WorldWideScience

Sample records for cells secrete neurotrophic

  1. Secretion of nerve growth factor, brain-derived neurotrophic factor, and glial cell-line derived neurotrophic factor in co-culture of four cell types in cerebrospinal fluid-containing medium

    Institute of Scientific and Technical Information of China (English)

    Sanjiang Feng; Minghua Zhuang; Rui Wu

    2012-01-01

    The present study co-cultured human embryonic olfactory ensheathing cells, human Schwann cells, human amniotic epithelial cells and human vascular endothelial cells in complete culture medium- containing cerebrospinal fluid. Enzyme linked immunosorbent assay was used to detect nerve growth factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor secretion in the supernatant of co-cultured cells. Results showed that the number of all cell types reached a peak at 7–10 days, and the expression of nerve growth factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor peaked at 9 days. Levels of secreted nerve growth factor were four-fold higher than brain-derived neurotrophic factor, which was three-fold higher than glial cell line-derived neurotrophic factor. Increasing concentrations of cerebrospinal fluid (10%, 20% and 30%) in the growth medium caused a decrease of neurotrophic factor secretion. Results indicated co-culture of human embryonic olfactory ensheathing cells, human Schwann cells, human amniotic epithelial cells and human vascular endothelial cells improved the expression of nerve growth factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor. The reduction of cerebrospinal fluid extravasation at the transplant site after spinal cord injury is beneficial for the survival and secretion of neurotrophic factors from transplanted cells.

  2. Light-induced retinal injury enhanced neurotrophins secretion and neurotrophic effect of mesenchymal stem cells in vitro

    Directory of Open Access Journals (Sweden)

    Wei Xu

    2013-04-01

    Full Text Available PURPOSE: To investigate neurotrophins expression and neurotrophic effect change in mesenchymal stem cells (MSCs under different types of stimulation. METHODS: Rats were exposed in 10,000 lux white light to develop light-induced retinal injury. Supernatants of homogenized retina (SHR, either from normal or light-injured retina, were used to stimulate MSCs. Quantitative real time for polymerase chain reaction (RT-PCR and enzyme-linked immunosorbent assay (ELISA were conducted for analysis the expression change in basic fibroblast growth factor (bFGF, brain-derived neurotrophic factor (BDNF and ciliary neurotrophic factor (CNTF in MSCs after stimulation. Conditioned medium from SHR-stimulated MSCs and control MSCs were collected for evaluation their effect on retinal explants. RESULTS: Supernatants of homogenized retina from light-injured rats significantly promoted neurotrophins secretion from MSCs (p<0.01. Conditioned medium from mesenchymal stem cells stimulated by light-injured SHR significantly reduced DNA fragmentation (p<0.01, up-regulated bcl-2 (p<0.01 and down-regulated bax (p<0.01 in retinal explants, displaying enhanced protective effect. CONCLUSIONS: Light-induced retinal injury is able to enhance neurotrophins secretion from mesenchymal stem cells and promote the neurotrophic effect of mesenchymal stem cells.

  3. Protective effects of neurotrophic factor-secreting cells in a 6-OHDA rat model of Parkinson disease.

    Science.gov (United States)

    Sadan, Ofer; Bahat-Stromza, Merav; Barhum, Yael; Levy, Yossef S; Pisnevsky, Anat; Peretz, Hagit; Ilan, Avihay Bar; Bulvik, Shlomo; Shemesh, Noam; Krepel, Dana; Cohen, Yoram; Melamed, Eldad; Offen, Daniel

    2009-10-01

    Stem cell-based therapy is a promising treatment for neurodegenerative diseases. In our laboratory, a novel protocol has been developed to induce bone marrow-derived mesenchymal stem cells (MSC) into neurotrophic factors- secreting cells (NTF-SC), thus combining stem cell-based therapy with the NTF-based neuroprotection. These cells produce and secrete factors such as brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor. Conditioned medium of the NTF-SC that was applied to a neuroblastoma cell line (SH-SY5Y) 1 h before exposure to the neurotoxin 6-hydroxydopamine (6-OHDA) demonstrated marked protection. An efficacy study was conducted on the 6-OHDA-induced lesion, a rat model of Parkinson's disease. The cells, either MSC or NTF-SC, were transplanted on the day of 6-OHDA administration and amphetamine-induced rotations were measured as a primary behavior index. We demonstrated that when transplanted posterior to the 6-OHDA lesion, the NTF-SC ameliorated amphetamine-induced rotations by 45%. HPLC analysis demonstrated that 6-OHDA induced dopamine depletion to a level of 21% compared to the untreated striatum. NTF-SC inhibited dopamine depletion to a level of 72% of the contralateral striatum. Moreover, an MRI study conducted with iron-labeled cells, followed by histological verification, revealed that the engrafted cells migrated toward the lesion. In a histological assessment, we found that the cells induced regeneration in the damaged striatal dopaminergic nerve terminal network. We therefore conclude that the induced MSC have a therapeutic potential for neurodegenerative processes and diseases, both by the NTFs secretion and by the migratory trait toward the diseased tissue.

