haptoglobin (Hpt binds ApoE, and influences its function in blood cholesterol homeostasis. Major aim of this study was to investigate whether Hpt influences the mechanisms by which cholesterol is shuttled from astrocytes to neurons. In detail it was studied Hpt effect on ApoE-dependent cholesterol efflux from astrocytes and ApoE-mediated cholesterol incorporation in neurons. We report here that Hpt impairs ApoE-mediated cholesterol uptake in human neuroblastoma cell line SH-SY5Y, and limits the toxicity of a massive concentration of cholesterol for these cells, while it does not affect cholesterol efflux from the human glioblastoma-astrocytoma cell line U-87 MG. As aging is the most important nongenetic risk factor for various neurodegenerative disorders, and our results suggest that Hpt modulates ApoE functions, we evaluated the Hpt and ApoE expression profiles in cerebral cortex and hippocampus of adolescent (2 months, adult (5 and 8 months, and middle-aged (16 months rats. Hpt mRNA level was higher in hippocampus of 8 and 16 month-old than in 2-month old rats (p<0.05, and Hpt concentration increased with the age from adolescence to middle-age (p<0.001. ApoE concentration, in hippocampus, was higher (p<0.001 in 5 month-old rats compared to 2 month but did not further change with aging. No age-related changes of Hpt (protein and mRNA were found in the cortex. Our results suggest that aging is associated with changes, particularly in the hippocampus, in the Hpt/ApoE ratio. Age-related changes in the concentration of Hpt were also found in human cerebrospinal fluids.The age-related changes might affect neuronal function and survival in brain, and have important implications in

  14. Antiproliferative effect of Tualang honey on oral squamous cell carcinoma and osteosarcoma cell lines

    Directory of Open Access Journals (Sweden)

    Ismail Noorliza M

    2010-09-01

    Full Text Available Abstract Background The treatment of oral squamous cell carcinomas (OSCC and human osteosarcoma (HOS includes surgery and/or radiotherapy which often lead to reduced quality of life. This study was aimed to study the antiproliferative activity of local honey (Tualang on OSCC and HOS cell lines. Methods Several concentrations of Tualang honey (1% - 20% were applied on OSCC and HOS cell lines for 3, 6, 12, 24, 48 and 72 hours. Morphological characteristics were observed under light and fluorescent microscope. Cell viability was assessed using MTT assay and the optical density for absorbance values in each experiment was measured at 570 nm by an ELISA reader. Detection of cellular apoptosis was done using the Annexin V-FITC Apoptosis Detection Kit. Results Morphological appearance showed apoptotic cellular changes like becoming rounded, reduction in cell number, blebbed membrane and apoptotic nuclear changes like nuclear shrinkage, chromatin condensation and fragmented nucleus on OSCC and HOS cell lines. Cell viability assay showed a time and dose-dependent inhibitory effect of honey on both cell lines. The 50% inhibitory concentration (IC50 for OSCC and HOS cell lines was found to be 4% and 3.5% respectively. The maximum inhibition of cell growth of ≥80% was obtained at 15% for both cell lines. Early apoptosis was evident by flow cytometry where percentage of early apoptotic cells increased in dose and time dependent manner. Conclusion Tualang honey showed antiproliferative effect on OSCC and HOS cell lines by inducing early apoptosis.

  15. Simultaneous measurement of natural killer cell cytotoxicity against each of three different target cell lines.

    Science.gov (United States)

    Blomberg, K

    1994-02-10

    A time-resolved fluorometric assay for the simultaneous measurement of natural killer cell activity against three different lanthanide diethylenetriaminopentaacetate (LaDTPA) labelled target cell lines is described. The target cell line K-562 was labelled with SmDTPA, the cell line Molt with TbDTPA and the cell line Raji with EuDTPA. After co-incubation of the three target cell lines with effector cells the fluorescence of the lanthanides released from the lysed target cells was measured in an enhancer solution in which they formed highly fluorescent complexes. It was possible to differentiate the specific release from the three target cell lines because the emission lines of the europium, samarium and terbium complexes formed in the enhancer solution are well separated from each other. The autofluorescence from culture media supplemented with serum was avoided by the use of time-resolved fluorometry. The results show that applying fluorometry based on the combination of spectral and temporal resolution to natural killer cell assays, makes possible the simultaneous determination of lysis in up to three target cell lines in complex culture medium. PMID:8308301

  16. Oral bioavailability of glyphosate: studies using two intestinal cell lines.

    Science.gov (United States)

    Vasiluk, Luba; Pinto, Linda J; Moore, Margo M

    2005-01-01

    Glyphosate is a commonly used nonselective herbicide that inhibits plant growth through interference with the production of essential aromatic amino acids. In vivo studies in mammals with radiolabeled glyphosate have shown that 34% of radioactivity was associated with intestinal tissue 2 h after oral administration. The aim of our research was to investigate the transport, binding, and toxicity of glyphosate to the cultured human intestinal epithelial cell line, Caco-2, and the rat small intestinal crypt-derived cell line, ileum epithelial cells-18 (IEC-18). An in vitro analysis of the transport kinetics of [14C]-glyphosate showed that 4 h after exposure, approximately 8% of radiolabeled glyphosate moved through the Caco-2 monolayer in a dose-dependent manner. Binding of glyphosate to cells was saturable and approximately 4 x 10(11) binding sites/cell were estimated from bound [14C]. Exposure of Caco-2 cells to > or =10 mg/ml glyphosate reduced transmembrane electrical resistance (TEER) by 82 to 96% and increased permeability to [3H]-mannitol, indicating that paracellular permeability increased in glyphosate-treated cells. At 10-mg/ml glyphosate, both IEC-18 and Caco-2 cells showed disruption in the actin cytoskeleton. In Caco-2 cells, significant lactate dehydrogenase leakage was observed when cells were exposed to 15 mg/ml of glyphosate. These data indicate that at doses >10 mg/ml, glyphosate significantly disrupts the barrier properties of cultured intestinal cells.

  17. Derivation of a Homozygous Human Androgenetic Embryonic Stem Cell Line.

    Science.gov (United States)

    Ding, Chenhui; Huang, Sunxing; Qi, Quan; Fu, Rui; Zhu, Wanwan; Cai, Bing; Hong, Pingping; Liu, Zhengxin; Gu, Tiantian; Zeng, Yanhong; Wang, Jing; Xu, Yanwen; Zhao, Xiaoyang; Zhou, Qi; Zhou, Canquan

    2015-10-01

    Human embryonic stem cells (hESCs) have long been considered as a promising source for cell replacement therapy. However, one major obstacle for the use of these cells is immune compatibility. Histocompatible human parthenogenetic ESCs have been reported as a new method for generating human leukocyte antigen (HLA)-matched hESCs. To further investigate the possibility of obtaining histocompatible stem cells from uniparental embryos, we tried to produce androgenetic haploid human embryos by injecting a single spermatozoon into enucleated human oocyte, and establish human androgenetic embryonic stem (hAGES) cell lines from androgenetic embryos. In the present study, a diploid hAGES cell line has been established, which exhibits typical features of human ESCs, including the expression of pluripotency markers, having differentiation potential in vitro and in vivo, and stable propagation in an undifferentiated state (>P40). Bisulfite sequencing of the H19, Snrpn, Meg3, and Kv imprinting control regions suggested that hAGES cells maintained to a certain extent a sperm methylation pattern. Genome-wide single nucleotide polymorphism, short tandem repeat, and HLA analyses revealed that the hAGES cell genome was highly homozygous. These results suggest that hAGES cells from spermatozoon could serve as a useful tool for studying the mechanisms underlying genomic imprinting in humans. It might also be used as a potential resource for cell replacement therapy as parthenogenetic stem cells.

  18. Characterization of cloned cells from an immortalized fetal pulmonary type II cell line

    Energy Technology Data Exchange (ETDEWEB)

    Henderson, R.F.; Waide, J.J.; Lechner, J.F.

    1995-12-01

    A cultured cell line that maintained expression of pulmonary type II cell markers of differentiation would be advantageous to generate a large number of homogenous cells in which to study the biochemical functions of type II cells. Type II epithelial cells are the source of pulmonary surfactant and a cell of origin for pulmonary adenomas. Last year our laboratory reported the induction of expression of two phenotypic markers of pulmonary type II cells (alkaline phosphatase activity and surfactant lipid synthesis) in cultured fetal rat lung epithelial (FRLE) cells, a spontaneously immortalized cell line of fetal rat lung type II cell origin. Subsequently, the induction of the ability to synthesize surfactant lipid became difficult to repeat. We hypothesized that the cell line was heterogenuous and some cells were more like type II cells than others. The purpose of this study was to test this hypothesis and to obtain a cultured cell line with type II cell phenotypic markers by cloning several FRLE cells and characterizing them for phenotypic markers of type II cells (alkaline phosphatase activity and presence of surfactant lipids). Thirty cloned cell lines were analyzed for induced alkaline phosphatase activity (on x-axis) and for percent of phospholipids that were disaturated (i.e., surfactant).

  19. Alloreactive cloned T cell lines. I. Interactions between cloned amplifier and cytolytic T cell lines

    OpenAIRE

    1980-01-01

    Several T cell clones have been derived by limiting dilution of secondary mixed leukocyte culture cells stimulated by H-2- and M locus (Mls)-disparate spleen cells. When examined for the expression of cytolytic activity and the ability to proliferate, these cell clones can be classified into two major categories. One type of cell is noncytolytic; when cultured with irradiated spleen cells, such clones proliferate in response to Mls determinants. Some, but not all, of these clones express Lyt-...