  4. Carbon nanotube-collagen three-dimensional culture of mesenchymal stem cells promotes expression of neural phenotypes and secretion of neurotrophic factors.

    Science.gov (United States)

    Lee, Jae Ho; Lee, Ja-Yeon; Yang, Sung Hee; Lee, Eun-Jung; Kim, Hae-Won

    2014-10-01

    Microenvironments provided by three-dimensional (3-D) hydrogels mimic native tissue conditions, supplying appropriate physical cues for regulating stem cell behaviors. Here, we focused on carbon nanotubes (CNTs) dispersed within collagen hydrogels to provide 3-D microenvironmental conditions for mesenchymal stem cells (MSCs) in stimulating biological functions for neural regeneration. Small concentrations of CNTs (0.1-1wt.%) did not induce toxicity to MSCs, and even improved the proliferative potential of the cells. MSCs cultured within the CNT-collagen hydrogel expressed considerable levels of neural markers, including GAP43 and βIII tubulin proteins by immunostaining as well as GAP43 and synapse I genes by reverse transcriptase polymerase chain reaction (RT-PCR). Of note was that neurotrophic factors, particularly nerve growth factor and brain derived neurotrophic factor, were significantly promoted by the incorporation of CNTs as confirmed by RT-PCR and Western blot analysis. A model experiment involving neuritogenesis of PC12 cells influenced by those releasing neurotrophic factors from MSCs cultured within the CNT-collagen hydrogel demonstrated the significant enhancement in neurite outgrowth behaviors. Taken together, collagen hydrogel provides excellent 3-D conditions for MSC growth, and a small incorporation of CNTs within the hydrogel significantly stimulates MSC expression of neural markers and secretion of neurotrophic factors.

  5. Retinal pigment epithelial cells secrete neurotrophic factors and synthesize dopamine: possible contribution to therapeutic effects of RPE cell transplantation in Parkinson's disease

    Directory of Open Access Journals (Sweden)

    Gu Qing

    2009-06-01

    Full Text Available Abstract Background New strategies for the treatment of Parkinson's disease (PD are shifted from dopamine (DA replacement to regeneration or restoration of the nigro-striatal system. A cell therapy using human retinal pigment epithelial (RPE cells as substitution for degenerated dopaminergic (DAergic neurons has been developed and showed promising prospect in clinical treatment of PD, but the exact mechanism underlying this therapy is not fully elucidated. In the present study, we investigated whether the beneficial effects of this therapy are related to the trophic properties of RPE cells and their ability to synthesize DA. Methods We evaluated the protective effects of conditioned medium (CM from cultured RPE cells on the DAergic cells against 6-hydroxydopamine (6-OHDA- and rotenone-induced neurotoxicity and determined the levels of glial cell derived neurotrophic factor (GDNF and brain derived neurotrophic factor (BDNF released by RPE cells. We also measured the DA synthesis and release. Finally we transplanted microcarriers-RPE cells into 6-OHDA lesioned rats and observed the improvement in apomorphine-induced rotations (AIR. Results We report here: (1 CM from RPE cells can secret trophic factors GDNF and BDNF, and protect DAergic neurons against the 6-OHDA- and rotenone-induced cell injury; (2 cultured RPE cells express L-dopa decarboxylase (DDC and synthesize DA; (3 RPE cells attached to microcarriers can survive in the host striatum and improve the AIR in 6-OHDA-lesioned animal model of PD; (4 GDNF and BDNF levels are found significantly higher in the RPE cell-grafted tissues. Conclusion These findings indicate the RPE cells have the ability to secret GDNF and BDNF, and synthesize DA, which probably contribute to the therapeutic effects of RPE cell transplantation in PD.

  6. Gastrodin promotes the secretion of brain-derived neurotrophic factor in the injured spinal cord

    Institute of Scientific and Technical Information of China (English)

    Changwei Song; Shiqiang Fang; Gang Lv; Xifan Mei

    2013-01-01

    Gastrodin, an active component of tall gastrodia tuber, is widely used in the treatment of dizziness, paralysis, epilepsy, stroke and dementia, and exhibits a neuroprotective effect. A rat model of spinal cord injury was established using Allen's method, and gastrodin was administered via the subarachnoid cavity and by intraperitoneal injection for 7 days. Results show that gastrodin promoted the secretion of brain-derived neurotrophic factor in rats with spinal cord injury. After gastrodin treatment, the maximum angle of the inclined plane test, and the Basso, Beattie and Bresnahan scores increased. Moreover, gastrodin improved neural tissue recovery in the injured spinal cord. These results demonstrate that gastrodin promotes the secretion of brain-derived neurotrophic factor, contributes to the recovery of neurological function, and protects neural cells against injury.

  7. Variant brain-derived neurotrophic factor (BDNF) (Met66) alters the intracellular trafficking and activity-dependent secretion of wild-type BDNF in neurosecretory cells and cortical neurons.

    Science.gov (United States)

    Chen, Zhe-Yu; Patel, Paresh D; Sant, Gayatree; Meng, Chui-Xiang; Teng, Kenneth K; Hempstead, Barbara L; Lee, Francis S

    2004-05-05

    Brain-derived neurotrophic factor (BDNF) plays a critical role in nervous system and cardiovascular development and function. Recently, a common single nucleotide polymorphism in the bdnf gene, resulting in a valine to methionine substitution in the prodomain (BDNF(Met)), has been shown to lead to memory impairment and susceptibility to neuropsychiatric disorders in humans heterozygous for the variant BDNF. When expressed by itself in hippocampal neurons, less BDNF(Met) is secreted in an activity-dependent manner. The nature of the cellular defect when both BDNF(Met) and wild-type BDNF (BDNF(Val)) are present in the same cell is not known. Given that this is the predominant expression profile in humans, we examined the effect of coexpressed BDNF(Met) on BDNF(Val) intracellular trafficking and processing. Our data indicate that abnormal trafficking of BDNF(Met) occurred only in neuronal and neurosecretory cells and that BDNF(Met) could alter the intracellular distribution and activity-dependent secretion of BDNF(Val). We determined that, when coexpressed in the same cell, approximately 70% of the variant BDNF forms BDNF(Val).BDNF(Met) heterodimers, which are inefficiently sorted into secretory granules resulting in a quantitative decreased secretion. Finally, we determined the form of BDNF secreted in an activity-dependent manner and observed no differences in the forms of BDNF(Met) or the BDNF(Val).BDNF(Met) heterodimer compared with BDNF(Val). Together, these findings indicate that components of the regulated secretory machinery interacts specifically with a signal in the BDNF prodomain and that perturbations in BDNF trafficking may lead to selective impairment in CNS function.

  8. Neurotrophic Effect of Adipose Tissue-Derived Stem Cells on Erectile Function Recovery by Pigment Epithelium-Derived Factor Secretion in a Rat Model of Cavernous Nerve Injury

    Directory of Open Access Journals (Sweden)

    Xin Chen

    2016-01-01

    Full Text Available The paracrine effect is the major mechanism of stem cell therapy. However, the details of the effect’s mechanism remain unknown. The aim of this study is to investigate whether adipose tissue-derived stem cells (ADSCs can ameliorate cavernous nerve injury-induced erectile dysfunction (CNIED rats and to determine its mechanism. Twenty-eight days after intracavernous injection of 5-ethynyl-2-deoxyuridine- (EdU- labeled ADSCs, the erectile function of all the rats was evaluated by intracavernosal pressure (ICP. The ADSCs steadily secreted detectable pigment epithelium-derived factor (PEDF in vitro. The expression of PEDF increased in the penis of the bilateral cavernous nerve injury (BCNI group for 14 days and then gradually decreased. On day 28 after the intracavernous injection, the ADSCs group exhibited a significantly increased ICP compared with the phosphate buffered saline- (PBS- treated group. Moreover, the neuronal nitric oxide synthase (nNOS and S100 expression in penile dorsal nerves and the smooth muscle content to collagen ratio in penile tissues significantly increased. Furthermore, elevated PEDF, p-Akt, and p-eNOS were identified in the ADSCs group. This study demonstrated that intracavernous injection of ADSCs improved erectile function, repaired the nerve, and corrected penile fibrosis. One potential mechanism is the PEDF secretion of ADSCs and subsequent PI3K/Akt pathway activation.