  20. Direct Conversion of Fibroblasts into Functional Astrocytes by Defined Transcription Factors

    OpenAIRE

    Massimiliano Caiazzo; Serena Giannelli; Pierluigi Valente; Gabriele Lignani; Annamaria Carissimo; Alessandro Sessa; Gaia Colasante; Rosa Bartolomeo; Luca Massimino; Stefano Ferroni; Carmine Settembre; Fabio Benfenati; Vania Broccoli

    2014-01-01

    Summary Direct cell reprogramming enables direct conversion of fibroblasts into functional neurons and oligodendrocytes using a minimal set of cell-lineage-specific transcription factors. This approach is rapid and simple, generating the cell types of interest in one step. However, it remains unknown whether this technology can be applied to convert fibroblasts into astrocytes, the third neural lineage. Astrocytes play crucial roles in neuronal homeostasis, and their dysfunctions contribute t...

  1. GSK-3β-induced Tau pathology drives hippocampal neuronal cell death in Huntington's disease: involvement of astrocyte-neuron interactions.

    Science.gov (United States)

    L'Episcopo, F; Drouin-Ouellet, J; Tirolo, C; Pulvirenti, A; Giugno, R; Testa, N; Caniglia, S; Serapide, M F; Cisbani, G; Barker, R A; Cicchetti, F; Marchetti, B

    2016-01-01

    Glycogen synthase kinase-3β (GSK-3β) has emerged as a critical factor in several pathways involved in hippocampal neuronal maintenance and function. In Huntington's disease (HD), there are early hippocampal deficits both in patients and transgenic mouse models, which prompted us to investigate whether disease-specific changes in GSK-3β expression may underlie these abnormalities. Thirty-three postmortem hippocampal samples from HD patients (neuropathological grades 2-4) and age- and sex-matched normal control cases were analyzed using real-time quantitative reverse transcription PCRs (qPCRs) and immunohistochemistry. In vitro and in vivo studies looking at hippocampal pathology and GSK-3β were also undertaken in transgenic R6/2 and wild-type mice. We identified a disease and stage-dependent upregulation of GSK-3β mRNA and protein levels in the HD hippocampus, with the active isoform pGSK-3β-Tyr(216) being strongly expressed in dentate gyrus (DG) neurons and astrocytes at a time when phosphorylation of Tau at the AT8 epitope was also present in these same neurons. This upregulation of pGSK-3β-Tyr(216) was also found in the R6/2 hippocampus in vivo and linked to the increased vulnerability of primary hippocampal neurons in vitro. In addition, the increased expression of GSK-3β in the astrocytes of R6/2 mice appeared to be the main driver of Tau phosphorylation and caspase3 activation-induced neuronal death, at least in part via an exacerbated production of major proinflammatory mediators. This stage-dependent overactivation of GSK-3β in HD-affected hippocampal neurons and astrocytes therefore points to GSK-3β as being a critical factor in the pathological development of this condition. As such, therapeutic targeting of this pathway may help ameliorate neuronal dysfunction in HD. PMID:27124580

  2. LINEing germ and embryonic stem cells' silencing of retrotransposons.

    Science.gov (United States)

    Ishiuchi, Takashi; Torres-Padilla, Maria-Elena

    2014-07-01

    Almost half of our genome is occupied by transposable elements. Although most of them are inactive, one type of non-long terminal repeat (LTR) retrotransposon, long interspersed nuclear element 1 (LINE1), is capable of retrotransposition. Two studies in this issue, Pezic and colleagues (pp. 1410-1428) and Castro-Diaz and colleagues (pp. 1397-1409), provide novel insight into the regulation of LINE1s in human embryonic stem cells and mouse germ cells and shed new light on the conservation of complex mechanisms to ensure silencing of transposable elements in mammals.

  3. Building bridges with astrocytes for spinal cord repair

    OpenAIRE

    Miller, Robert H.

    2006-01-01

    Simultaneous suppression of glial scarring and a general enhancement of axonal outgrowth has now been accomplished in an adult rat model of spinal cord transection. Transplantation of a novel astrocyte cell type derived from glial-restricted precursors in vitro raise the eventual possibility of cellular therapy for spinal cord injury.

  4. Protective Effects of Gastrodin Against Autophagy-Mediated Astrocyte Death.

    Science.gov (United States)

    Wang, Xin-shang; Tian, Zhen; Zhang, Nan; Han, Jing; Guo, Hong-liang; Zhao, Ming-gao; Liu, Shui-bing

    2016-03-01

    Gastrodin is an active ingredient derived from the rhizome of Gastrodia elata. This compound is usually used to treat convulsive illness, dizziness, vertigo, and headache. This study aimed to investigate the effect of gastrodin on the autophagy of glial cells exposed to lipopolysaccharides (LPS, 1 µg/mL). Autophagy is a form of programmed cell death, although it also promotes cell survival. In cultured astrocytes, LPS exposure induced excessive autophagy and apoptosis, which were significantly prevented by the pretreatment cells with gastrodin (10 μM). The protective effects of gastrodin via autophagy inhibition were verified by the decreased levels of LC3-II, P62, and Beclin-1, which are classical markers for autophagy. Furthermore, gastrodin protected astrocytes from apoptosis through Bcl-2 and Bax signaling pathway. The treatment of astrocytes with rapamycin (500 nM), wortmannin (100 nM), and LY294002 (10 μM), which are inhibitors of mTOR and PI3K, respectively, eliminated the known effects of gastrodin on the inhibited Beclin-1 expression. Furthermore, gastrodin blocked the down-regulation of glutamine synthetase induced by LPS exposure in astrocytes. Our results suggest that gastrodin can be used as a preventive agent for the excessive autophagy induced by LPS. PMID:26643508

  5. Megalencephalic leukoencephalopathy with subcortical cysts protein-1 regulates epidermal growth factor receptor signaling in astrocytes.

    Science.gov (United States)

    Lanciotti, Angela; Brignone, Maria Stefania; Visentin, Sergio; De Nuccio, Chiara; Catacuzzeno, Luigi; Mallozzi, Cinzia; Petrini, Stefania; Caramia, Martino; Veroni, Caterina; Minnone, Gaetana; Bernardo, Antonietta; Franciolini, Fabio; Pessia, Mauro; Bertini, Enrico; Petrucci, Tamara Corinna; Ambrosini, Elena

    2016-04-15

    Mutations in the MLC1 gene, which encodes a protein expressed in brain astrocytes, are the leading cause of MLC, a rare leukodystrophy characterized by macrocephaly, brain edema, subcortical cysts, myelin and astrocyte vacuolation. Although recent studies indicate that MLC1 protein is implicated in the regulation of cell volume changes, the exact role of MLC1 in brain physiology and in the pathogenesis of MLC disease remains to be clarified. In preliminary experiments, we observed that MLC1 was poorly expressed in highly proliferating astrocytoma cells when compared with primary astrocytes, and that modulation of MLC1 expression influenced astrocyte growth. Because volume changes are key events in cell proliferation and during brain development MLC1 expression is inversely correlated to astrocyte progenitor proliferation levels, we investigated the possible role for MLC1 in the control of astrocyte proliferation. We found that overexpression of wild type but not mutant MLC1 in human astrocytoma cells hampered cell growth by favoring epidermal growth factor receptor (EGFR) degradation and by inhibiting EGF-induced Ca(+) entry, ERK1/2 and PLCγ1 activation, and calcium-activated KCa3.1 potassium channel function, all molecular pathways involved in astrocyte proliferation stimulation. Interestingly, MLC1 did not influence AKT, an EGFR-stimulated kinase involved in cell survival. Moreover, EGFR expression was higher in macrophages derived from MLC patients than from healthy individuals. Since reactive astrocytes proliferate and re-express EGFR in response to different pathological stimuli, the present findings provide new information on MLC pathogenesis and unravel an important role for MLC1 in other brain pathological conditions where astrocyte activation occurs. PMID:26908604

  6. Establishment of a luciferase assay-based screening system: Fumitremorgin C selectively inhibits cellular proliferation of immortalized astrocytes expressing an active form of AKT

    International Nuclear Information System (INIS)

    The AKT pathway is frequently activated in glioblastoma, and as such, inhibitors of this pathway could prove very useful as anti-glioblastoma therapies. Here we established immortalized astrocytes expressing Renilla luciferase as well as those expressing both an active form of AKT and firefly luciferase. Since both luciferase activities represent the numbers of corresponding cell lines, novel inhibitors of the AKT pathway can be identified by treating co-cultures containing the two types of luciferase-expressing cells with individual compounds. Indeed, such a screening system succeeded in identifying fumitremorgin C as an efficient inhibitor of the AKT pathway, which was further confirmed by the ability of fumitremorgin C to selectively inhibit the growth of immortalized astrocytes expressing an active form of AKT. The present study proposes a broadly applicable approach for identifying therapeutic agents that target the pathways and/or molecules responsible for cancer development

  7. Neuron to astrocyte communication via cannabinoid receptors is necessary for sustained epileptiform activity in rat hippocampus.