  9. Flavonoids Induce the Synthesis and Secretion of Neurotrophic Factors in Cultured Rat Astrocytes: A Signaling Response Mediated by Estrogen Receptor

    Directory of Open Access Journals (Sweden)

    Sherry L. Xu

    2013-01-01

    Full Text Available Neurotrophic factors are playing vital roles in survival, growth, and function of neurons. Regulation of neurotrophic factors in the brain has been considered as one of the targets in developing drug or therapy against neuronal disorders. Flavonoids, a family of multifunctional natural compounds, are well known for their neuronal beneficial effects. Here, the effects of flavonoids on regulating neurotrophic factors were analyzed in cultured rat astrocytes. Astrocyte is a major secreting source of neurotrophic factors in the brain. Thirty-three flavonoids were screened in the cultures, and calycosin, isorhamnetin, luteolin, and genistein were identified to be highly active in inducing the synthesis and secretion of neurotrophic factors, including nerve growth factor (NGF, glial-derived neurotrophic factor (GDNF, and brain-derived neurotrophic factor (BDNF. The inductions were in time- and dose-dependent manners. In cultured astrocytes, the phosphorylation of estrogen receptor was triggered by application of flavonoids. The phosphorylation was blocked by an inhibitor of estrogen receptor, which in parallel reduced the flavonoid-induced expression of neurotrophic factors. The results proposed the role of flavonoids in protecting brain diseases, and therefore these flavonoids could be developed for health food supplement for patients suffering from neurodegenerative diseases.

  10. cAMP-mediated secretion of brain-derived neurotrophic factor in developing airway smooth muscle.

    Science.gov (United States)

    Thompson, Michael A; Britt, Rodney D; Kuipers, Ine; Stewart, Alecia; Thu, James; Pandya, Hitesh C; MacFarlane, Peter; Pabelick, Christina M; Martin, Richard J; Prakash, Y S

    2015-10-01

    Moderate hyperoxic exposure in preterm infants contributes to subsequent airway dysfunction and to risk of developing recurrent wheeze and asthma. The regulatory mechanisms that can contribute to hyperoxia-induced airway dysfunction are still under investigation. Recent studies in mice show that hyperoxia increases brain-derived neurotrophic factor (BDNF), a growth factor that increases airway smooth muscle (ASM) proliferation and contractility. We assessed the mechanisms underlying effects of moderate hyperoxia (50% O2) on BDNF expression and secretion in developing human ASM. Hyperoxia increased BDNF secretion, but did not alter endogenous BDNF mRNA or intracellular protein levels. Exposure to hyperoxia significantly increased [Ca2+]i responses to histamine, an effect blunted by the BDNF chelator TrkB-Fc. Hyperoxia also increased ASM cAMP levels, associated with reduced PDE4 activity, but did not alter protein kinase A (PKA) activity or adenylyl cyclase mRNA levels. However, 50% O2 increased expression of Epac2, which is activated by cAMP and can regulate protein secretion. Silencing RNA studies indicated that Epac2, but not Epac1, is important for hyperoxia-induced BDNF secretion, while PKA inhibition did not influence BDNF secretion. In turn, BDNF had autocrine effects of enhancing ASM cAMP levels, an effect inhibited by TrkB and BDNF siRNAs. Together, these novel studies suggest that hyperoxia can modulate BDNF secretion, via cAMP-mediated Epac2 activation in ASM, resulting in a positive feedback effect of BDNF-mediated elevation in cAMP levels. The potential functional role of this pathway is to sustain BDNF secretion following hyperoxic stimulus, leading to enhanced ASM contractility and proliferation.

  11. Pro-region engineering for improved yeast display and secretion of brain derived neurotrophic factor.

    Science.gov (United States)

    Burns, Michael L; Malott, Thomas M; Metcalf, Kevin J; Puguh, Arthya; Chan, Jonah R; Shusta, Eric V

    2016-03-01

    Brain derived neurotrophic factor (BDNF) is a promising therapeutic candidate for a variety of neurological diseases. However, it is difficult to produce as a recombinant protein. In its native mammalian context, BDNF is first produced as a pro-protein with subsequent proteolytic removal of the pro-region to yield mature BDNF protein. Therefore, in an attempt to improve yeast as a host for heterologous BDNF production, the BDNF pro-region was first evaluated for its effects on BDNF surface display and secretion. Addition of the wild-type pro-region to yeast BDNF production constructs improved BDNF folding both as a surface-displayed and secreted protein in terms of binding its natural receptors TrkB and p75, but titers remained low. Looking to further enhance the chaperone-like functions provided by the pro-region, two rounds of directed evolution were performed, yielding mutated pro-regions that further improved the display and secretion properties of BDNF. Subsequent optimization of the protease recognition site was used to control whether the produced protein was in pro- or mature BDNF forms. Taken together, we have demonstrated an effective strategy for improving BDNF compatibility with yeast protein engineering and secretion platforms.

  12. Pleiotrophin promotes microglia proliferation and secretion of neurotrophic factors by activating extracellular signal-regulated kinase 1/2 pathway.

    Science.gov (United States)

    Miao, Jiayin; Ding, Minghui; Zhang, Aiwu; Xiao, Zijian; Qi, Weiwei; Luo, Ning; Di, Wei; Tao, Yuqian; Fang, Yannan

    2012-12-01

    Pleiotrophin (PTN) is an effective neuroprotective factor and its expression is strikingly increased in microglia after ischemia/reperfusion injury. However, whether PTN could provide neurotrophic support to neurons by regulating microglia function is not clear. In this study, we demonstrated that the expression of PTN was induced in microglia after oxygen-glucose deprivation/reperfusion. PTN promoted the proliferation of microglia by enhancing the G1 to S phase transition. PTN also stimulated the secretion of brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) and nerve growth factor (NGF) in microglia, but did not upregulate the expression of proinflammatory factors such as TNF-α, IL-1β and iNOS. Mechanistically, we found that PTN increased the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 in microglia in both concentration-dependent and time-dependent manners. In addition, ERK1/2 inhibitor U0126 abolished the proliferation and G1 to S phase transition of microglia stimulated by PTN, and inhibited the production of BDNF, CNTF and NGF induced by PTN. In conclusion, our results demonstrated that PTN-ERK1/2 pathway plays important role in regulating microglia growth and secretion of neurotrophic factors. These findings provide new insight into the neuroprotective role of PTN and suggest that PTN is a new target for therapeutic intervention of stroke.

  13. Effects of acetylcholine and electrical stimulation on glial cell line-derived neurotrophic factor production in skeletal muscle cells.

    Science.gov (United States)

    Vianney, John-Mary; Miller, Damon A; Spitsbergen, John M

    2014-11-01

    Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor required for survival of neurons in the central and peripheral nervous system. Specifically, GDNF has been characterized as a survival factor for spinal motor neurons. GDNF is synthesized and secreted by neuronal target tissues, including skeletal muscle in the peripheral nervous system; however, the mechanisms by which GDNF is synthesized and released by skeletal muscle are not fully understood. Previous results suggested that cholinergic neurons regulate secretion of GDNF by skeletal muscle. In the current study, GDNF production by skeletal muscle myotubes following treatment with acetylcholine was examined. Acetylcholine receptors on myotubes were identified with labeled alpha-bungarotoxin and were blocked using unlabeled alpha-bungarotoxin. The question of whether electrical stimulation has a similar effect to that of acetylcholine was also investigated. Cells were stimulated with voltage pulses; at 1 and 5 Hz frequencies for times ranging from 30 min to 48 h. GDNF content in myotubes and GDNF in conditioned culture medium were quantified by enzyme-linked immunosorbant assay. Results suggest that acetylcholine and short-term electrical stimulation reduce GDNF secretion, while treatment with carbachol or long-term electrical stimulation enhances GDNF production by skeletal muscle.

  14. Calcitonin gene-related peptide regulation of glial cell-line derived neurotrophic factor in differentiated rat myotubes.