    Directory of Open Access Journals (Sweden)

    Guyllaume Coiret

    Full Text Available Astrocytes are integral functional components of synapses, regulating transmission and plasticity. They have also been implicated in the pathogenesis of epilepsy, although their precise roles have not been comprehensively characterized. Astrocytes integrate activity from neighboring synapses by responding to neuronally released neurotransmitters such as glutamate and ATP. Strong activation of astrocytes mediated by these neurotransmitters can promote seizure-like activity by initiating a positive feedback loop that induces excessive neuronal discharge. Recent work has demonstrated that astrocytes express cannabinoid 1 (CB1 receptors, which are sensitive to endocannabinoids released by nearby pyramidal cells. In this study, we tested whether this mechanism also contributes to epileptiform activity. In a model of 4-aminopyridine induced epileptic-like activity in hippocampal slice cultures, we show that pharmacological blockade of astrocyte CB1 receptors did not modify the initiation, but significantly reduced the maintenance of epileptiform discharge. When communication in astrocytic networks was disrupted by chelating astrocytic calcium, this CB1 receptor-mediated modulation of epileptiform activity was no longer observed. Thus, endocannabinoid signaling from neurons to astrocytes represents an additional significant factor in the maintenance of epileptiform activity in the hippocampus.

  8. In Vivo Evidence for a Lactate Gradient from Astrocytes to Neurons

    KAUST Repository

    Mächler, Philipp

    2015-11-19

    Investigating lactate dynamics in brain tissue is challenging, partly because in vivo data at cellular resolution are not available. We monitored lactate in cortical astrocytes and neurons of mice using the genetically encoded FRET sensor Laconic in combination with two-photon microscopy. An intravenous lactate injection rapidly increased the Laconic signal in both astrocytes and neurons, demonstrating high lactate permeability across tissue. The signal increase was significantly smaller in astrocytes, pointing to higher basal lactate levels in these cells, confirmed by a one-point calibration protocol. Trans-acceleration of the monocarboxylate transporter with pyruvate was able to reduce intracellular lactate in astrocytes but not in neurons. Collectively, these data provide in vivo evidence for a lactate gradient from astrocytes to neurons. This gradient is a prerequisite for a carrier-mediated lactate flux from astrocytes to neurons and thus supports the astrocyte-neuron lactate shuttle model, in which astrocyte-derived lactate acts as an energy substrate for neurons. © 2016 Elsevier Inc.

  9. Exercise Counteracts Aging-Related Memory Impairment: A Potential Role for the Astrocytic Metabolic Shuttle.

    Science.gov (United States)

    Tsai, Sheng-Feng; Chen, Pei-Chun; Calkins, Marcus J; Wu, Shih-Ying; Kuo, Yu-Min

    2016-01-01

    Age-related cognitive impairment has become one of the most common health threats in many countries. The biological substrate of cognition is the interconnection of neurons to form complex information processing networks. Experience-based alterations in the activities of these information processing networks lead to neuroadaptation, which is physically represented at the cellular level as synaptic plasticity. Although synaptic plasticity is known to be affected by aging, the underlying molecular mechanisms are not well described. Astrocytes, a glial cell type that is infrequently investigated in cognitive science, have emerged as energy suppliers which are necessary for meeting the abundant energy demand resulting from glutamatergic synaptic activity. Moreover, the concerted action of an astrocyte-neuron metabolic shuttle is essential for cognitive function; whereas, energetic incoordination between astrocytes and neurons may contribute to cognitive impairment. Whether altered function of the astrocyte-neuron metabolic shuttle links aging to reduced synaptic plasticity is unexplored. However, accumulated evidence documents significant beneficial effects of long-term, regular exercise on cognition and synaptic plasticity. Furthermore, exercise increases the effectiveness of astrocyte-neuron metabolic shuttle by upregulation of astrocytic lactate transporter levels. This review summarizes previous findings related to the neuronal activity-dependent astrocyte-neuron metabolic shuttle. Moreover, we discuss how aging and exercise may shape the astrocyte-neuron metabolic shuttle in cognition-associated brain areas. PMID:27047373

  10. CCL2 modulates cytokine production in cultured mouse astrocytes

    Directory of Open Access Journals (Sweden)

    Frugier Tony

    2010-10-01

    Full Text Available Abstract Background The chemokine CCL2 (also known as monocyte chemoattractant protein-1, or MCP-1 is upregulated in patients and rodent models of traumatic brain injury (TBI, contributing to post-traumatic neuroinflammation and degeneration by directing the infiltration of blood-derived macrophages into the injured brain. Our laboratory has previously reported that Ccl2-/- mice show reduced macrophage accumulation and tissue damage, corresponding to improved motor recovery, following experimental TBI. Surprisingly, Ccl2-deficient mice also exhibited delayed but exacerbated secretion of key proinflammatory cytokines in the injured cortex. Thus we sought to further characterise CCL2's potential ability to modulate immunoactivation of astrocytes in vitro. Methods Primary astrocytes were isolated from neonatal wild-type and Ccl2-deficient mice. Established astrocyte cultures were stimulated with various concentrations of lipopolysaccharide (LPS and interleukin (IL-1β for up to 24 hours. Separate experiments involved pre-incubation with mouse recombinant (rCCL2 prior to IL-1β stimulation in wild-type cells. Following stimulation, cytokine secretion was measured in culture supernatant by immunoassays, whilst cytokine gene expression was quantified by real-time reverse transcriptase polymerase chain reaction. Results LPS (0.1-100 μg/ml; 8 h induced the significantly greater secretion of five key cytokines and chemokines in Ccl2-/- astrocytes compared to wild-type cells. Consistently, IL-6 mRNA levels were 2-fold higher in Ccl2-deficient cells. IL-1β (10 and 50 ng/ml; 2-24 h also resulted in exacerbated IL-6 production from Ccl2-/- cultures. Despite this, treatment of wild-type cultures with rCCL2 alone (50-500 ng/ml did not induce cytokine/chemokine production by astrocytes. However, pre-incubation of wild-type astrocytes with rCCL2 (250 ng/ml, 12 h prior to stimulation with IL-1β (10 ng/ml, 8 h significantly reduced IL-6 protein and gene

  11. Cellular and Phenotypic Characterization of Canine Osteosarcoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Marie E. Legare, Jamie Bush, Amanda K. Ashley, Taka Kato, William H. Hanneman

    2011-01-01

    Full Text Available Canine and human osteosarcoma (OSA have many similarities, with the majority of reported cases occurring in the appendicular skeleton, gender predominance noted, high rate of metastasis at the time of presentation, and a lack of known etiology for this devastating disease. Due to poor understanding of the molecular mechanisms underlying OSA, we have characterized seven different OSA canine cell lines: Abrams, D17, Grey, Hughes, Ingles, Jarques, and Marisco and compared them to U2, a human OSA cell line, for the following parameters: morphology, growth, contact inhibition, migrational tendencies, alkaline phosphatase staining, heterologous tumor growth, double-strand DNA breaks, and oxidative damage. All results demonstrated the positive characteristics of the Abrams cell line for use in future studies of OSA. Of particular interest, the robust growth of a subcutaneous tumor and rapid pulmonary metastasis of the Abrams cell line in an immunocompromised mouse shows incredible potential for the future use of Abrams as a canine OSA model. Further investigations utilizing a canine cell model of OSA, such as Abrams, will be invaluable to understanding the molecular events underlying OSA, pharmaceutical inhibition of metastasis, and eventual prevention of this devastating disease.

  12. Reactive protoplasmic and fibrous astrocytes contain high levels of calpain-cleaved alpha 2 spectrin.

    Science.gov (United States)

    Kim, Jung H; Kwon, Soojung J; Stankewich, Michael C; Huh, Gi-Yeong; Glantz, Susan B; Morrow, Jon S

    2016-02-01

    Calpain, a family of calcium-dependent neutral proteases, plays important roles in neurophysiology and pathology through the proteolytic modification of cytoskeletal proteins, receptors and kinases. Alpha 2 spectrin (αII spectrin) is a major substrate for this protease family, and the presence of the αII spectrin breakdown product (αΙΙ spectrin BDP) in a cell is evidence of calpain activity triggered by enhanced intracytoplasmic Ca(2+) concentrations. Astrocytes, the most dynamic CNS cells, respond to micro-environmental changes or noxious stimuli by elevating intracytoplasmic Ca(2+) concentration to become activated. As one measure of whether calpains are involved with reactive glial transformation, we examined paraffin sections of the human cerebral cortex and white matter by immunohistochemistry with an antibody specific for the calpain-mediated αΙΙ spectrin BDP. We also performed conventional double immunohistochemistry as well as immunofluorescent studies utilizing antibodies against αΙΙ spectrin BDP as well as glial fibrillary acidic protein (GFAP). We found strong immunopositivity in selected protoplasmic and fibrous astrocytes, and in transitional forms that raise the possibility of some of fibrous astrocytes emerging from protoplasmic astrocytes. Immunoreactive astrocytes were numerous in brain sections from cases with severe cardiac and/or respiratory diseases in the current study as opposed to our previous study of cases without significant clinical conditions that failed to reveal such remarkable immunohistochemical alterations. Our study suggests that astrocytes become αΙΙ spectrin BDP immunopositive in various stages of activation, and that spectrin cleavage product persists even in fully reactive astrocytes. Immunohistochemistry for αΙΙ spectrin BDP thus marks reactive astrocytes, and highlights the likelihood that calpains and their proteolytic processing of spectrin participate in the morphologic and physiologic transition from

  13. Human astrocytic grid networks patterned in parylene-C inlayed SiO2 trenches.

    Science.gov (United States)

    Jordan, M D; Raos, B J; Bunting, A S; Murray, A F; Graham, E S; Unsworth, C P

    2016-10-01

    Recent literature suggests that glia, and in particular astrocytes, should be studied as organised networks which communicate through gap junctions. Astrocytes, however, adhere to most surfaces and are highly mobile cells. In order to study, such organised networks effectively in vitro it is necessary to influence them to pattern to certain substrates whilst being repelled from others and to immobilise the astrocytes sufficiently such that they do not continue to migrate further whilst under study. In this article, we demonstrate for the first time how it is possible to facilitate the study of organised patterned human astrocytic networks using hNT astrocytes in a SiO2 trench grid network that is inlayed with the biocompatible material, parylene-C. We demonstrate how the immobilisation of astrocytes lies in the depth of the SiO2 trench, determining an optimum trench depth and that the optimum patterning of astrocytes is a consequence of the parylene-C inlay and the grid node spacing. We demonstrate high fidelity of the astrocytic networks and demonstrate that functionality of the hNT astrocytes through ATP evoked calcium signalling is also dependent on the grid node spacing. Finally, we demonstrate that the location of the nuclei on the grid nodes is also a function of the grid node spacing. The significance of this work, is to describe a suitable platform to facilitate the study of hNT astrocytes from the single cell level to the network level to improve knowledge and understanding of how communication links to spatial organisation at these higher order scales and trigger in vitro research further in this area with clinical applications in the area of epilepsy, stroke and focal cerebral ischemia. PMID:27521614

  14. Osmotic stress affects functional properties of human melanoma cell lines

    CERN Document Server

    La Porta, Caterina A M; Pasini, Maria; Laurson, Lasse; Alava, Mikko J; Zapperi, Stefano; Amar, Martine Ben

    2015-01-01

    Understanding the role of microenvironment in cancer growth and metastasis is a key issue for cancer research. Here, we study the effect of osmotic pressure on the functional properties of primary and metastatic melanoma cell lines. In particular, we experimentally quantify individual cell motility and transmigration capability. We then perform a circular scratch assay to study how a cancer cell front invades an empty space. Our results show that primary melanoma cells are sensitive to a low osmotic pressure, while metastatic cells are less. To better understand the experimental results, we introduce and study a continuous model for the dynamics of a cell layer and a stochastic discrete model for cell proliferation and diffusion. The two models capture essential features of the experimental results and allow to make predictions for a wide range of experimentally measurable parameters.

  15. Cell membrane fatty acid composition differs between normal and malignant cell lines.

    Science.gov (United States)

    Meng, Xialong; Riordan, Neil H; Riordan, Hugh D; Mikirova, Nina; Jackson, James; González, Michael J; Miranda-Massari, Jorge R; Mora, Edna; Trinidad Castillo, Waleska

    2004-06-01

    Twenty-eight fatty acids (C8:0 to C24:l n-9) were measured by gas chromatography in four normal cell lines (C3H / 10T1 / 2, CCD-18Co, CCD-25SK and CCD-37Lu) and seven cancer cell lines (C-41, Caov-3, LS-180, PC-3, SK-MEL-28, SK-MES-1 and U-87 MG). Results show differences in the content and proportions of fatty acids when comparing cancer cell lines with their normal counterparts. Cancer cell lines showed lower C20: 4 n-6, C24:1 n-9, polyunsaturated fatty acids (PUFA's) and ratios of C20:4 n-6 to C20:5 n-3 and C16:0 to C18:1 n-9 and stearic to oleic (SA/OA) than their normal counterparts. All cancer cell lines had SA/OA ratios lower than 7.0 while normal cell lines had ratios greater than 0.7 (p<0.05). In addition, the ratios of total saturated fatty acids (SFA) to PUFA'S and the concentration of C18:1 n-9, C18:2 n-6, C20:5 n-3 were higher in cancer cell lines as compared to normal cell lines. A positive correlation was detected between C16:0 and longer SFA'S (r = +0.511, p<0.05) in normal cell lines whereas a negative correlation (r=0.608, p<0.05) was obtained for malignant cell lines. Moreover, cancerous cell lines exhibited a particular desaturation defect and an abnormal incorporation of C18:2 n-6 and C20-4 n-6 fatty acids. PMID:15377057

  16. UOK 268 Cell Line for Hereditary Leiomyomatosis and Renal Cell Carcinoma | NCI Technology Transfer Center | TTC

    Science.gov (United States)

    The National Cancer Institute’s Urologic Oncology Branch seeks parties to co-develop the UOK 262 immortalized cell line as research tool to study aggressive hereditary leiomyomatosis and renal cell carcinoma (HLRCC)-associated recurring kidney cancer.

  17. Adhesion of Actinobacillus actinomycetemcomitans to a human oral cell line.

    OpenAIRE

    Mintz, K. P.; Fives-Taylor, P M

    1994-01-01

    Two quantitative, rapid assays were developed to study the adhesion of Actinobacillus actinomycetemcomitans, an oral bacterium associated with periodontal disease, to human epithelial cells. The human oral carcinoma cell line KB was grown in microtiter plates, and adherent bacteria were detected by an enzyme-linked immunosorbent assay with purified anti-A. actinomycetemcomitans serum and horseradish peroxidase-conjugated secondary antibody or [3H]thymidine-labeled bacteria. Adhesion was found...

  18. Cysteine modified polyaniline films improve biocompatibility for two cell lines

    International Nuclear Information System (INIS)

    This work focuses on one of the most exciting application areas of conjugated conducting polymers, which is cell culture and tissue engineering. To improve the biocompatibility of conducting polymers we present an easy method that involves the modification of the polymer backbone using L-cysteine. In this publication, we show the synthesis of polyaniline (PANI) films supported onto Polyethylene terephthalate (PET) films, and modified using cysteine (PANI-Cys) in order to generate a biocompatible substrate for cell culture. The PANI-Cys films are characterized by Fourier Transform infrared and UV–visible spectroscopy. The changes in the hydrophilicity of the polymer films after and before the modification were tested using contact angle measurements. After modification the contact angle changes from 86° ± 1 to 90° ± 1, suggesting a more hydrophylic surface. The adhesion properties of LM2 and HaCaT cell lines on the surface of PANI-Cys films in comparison with tissue culture plastic (TCP) are studied. The PANI-Cys film shows better biocompatibility than PANI film for both cell lines. The cell morphologies on the TCP and PANI-Cys film were examined by florescence and Atomic Force Microscopy (AFM). Microscopic observations show normal cellular behavior when PANI-Cys is used as a substrate of both cell lines (HaCaT and LM2) as when they are cultured on TCP. The ability of these PANI-Cys films to support cell attachment and growth indicates their potential use as biocompatible surfaces and in tissue engineering. - Highlights: • A new surface PANI-Cys was produced on films of polyethylene terephthalate. • The relationship between surface characteristics and biocompatibility is analyzed. • The PANI-Cys film presents good biocompatibility for two cell lines

  19. Cysteine modified polyaniline films improve biocompatibility for two cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Yslas, Edith I., E-mail: eyslas@exa.unrc.edu.ar [Departamento de Biología Molecular, Universidad Nacional de Río Cuarto, Agencia Postal Nro3, X580BYA Río Cuarto (Argentina); Cavallo, Pablo; Acevedo, Diego F.; Barbero, César A. [Departamento de Química, Universidad Nacional de Río Cuarto, Agencia Postal Nro3, X580BYA Río Cuarto (Argentina); Rivarola, Viviana A. [Departamento de Biología Molecular, Universidad Nacional de Río Cuarto, Agencia Postal Nro3, X580BYA Río Cuarto (Argentina)

    2015-06-01

    This work focuses on one of the most exciting application areas of conjugated conducting polymers, which is cell culture and tissue engineering. To improve the biocompatibility of conducting polymers we present an easy method that involves the modification of the polymer backbone using L-cysteine. In this publication, we show the synthesis of polyaniline (PANI) films supported onto Polyethylene terephthalate (PET) films, and modified using cysteine (PANI-Cys) in order to generate a biocompatible substrate for cell culture. The PANI-Cys films are characterized by Fourier Transform infrared and UV–visible spectroscopy. The changes in the hydrophilicity of the polymer films after and before the modification were tested using contact angle measurements. After modification the contact angle changes from 86° ± 1 to 90° ± 1, suggesting a more hydrophylic surface. The adhesion properties of LM2 and HaCaT cell lines on the surface of PANI-Cys films in comparison with tissue culture plastic (TCP) are studied. The PANI-Cys film shows better biocompatibility than PANI film for both cell lines. The cell morphologies on the TCP and PANI-Cys film were examined by florescence and Atomic Force Microscopy (AFM). Microscopic observations show normal cellular behavior when PANI-Cys is used as a substrate of both cell lines (HaCaT and LM2) as when they are cultured on TCP. The ability of these PANI-Cys films to support cell attachment and growth indicates their potential use as biocompatible surfaces and in tissue engineering. - Highlights: • A new surface PANI-Cys was produced on films of polyethylene terephthalate. • The relationship between surface characteristics and biocompatibility is analyzed. • The PANI-Cys film presents good biocompatibility for two cell lines.

  20. 2-DE analysis of breast cancer cell lines 1833 and 4175 with distinct metastatic organ-specific potentials: comparison with parental cell line MDA-MB-231.

    Science.gov (United States)

    Selicharova, Irena; Sanda, Miloslav; Mladkova, Jana; Ohri, Sujata Saraswat; Vashishta, Aruna; Fusek, Martin; Jiracek, Jiri; Vetvicka, Vaclav

    2008-05-01

    Human MDA-MB-231 derived breast cancer cell lines 1833 and 4175 have different metastatic potentials in terms of their tissue tropisms and aggressiveness. Cell line 1833 is specifically metastatic to the bone. The highly aggressive cell line 4175 is specific to the lung. We performed 2-DE analysis of the cell lines. We found 16 significantly changed protein spots, 14 protein spots were identified. Expression of cathepsin D, triosephosphate isomerase, phosphoglycerate kinase 1, heme binding protein 1 and annexin 2 could be correlated with the in vitro aggressiveness of the respective cell lines. Interstitial collagenase and dimethylargininase 2 were exclusive to the cell line 1833 and might contribute to its bone specificity. Serpin B9, cathepsin B chain b, galectin 3 and HSP 27 were changed in the lung specific cell line 4175. The possible contribution of identified proteins to differences in metastatic behavior of the cell lines is discussed. PMID:18425382

  1. Establishment and applications of male germ cell and Sertoli cell lines.

    Science.gov (United States)

    Wang, Hong; Wen, Liping; Yuan, Qingqing; Sun, Min; Niu, Minghui; He, Zuping

    2016-08-01

    Within the seminiferous tubules there are two major cell types, namely male germ cells and Sertoli cells. Recent studies have demonstrated that male germ cells and Sertoli cells can have significant applications in treating male infertility and other diseases. However, primary male germ cells are hard to proliferate in vitro and the number of spermatogonial stem cells is scarce. Therefore, methods that promote the expansion of these cell populations are essential for their use from the bench to the bed side. Notably, a number of cell lines for rodent spermatogonia, spermatocytes and Sertoli cells have been developed, and significantly we have successfully established a human spermatogonial stem cell line with an unlimited proliferation potential and no tumor formation. This newly developed cell line could provide an abundant source of cells for uncovering molecular mechanisms underlying human spermatogenesis and for their utilization in the field of reproductive and regenerative medicine. In this review, we discuss the methods for establishing spermatogonial, spermatocyte and Sertoli cell lines using various kinds of approaches, including spontaneity, transgenic animals with oncogenes, simian virus 40 (SV40) large T antigen, the gene coding for a temperature-sensitive mutant of p53, telomerase reverse gene (Tert), and the specific promoter-based selection strategy. We further highlight the essential applications of these cell lines in basic research and translation medicine. PMID:27069011

  2. Astrocyte signaling in the presence of spatial inhomogeneities

    Science.gov (United States)

    Stamatakis, Michail; Mantzaris, Nikos V.

    2007-09-01

    Astrocytes, a special type of glial cells, were considered to have just a supporting role in information processing in the brain. However, several recent studies have shown that they can be chemically stimulated by various neurotransmitters, such as ATP, and can generate Ca2+ and ATP waves, which can propagate over many cell lengths before being blocked. Although pathological conditions, such as spreading depression and epilepsy, have been linked to abnormal wave propagation in astrocytic cellular networks, a quantitative understanding of the underlying characteristics is still lacking. Astrocytic cellular networks are inhomogeneous, in the sense that the domain they occupy contains passive regions or gaps, which are unable to support wave propagation. Thus, this work focuses on understanding the complex interplay between single-cell signal transduction, domain inhomogeneity, and the characteristics of wave propagation and blocking in astrocytic cellular networks. The single-cell signal transduction model that was employed accounts for ATP-mediated IP3 production, the subsequent Ca2+ release from the ER, and ATP release into the extracellular space. The model is excitable and thus an infinite range of wave propagation is observed if the domain of propagation is homogeneous. This is not always the case for inhomogeneous domains. To model wave propagation in inhomogeneous astrocytic networks, a reaction-diffusion framework was developed and one-gap as well as multiple-gap cases were simulated using an efficient finite-element algorithm. The minimum gap length that blocks the wave was computed as a function of excitability levels and geometric characteristics of the inhomogeneous network, such as the length of the active regions (cells). Complex transient patterns, such as wave reflection, wave trapping, and generation of echo waves, were also predicted by the model, and their relationship to the geometric characteristics of the network was evaluated. Therefore, the

  3. Astrocytes Underlie Neuroinflammatory Memory Impairment

    OpenAIRE

    Osso, LA; Chan, JR

    2015-01-01

    © 2015 Elsevier Inc. All rights reserved. Neuroinflammation is being increasingly recognized as a potential mediator of cognitive impairments in various neurological conditions. Habbas et al. demonstrate that the pro-inflammatory cytokine tumor necrosis factor alpha signals through astrocytes to alter synaptic transmission and impair cognition in a mouse model of multiple sclerosis.

  4. 76 FR 16609 - Proposed Information Collection; Comment Request; Identification of Human Cell Lines Project

    Science.gov (United States)

    2011-03-24

    ...; Identification of Human Cell Lines Project AGENCY: National Institute of Standards and Technology (NIST...) profiling up to 1500 human cell line samples as part of the Identification of Human Cell Lines Project. All... for Biotechnology Information (NCBI) and will be used to differentiate among cell lines, as...

  5. Silicon Carbide Tiles for Sidewall Lining in Aluminium Electrolysis Cells

    Institute of Scientific and Technical Information of China (English)

    RUANBo; ZHAOJunguo; 等

    1999-01-01

    The paper introduces the nitride bonded silicon carbide used for sidewall lining in aluminium eletrolysis cells ,including technical process,main properties and application results.Comparison tests on various physical properties of silicon carbide products made by LIRR and other producers worldwide have also been conducted in an independent laboratory.

  6. Third-line chemotherapy for small cell lung cancer

    NARCIS (Netherlands)

    de Jong, WK; ten Hacken, NHT; Groen, HJM

    2006-01-01

    Efficacy of third-line chemotherapy treatment for small cell lung cancer (SCLC) is unknown. We present our experience with third-tine chemotherapy for recurrent SCLC. Between January 1996 and July 2004 all. consecutive patients treated for SCLC were retrospectively studied. We recorded patient chara

  7. RADIATION-INDUCED APOPTOSIS OF TWO NASOPHARANGEAL CARCINOMA CELL LINES

    Institute of Scientific and Technical Information of China (English)

    WANG Feng-wei; LIANG Ke; YIN Wei-bo; SHEN Yu; SHENG Xiu-gui

    1999-01-01

    Objective: To study apoptosis induced by radiation in two nasopharyngeal carcinoma (NPC) cell lines, CNE and CNE-2. Methods: Hoechst 33342 staining, immunohistochemical staining, RT-PCR, DNA dot blotting and Southern blotting were used to identify apoptosis.Results: A single dose of X-irradiation resulted in apoptosis, the apoptotic index (AI) was time- and dosedependent. Different apoptotic responses existed in the two cell lines. Immunohistochemical staining showed that bcl-2 protein was strongly positive in CNE but negative in CNE-2. However, RT-PCR revealed p53mRNA in CNE-2 but not in CNE. P53 and bcl-2 genes were both present in the two cell lines as shown by DNA blotting, but the 2.8 kb fragment of the p53 gene was much lower than the 5.6 kb fragment on CNE which was clearly shown in Southern hybridization, suggestive of partial deletion of p53 gene in CNE. Conclusion:Apoptotic response to radiation is different in two NPC cell lines. CNE is more radioresistant than CNE-2.Overexpression of bcl-2 protein and partial deletion of p53 gene may explain their difference in radiosensitivity.

  8. Cryopreservation of specialized chicken lines using cultured primordial germ cells.

    Science.gov (United States)

    Nandi, S; Whyte, J; Taylor, L; Sherman, A; Nair, V; Kaiser, P; McGrew, M J

    2016-08-01

    Biosecurity and sustainability in poultry production requires reliable germplasm conservation. Germplasm conservation in poultry is more challenging in comparison to other livestock species. Embryo cryopreservation is not feasible for egg-laying animals, and chicken semen conservation has variable success for different chicken breeds. A potential solution is the cryopreservation of the committed diploid stem cell precursors to the gametes, the primordial germ cells ( PGCS: ). Primordial germ cells are the lineage-restricted cells found at early embryonic stages in birds and form the sperm and eggs. We demonstrate here, using flocks of partially inbred, lower-fertility, major histocompatibility complex- ( MHC-: ) restricted lines of chicken, that we can easily derive and cryopreserve a sufficient number of independent lines of male and female PGCs that would be sufficient to reconstitute a poultry breed. We demonstrate that germ-line transmission can be attained from these PGCs using a commercial layer line of chickens as a surrogate host. This research is a major step in developing and demonstrating that cryopreserved PGCs could be used for the biobanking of specialized flocks of birds used in research settings. The prospective application of this technology to poultry production will further increase sustainability to meet current and future production needs. PMID:27099306

  9. DIVERSITY OF ARSENIC METABOLISM IN CULTURED HUMAN CANCER CELL LINES

    Science.gov (United States)

    Diversity of arsenic metabolism in cultured human cancer cell lines. Arsenic has been known to cause a variety of malignancies in human. Pentavalent As (As 5+) is reduced to trivalent As (As3+) which is further methylated by arsenic methyltransferase(s) to monomethylarson...

  10. α7 Nicotinic Receptor Promotes the Neuroprotective Functions of Astrocytes against Oxaliplatin Neurotoxicity

    Directory of Open Access Journals (Sweden)

    Lorenzo Di Cesare Mannelli

    2015-01-01

    Full Text Available Neuropathies are characterized by a complex response of the central nervous system to injuries. Glial cells are recruited to maintain neuronal homeostasis but dysregulated activation leads to pain signaling amplification and reduces the glial neuroprotective power. Recently, we highlighted the property of α7 nicotinic-acetylcholine-receptor (nAChR agonists to relieve pain and induce neuroprotection simultaneously with a strong increase in astrocyte density. Aimed to study the role of α7 nAChR in the neuron-glia cross-talk, we treated primary rat neurons and astrocytes with the neurotoxic anticancer drug oxaliplatin evaluating the effect of the α7 nAChR agonist PNU-282987 (PNU. Oxaliplatin (1 μM, 48 h reduced cell viability and increased caspase-3 activity of neuron monocultures without damaging astrocytes. In cocultures, astrocytes were not able to protect neurons by oxaliplatin even if glial cell metabolism was stimulated (pyruvate increase. On the contrary, the coculture incubation with 10 μM PNU improved neuron viability and inhibited apoptosis. In the absence of astrocytes, the protection disappeared. Furthermore, PNU promoted the release of the anti-inflammatory cytokine TGF-β1 and the expression of the glutamate-detoxifying enzyme glutamine synthetase. The α7 nAChR stimulation protects neurons from oxaliplatin toxicity through an astrocyte-mediated mechanism. α7 nAChR is suggested for recovering the homeostatic role of astrocytes.

  11. Sphere-forming cell subpopulations with cancer stem cell properties in human hepatoma cell lines

    Directory of Open Access Journals (Sweden)

    Chen Lei

    2011-06-01

    Full Text Available Abstract Background Cancer stem cells (CSCs are regarded as the cause of tumor formation and recurrence. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs. Methods Human hepatoma cell lines were plated in stem cell conditioned culture system allowed for sphere forming. To evaluate the stemness characteristics of spheres, the self-renewal, proliferation, chemoresistance, tumorigenicity of the PLC/PRF/5 sphere-forming cells, and the expression levels of stem cell related proteins in the PLC/PRF/5 sphere-forming cells were assessed, comparing with the parental cells. The stem cell RT-PCR array was performed to further explore the biological properties of liver CSCs. Results The PLC/PRF/5, MHCC97H and HepG2 cells could form clonal nonadherent 3-D spheres and be serially passaged. The PLC/PRF/5 sphere-forming cells possessed a key criteria that define CSCs: persistent self-renewal, extensive proliferation, drug resistance, overexpression of liver CSCs related proteins (Oct3/4, OV6, EpCAM, CD133 and CD44. Even 500 sphere-forming cells were able to form tumors in NOD/SCID mice, and the tumor initiating capability was not decreased when spheres were passaged. Besides, downstream proteins DTX1 and Ep300 of the CSL (CBF1 in humans, Suppressor of hairless in Drosophila and LAG1 in C. elegans -independent Notch signaling pathway were highly expressed in the spheres, and a gamma-secretase inhibitor MRK003 could significantly inhibit the sphere formation ability. Conclusions Nonadherent tumor spheres from hepatoma cell lines cultured in stem cell conditioned medium possess liver CSC properties, and the CSL-independent Notch signaling pathway may play a role in liver CSCs.

  12. Ketogenic diet and astrocyte/neuron metabolic interactions

    Directory of Open Access Journals (Sweden)

    Vamecq Joseph

    2007-05-01

    Full Text Available The ketogenic diet is an anticonvulsant diet enriched in fat. It provides the body with a minimal protein requirement and a restricted carbohydrate supply, the vast majority of calories (more than 80-90% being given by fat. Though anticonvulsant activity of ketogenic diet has been well documented by a large number of experimental and clinical studies, underlying mechanisms still remain partially unclear. Astrocyte-neuron interactions, among which metabolic shuttles, may influence synaptic activity and hence anticonvulsant protection. The astrocyte-neuron metabolic shuttles may be themselves influenced by the availability in energetic substrates such as hydrates of carbon and fats. Historically, ketogenic diet had been designed to mimic changes such as ketosis occurring upon starvation, a physiological state already known to exhibit anticonvulsant protection and sometimes referred to as “water diet”. For this reason, a special attention should be paid to metabolic features shared in common by ketogenic diet and starvation and especially those features that might result in anticonvulsant protection. Compared to feeding by usual mixed diet, starvation and ketogenic diet are both characterised by increased fat, lowered glucose and aminoacid supplies to cells. The resulting impact of these changes in energetic substrates on astrocyte/neuron metabolic shuttles might have anticonvulsant and/or neuroprotective properties. This is the aim of this communication to review some important astrocyte/neuron metabolic interactions (astrocyte/neuron lactate shuttle, glutamateinduced astrocytic glycolysis activation, glutamate/glutamine cycle along with the neurovascular coupling and the extent to which the way of their alteration by starvation and/or ketogenic diet might result in seizure and/or brain protection.

  13. Cytochrome c dysregulation induced by HIV infection of astrocytes results in bystander apoptosis of uninfected astrocytes by an IP3 and calcium-dependent mechanism

    OpenAIRE

    Eugenin, Eliseo A.; Berman, Joan W.

    2013-01-01

    HIV entry into the CNS is an early event after peripheral infection, resulting in neurologic dysfunction in a significant number of individuals despite successful anti-retroviral therapy. The mechanisms by which HIV mediates CNS dysfunction are not well understood. Our group recently demonstrated that HIV infection of astrocytes results in survival of HIV infected cells and apoptosis of surrounding uninfected astrocytes by the transmission of toxic intracellular signals through gap junctions....

  14. Choosing the right chondrocyte cell line: Focus on nitric oxide.

    Science.gov (United States)

    Santoro, Anna; Conde, Javier; Scotece, Morena; Abella, Vanessa; López, Verónica; Pino, Jesús; Gómez, Rodolfo; Gómez-Reino, Juan Jesús; Gualillo, Oreste

    2015-12-01

    Nitric oxide (NO) has been considered a catabolic factor that contributes to OA pathology by inducing chondrocytes apoptosis, matrix metalloproteinases synthesis, and pro-inflammatory cytokines expression. Thus, the research on NO regulation in chondrocytes represents a relevant field which needs to be explored in depth. However, to date, only the murine ATDC-5 cell line and primary chondrocytes are well-established cells to study NO production in cartilage tissues. The goal of this study is to determine whether two commonly used human chondrocytic cell lines: SW-1353 and T/C-28a2 cell lines are good models to examine lipopolysaccharide and/or pro-inflammatory cytokine-driven NO release and iNOS expression. To this aim, we carefully examined NO production and iNOS protein expression in human T/C-28a2 and SW-1353 chondrocytes stimulated with LPS and interleukin (IL)-1 alone or in combination. We also use ATDC-5 cells as a positive control for NO production. NO accumulation has been determined by colorimetric Griess reaction, whereas NOS type II expression was determined by Western Blot analysis. Our results clearly demonstrated that neither human T/C-28a2 nor SW-1353 chondrocytes showed a detectable increase in NO production or iNOS expression after bacterial endotoxin or cytokines challenge with IL-1. Our study demonstrated that T/C-28a2 and SW-1353 human cell lines are not suitable for studying NO release and iNOS expression confirming that ATDC5 and human primary cultured chondrocytes are the best in vitro cell system to study the actions derived from this mediator. PMID:26016689

  15. THP-1 cell line: an in vitro cell model for immune-modulation approach : Review

    NARCIS (Netherlands)

    Chanput, W.; Mes, J.J.; Wichers, H.J.

    2014-01-01

    THP-1 is a human leukemia monocytic cell line, which has been extensively used to study monocyte/macrophage functions, mechanisms, signaling pathways, and nutrient and drug transport. This cell line has become a common model to estimate modulation of monocyte and macrophage activities. This review a

  16. Human Embryonic St me Cell Lines fromthe Chinese Population and Differentiation to Liver and Muscle Cell Types

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    We have established 6 hES cell lines from IVF surplus blastocysts. Characterization of these lines have shown that 4 of the 6 lines meet all of the criterion (Science) for hES cell lines and 2 of them display most characteristics of hES cells but do not form teratoma. In order to produce hES cell lines without using mouse feeders, we have produced a hES cell line using feeders derived from hES cells themselves, and showed that hES-derived feeders are capable of supporting the derivation of new hES cell line...

  17. Simultaneous measurement of NK cell cytotoxicity against two target cell lines labelled with fluorescent lanthanide chelates.

    Science.gov (United States)

    Lövgren, J; Blomberg, K

    1994-07-12

    We describe a cytotoxicity assay which permits the simultaneous measurement of natural killer cell activity against two different cell lines. The target cell lines are labelled either with a fluorescent europium chelate or with a fluorescent terbium chelate and cell death is quantified by measuring the chelate release. K-562, Molt4 and Daudi cell lines have been used as targets. The release of the two chelates from the target cells can be detected with the help of time resolved fluorometry. As the measurements are made after background fluorescence has decayed no additional steps are needed to correct for the background from the medium. The assay procedure used for measurement of cytotoxicity against two target cell lines is very similar to the widely used 51Cr release assay. PMID:8034979

  18. Every Single Cell Clones from Cancer Cell Lines Growing Tumors In Vivo May Not Invalidate the Cancer Stem Cell Concept

    OpenAIRE

    Li, Fengzhi

    2009-01-01

    We present the result of our research on the tumorigenic ability of single cell clones isolated from an aggressive murine breast cancer cell line in a matched allografting mouse model. Tumor formation is basically dependent on the cell numbers injected per location. We argue that in vivo tumor formation from single cell clones, isolated in vitro from cancer cell lines, may not provide conclusive evidence to disprove the cancer stem cell (CSC) theory without additional data.

  19. Neonatal astrocyte damage is sufficient to trigger progressive striatal degeneration in a rat model of glutaric acidemia-I.

    Directory of Open Access Journals (Sweden)

    Silvia Olivera-Bravo

    Full Text Available BACKGROUND: We have investigated whether an acute metabolic damage to astrocytes during the neonatal period may critically disrupt subsequent brain development, leading to neurodevelopmental disorders. Astrocytes are vulnerable to glutaric acid (GA, a dicarboxylic acid that accumulates in millimolar concentrations in Glutaric Acidemia I (GA-I, an inherited neurometabolic childhood disease characterized by degeneration of striatal neurons. While GA induces astrocyte mitochondrial dysfunction, oxidative stress and subsequent increased proliferation, it is presently unknown whether such astrocytic dysfunction is sufficient to trigger striatal neuronal loss. METHODOLOGY/PRINCIPAL FINDINGS: A single intracerebroventricular dose of GA was administered to rat pups at postnatal day 0 (P0 to induce an acute, transient rise of GA levels in the central nervous system (CNS. GA administration potently elicited proliferation of astrocytes expressing S100β followed by GFAP astrocytosis and nitrotyrosine staining lasting until P45. Remarkably, GA did not induce acute neuronal loss assessed by FluoroJade C and NeuN cell count. Instead, neuronal death appeared several days after GA treatment and progressively increased until P45, suggesting a delayed onset of striatal degeneration. The axonal bundles perforating the striatum were disorganized following GA administration. In cell cultures, GA did not affect survival of either striatal astrocytes or neurons, even at high concentrations. However, astrocytes activated by a short exposure to GA caused neuronal death through the production of soluble factors. Iron porphyrin antioxidants prevented GA-induced astrocyte proliferation and striatal degeneration in vivo, as well as astrocyte-mediated neuronal loss in vitro. CONCLUSIONS/SIGNIFICANCE: Taken together, these results indicate that a transient metabolic insult with GA induces long lasting phenotypic changes in astrocytes that cause them to promote striatal

  20. Effect of 8-Chloroadenosine on Undifferentiatied HL-60 Cell Line

    Institute of Scientific and Technical Information of China (English)

    CUIJing-rong; HUIYu; XIANGYou-qing; ZHANGLi-he

    2003-01-01

    Aim To study the effect of 8-chloroadenosine (8-CA)on undifferentiatied HL-60 cell line. Methods The IC50 of cancer cell proliferation was determined using a microculture plate reader at 570 nm (MTT) and 540 nm (SRB).Morphology of HL-60 cells was observed under a scanning electron microscope and a transmission electron microscope. The differentiation of HL-60 cells was examined by nitro blue tetrazolium reduction (NBT) and acid phosphatase assay. The cycle of HL-60 cells was analyzed by flow cytometry. Results 8-CA inhibited proliferation of eight human cancer cell lines.The IC50 ranked in the following order; KB (0.05 μmol·L-1 ) < HL-60 (0.25 μmol·L-1) < Bel-7402 (0.56μmol·L-1 )< MCF-7 (0.65μmol·L-1) < HCT (0.79 μmol·L-1) < HeLa (0.89μmol·L-1) < BGC-823 ( 1.149μmol·L-1) cell surface shortened, and the shape of HL-60 cells nuclei changed to kidney-shaped, horse shoe-shaped and bilob ated after treatment with 8-CA. Meanwhile, 8-CA promoted NBT reduction and increased activity of acid phosphatase in HL-60 ceils in a time and concentration-dependent manner. Flow cytometry analysis indicated that 8-CA induced an appreciable increase of the cell population in G1 phase with a marked reduction in S phase. Conclusion 8-CA can induce differentiation of HL-60 cells and block the cells at G1 phase, thus inhibiting proliferation of HL-60 cells.

  1. Control of the neurovascular coupling by nitric oxide-dependent regulation of astrocytic Ca2+ signaling

    Directory of Open Access Journals (Sweden)

    Manuel Francisco Muñoz

    2015-03-01

    Full Text Available Neuronal activity must be tightly coordinated with blood flow to keep proper brain function, which is achieved by a mechanism known as neurovascular coupling. Then, an increase in synaptic activity leads to a dilation of local parenchymal arterioles that matches the enhanced metabolic demand. Neurovascular coupling is orchestrated by astrocytes. These glial cells are located between neurons and the microvasculature, with the astrocytic endfeet ensheathing the vessels, which allows fine intercellular communication. The neurotransmitters released during neuronal activity reach astrocytic receptors and trigger a Ca2+ signaling that propagates to the endfeet, activating the release of vasoactive factors and arteriolar dilation. The astrocyte Ca2+ signaling is coordinated by gap junction channels and hemichannels formed by connexins (Cx43 and Cx30 and channels formed by pannexins (Panx-1. The neuronal activity-initiated Ca2+ waves are propagated among neighboring astrocytes directly via gap junctions or through ATP release via connexin hemichannels or pannexin channels. In addition, Ca2+ entry via connexin hemichannels or pannexin channels may participate in the regulation of the astrocyte signaling-mediated neurovascular coupling. Interestingly, nitric oxide (NO can activate connexin hemichannel by S-nitrosylation and the Ca2+-dependent NO-synthesizing enzymes endothelial NO synthase (eNOS and neuronal NOS (nNOS are expressed in astrocytes. Therefore, the astrocytic Ca2+ signaling triggered in neurovascular coupling may activate NO production, which, in turn, may lead to Ca2+ influx through hemichannel activation. Furthermore, NO release from the hemichannels located at astrocytic endfeet may contribute to the vasodilation of parenchymal arterioles. In this review, we discuss the mechanisms involved in the regulation of the astrocytic Ca2+ signaling that mediates neurovascular coupling, with a special emphasis in the possible participation of NO in

  2. A Computational Model to Investigate Astrocytic Glutamate Uptake Influence on Synaptic Transmission and Neuronal Spiking

    Directory of Open Access Journals (Sweden)

    Sushmita Lakshmi Allam

    2012-10-01

    Full Text Available Over the past decades, our view of astrocytes has switched from passive support cells to active processing elements in the brain. The current view is that astrocytes shape neuronal communication and also play an important role in many neurodegenerative diseases. Despite the growing awareness of the importance of astrocytes, the exact mechanisms underlying neuron-astrocyte communication and the physiological consequences of astrocytic-neuronal interactions remain largely unclear. In this work, we define a modeling framework that will permit to address unanswered questions regarding the role of astrocytes. Our computational model of a detailed glutamatergic synapse facilitates the analysis of neural system responses to various stimuli and conditions that are otherwise difficult to obtain experimentally, in particular the readouts at the sub-cellular level. In this paper, we extend a detailed glutamatergic synaptic model, to include astrocytic glutamate transporters. We demonstrate how these glial transporters, responsible for the majority of glutamate uptake, modulate synaptic transmission mediated by ionotropic AMPA and NMDA receptors at glutamatergic synapses. Furthermore, we investigate how these local signaling effects at the synaptic level are translated into varying spatio-temporal patterns of neuron firing. Paired pulse stimulation results reveal that the effect of astrocytic glutamate uptake is more apparent when the input inter-spike interval is sufficiently long to allow the receptors to recover from desensitization. These results suggest an important functional role of astrocytes in spike timing dependent processes and demand further investigation of the molecular basis of certain neurological diseases specifically related to alterations in astrocytic glutamate uptake, such as epilepsy.

  3. A tale of two stories: astrocyte regulation of synaptic depression and facilitation.

    Directory of Open Access Journals (Sweden)

    Maurizio De Pittà

    2011-12-01

    Full Text Available Short-term presynaptic plasticity designates variations of the amplitude of synaptic information transfer whereby the amount of neurotransmitter released upon presynaptic stimulation changes over seconds as a function of the neuronal firing activity. While a consensus has emerged that the resulting decrease (depression and/or increase (facilitation of the synapse strength are crucial to neuronal computations, their modes of expression in vivo remain unclear. Recent experimental studies have reported that glial cells, particularly astrocytes in the hippocampus, are able to modulate short-term plasticity but the mechanism of such a modulation is poorly understood. Here, we investigate the characteristics of short-term plasticity modulation by astrocytes using a biophysically realistic computational model. Mean-field analysis of the model, supported by intensive numerical simulations, unravels that astrocytes may mediate counterintuitive effects. Depending on the expressed presynaptic signaling pathways, astrocytes may globally inhibit or potentiate the synapse: the amount of released neurotransmitter in the presence of the astrocyte is transiently smaller or larger than in its absence. But this global effect usually coexists with the opposite local effect on paired pulses: with release-decreasing astrocytes most paired pulses become facilitated, namely the amount of neurotransmitter released upon spike i+1 is larger than that at spike i, while paired-pulse depression becomes prominent under release-increasing astrocytes. Moreover, we show that the frequency of astrocytic intracellular Ca(2+ oscillations controls the effects of the astrocyte on short-term synaptic plasticity. Our model explains several experimental observations yet unsolved, and uncovers astrocytic gliotransmission as a possible transient switch between short-term paired-pulse depression and facilitation. This possibility has deep implications on the processing of neuronal spikes

  4. Mitochondrial DNA sequence analysis of two mouse hepatocarcinoma cell lines

    Institute of Scientific and Technical Information of China (English)

    Ji-Gang Dai; Xia Lei; Jia-Xin Min; Guo-Qiang Zhang; Hong Wei

    2005-01-01

    AIM: To study genetic difference of mitochondrial DNA (mtDNA)between two hepatocarcinoma cell lines (Hca-F and Hca-P)with diverse metastatic characteristics and the relationship between mtDNA changes in cancer cells and their oncogenic phenotype.METHODS: Mitochondrial DNA D-loop, tRNAMet+Glu+Ile and ND3gene fragments from the hepatocarcinoma cell lines with 1100, 1126 and 534 bp in length respectively were analysed by PCR amplification and restriction fragment length polymorphism techniques. The D-loop 3' end sequence of the hepatocarcinoma cell lines was determined by sequencing.RESULTS: No amplification fragment length polymorphism and restriction fragment length polymorphism were observed in tRNAMet+Glu+Ile,ND3 and D-loop of mitochondrial DNA of the hepatocarcinoma cells. Sequence differences between Hca-F and Hca-P were found in mtDNA D-loop.CONCLUSION: Deletion mutations of mitochondrial DNA restriction fragment may not play a significant role in carcinogenesis. Genetic difference of mtDNA D-loop between Hca-F and Hca-P, which may reflect the environmental and genetic influences during tumor progression, could be linked to their tumorigenic phenotypes.

  5. Growth inhibition by tyrosine kinase inhibitors in mesothelioma cell lines.

    Science.gov (United States)

    Nutt, Joyce E; O'Toole, Kieran; Gonzalez, David; Lunec, John

    2009-06-01

    Clinical outcome following chemotherapy for malignant pleural mesothelioma is poor and improvements are needed. This preclinical study investigates the effect of five tyrosine kinase inhibitors (PTK787, ZD6474, ZD1839, SU6668 and SU11248) on the growth of three mesothelioma cell lines (NCI H226, NCI H28 and MSTO 211H), the presence of growth factor receptors and inhibition of their downstream signalling pathways. GI50 values were determined: ZD6474 and SU11248, mainly VEGFR2 inhibitors, gave the lowest GI50 across all cell lines (3.5-6.9 microM) whereas ZD1839 gave a GI50 in this range only in H28 cells. All cell lines were positive for EGFR, but only H226 cells were positive for VEGFR2 by Western blotting. ZD6474 and ZD1839 inhibited EGF-induced phosphorylation of EGFR, AKT and ERK, whereas VEGF-induced phosphorylation of VEGFR2 was completely inhibited with 0.1 microM SU11248. VEGFR2 was detected in tumour samples by immunohistochemistry. VEGFR2 tyrosine kinase inhibitors warrant further investigation in mesothelioma. PMID:19318229

  6. Cytotoxic studies of paclitaxel (Taxol) in human tumour cell lines.

    OpenAIRE

    Liebmann, J. E.; Cook, J. A.; Lipschultz, C.; Teague, D.; Fisher, J; Mitchell, J B

    1993-01-01

    The cytotoxicity of paclitaxel against eight human tumour cell lines has been studied with in vitro clonogenic assays. The fraction of surviving cells fell sharply after exposure for 24 h to paclitaxel concentrations ranging from 2 to 20 nM; the paclitaxel IC50 was found to range between 2.5 and 7.5 nM. Increasing the paclitaxel concentration above 50 nM, however, resulted in no additional cytotoxicity after a 24 h drug exposure. Cells incubated in very high concentrations of paclitaxel (10,0...

  7. Plasmids and packaging cell lines for use in phage display

    Science.gov (United States)

    Bradbury, Andrew M.

    2012-07-24

    The invention relates to a novel phagemid display system for packaging phagemid DNA into phagemid particles which completely avoids the use of helper phage. The system of the invention incorporates the use of bacterial packaging cell lines which have been transformed with helper plasmids containing all required phage proteins but not the packaging signals. The absence of packaging signals in these helper plasmids prevents their DNA from being packaged in the bacterial cell, which provides a number of significant advantages over the use of both standard and modified helper phage. Packaged phagemids expressing a protein or peptide of interest, in fusion with a phage coat protein such as g3p, are generated simply by transfecting phagemid into the packaging cell line.

  8. Cytotoxic effect of Plantago spp. on cancer cell lines.

    Science.gov (United States)

    Gálvez, Marina; Martín-Cordero, Carmen; López-Lázaro, Miguel; Cortés, Felipe; Ayuso, María Jesús

    2003-10-01

    Methanolic extracts from seven Plantago species used in traditional medicine for the treatment of cancer, were evaluated for cytotoxic activity against three human cancer cell lines recommended by the National Cancer Institute (NCI, USA). The results showed that Plantago species exhibited cytotoxic activity, showing a certain degree of selectivity against the tested cells in culture. Since the flavonoids are able to strongly inhibit the proliferation of human cancer cell lines, we have identified luteolin-7-O-beta-glucoside as major flavonoid present in most of the Plantago species. Also, we have evaluated this compound and its aglycon, luteolin, for their cytotoxic and DNA topoisomerase I poisons activities. These results could justify the traditional use of the Plantago species and topoisomerase-mediated DNA damage might be a possible mechanism by which flavonoids of Plantago exert their cytotoxicity potential. PMID:12963131

  9. Astrocytes protect neurons against methylmercury via ATP/P2Y(1 receptor-mediated pathways in astrocytes.

    Directory of Open Access Journals (Sweden)

    Yusuke Noguchi

    Full Text Available Methylmercury (MeHg is a well known environmental pollutant that induces serious neuronal damage. Although MeHg readily crosses the blood-brain barrier, and should affect both neurons and glial cells, how it affects glia or neuron-to-glia interactions has received only limited attention. Here, we report that MeHg triggers ATP/P2Y1 receptor signals in astrocytes, thereby protecting neurons against MeHg via interleukin-6 (IL-6-mediated pathways. MeHg increased several mRNAs in astrocytes, among which IL-6 was the highest. For this, ATP/P2Y1 receptor-mediated mechanisms were required because the IL-6 production was (i inhibited by a P2Y1 receptor antagonist, MRS2179, (ii abolished in astrocytes obtained from P2Y1 receptor-knockout mice, and (iii mimicked by exogenously applied ATP. In addition, (iv MeHg released ATP by exocytosis from astrocytes. As for the intracellular mechanisms responsible for IL-6 production, p38 MAP kinase was involved. MeHg-treated astrocyte-conditioned medium (ACM showed neuro-protective effects against MeHg, which was blocked by anti-IL-6 antibody and was mimicked by the application of recombinant IL-6. As for the mechanism of neuro-protection by IL-6, an adenosine A1 receptor-mediated pathway in neurons seems to be involved. Taken together, when astrocytes sense MeHg, they release ATP that autostimulates P2Y1 receptors to upregulate IL-6, thereby leading to A1 receptor-mediated neuro-protection against MeHg.

  10. Reciprocal neuron-astrocyte signaling in epileptic seizure generation and propagation

    OpenAIRE

    Cammarota, Mario

    2013-01-01

    The idea that astrocytes – the main population of glial cells in the brain – are active partners of neurons in many aspects of brain functions represented a Copernican Revolution in neurobiology. Astrocytes, which were for many years considered just like the cement (from Greek glia i.e. glue) that keeps neuronal cells together, have now been moved from the periphery to the centre of the universe of information processing in the brain providing a radically different point of obs...

  11. N-Cadherin and Integrins: Two Receptor Systems That Mediate Neuronal Process Outgrowth on Astrocyte Surfaces

    OpenAIRE

    Tomaselli, Kevin J.; Neugebauer, Karla M; Bixby, John L.; Lilien, Jack; Reichardt, Louis F.

    2008-01-01

    Receptor-mediated interactions between neurons and astroglia are likely to play a crucial role in the growth and guidance of CNS axons. Using antibodies to neuronal cell surface proteins, we identified two receptor systems mediating neurite outgrowth on cultured astrocytes. N-cadherin, a Ca2+-dependent cell adhesion molecule, functions prominently in the outgrowth of neurites on astrocytes by E8 and E14 chick ciliary ganglion (CC) neurons. β1-class integrin ECM receptor heterodimers function ...

  12. Survey of Differentially Methylated Promoters in Prostate Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Yipeng Wang

    2005-08-01

    Full Text Available DNA methylation, copy number in the genomes of three immortalized prostate epithelial, five cancer cell lines (LNCaP, PC3, PC3M, PC3M-Pro4, PC3MLN4 were compared using a microarray-based technique. Genomic DNA is cut with a methylation-sensitive enzyme Hpall, followed by linker ligation, polymerase chain reaction (PCR amplification, labeling, hybridization to an array of promoter sequences. Only those parts of the genomic DNA that have unmethylated restriction sites within a few hundred base pairs generate PCR products detectable on an array. Of 2732 promoter sequences on a test array, 504 (18.5% showed differential hybridization between immortalized prostate epithelial, cancer <