    Science.gov (United States)

    Rosa, Elyse; Cha, Jieun; Bain, James R; Fahnestock, Margaret

    2015-03-01

    Glial cell-line derived neurotrophic factor (GDNF) is the most potent trophic factor for motoneuron survival and neuromuscular junction formation. GDNF is upregulated in injured or denervated skeletal muscle and returns to normal levels following reinnervation. However, the mechanism by which GDNF is regulated in denervated muscle is not well understood. The nerve-derived neurotransmitter calcitonin gene-related peptide (CGRP) is upregulated following neuromuscular injury and is subsequently released from motoneurons at the neuromuscular junction. CGRP also promotes nerve regeneration, but the mechanism is not well understood. The current study investigates whether this increase in CGRP regulates GDNF, thus playing a key role in promoting regeneration of injured nerves. This study demonstrates that CGRP increases GDNF secretion without affecting its transcription or translation. Rat L6 myoblasts were differentiated into myotubes and subsequently treated with CGRP. GDNF mRNA expression levels were quantified by quantitative real-time reverse transcription-polymerase chain reaction, and secreted GDNF was quantified in the conditioned medium by ELISA. CGRP treatment increased secreted GDNF protein without altering GDNF mRNA levels. The translational inhibitor cycloheximide did not affect CGRP-induced GDNF secreted protein levels, whereas the secretional inhibitor brefeldin A blocked the CGRP-induced increase in GDNF. This study highlights the importance of injury-induced upregulation of CGRP by exposing its ability to increase GDNF levels and demonstrates a secretional mechanism for regulation of this key regeneration-promoting neurotrophic factor.

  15. New insight in expression, transport, and secretion of brain-derived neurotrophic factor: Implications in brainrelated diseases

    Institute of Scientific and Technical Information of China (English)

    Naoki; Adachi; Tadahiro; Numakawa; Misty; Richards; Shingo; Nakajima; Hiroshi; Kunugi

    2014-01-01

    Brain-derived neurotrophic factor(BDNF) attracts increasing attention from both research and clinical fields because of its important functions in the central nervous system. An adequate amount of BDNF is critical to develop and maintain normal neuronal circuits in the brain. Given that loss of BDNF function has beenreported in the brains of patients with neurodegenerative or psychiatric diseases, understanding basic properties of BDNF and associated intracellular processes is imperative. In this review, we revisit the gene structure, transcription, translation, transport and secretion mechanisms of BDNF. We also introduce implications of BDNF in several brain-related diseases including Alzheimer’s disease, Huntington’s disease, depression and schizophrenia.

  16. Transfection of the glial cell line-derived neurotrophic factor gene promotes neuronal differentiation

    Institute of Scientific and Technical Information of China (English)

    Jie Du; Xiaoqing Gao; Li Deng; Nengbin Chang; Huailin Xiong; Yu Zheng

    2014-01-01

    Glial cell line-derived neurotrophic factor recombinant adenovirus vector-transfected bone marrow mesenchymal stem cells were induced to differentiate into neuron-like cells using inductive medium containing retinoic acid and epidermal growth factor. Cell viability, micro-tubule-associated protein 2-positive cell ratio, and the expression levels of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43 protein in the su-pernatant were signiifcantly higher in glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells compared with empty virus plasmid-transfected bone marrow mes-enchymal stem cells. Furthermore, microtubule-associated protein 2, glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43 mRNA levels in cell pellets were statistically higher in glial cell line-derived neurotrophic factor/bone marrow mesen-chymal stem cells compared with empty virus plasmid-transfected bone marrow mesenchymal stem cells. These results suggest that glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells have a higher rate of induction into neuron-like cells, and this enhanced differentiation into neuron-like cells may be associated with up-regulated expression of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43.

  17. APP-dependent glial cell line-derived neurotrophic factor gene expression drives neuromuscular junction formation.

    Science.gov (United States)

    Stanga, Serena; Zanou, Nadège; Audouard, Emilie; Tasiaux, Bernadette; Contino, Sabrina; Vandermeulen, Gaëlle; René, Frédérique; Loeffler, Jean-Philippe; Clotman, Frédéric; Gailly, Philippe; Dewachter, Ilse; Octave, Jean-Noël; Kienlen-Campard, Pascal

    2016-05-01

    Besides its crucial role in the pathogenesis of Alzheimer's disease, the knowledge of amyloid precursor protein (APP) physiologic functions remains surprisingly scarce. Here, we show that APP regulates the transcription of the glial cell line-derived neurotrophic factor (GDNF). APP-dependent regulation of GDNF expression affects muscle strength, muscular trophy, and both neuronal and muscular differentiation fundamental for neuromuscular junction (NMJ) maturation in vivo In a nerve-muscle coculture model set up to modelize NMJ formation in vitro, silencing of muscular APP induces a 30% decrease in secreted GDNF levels and a 40% decrease in the total number of NMJs together with a significant reduction in the density of acetylcholine vesicles at the presynaptic site and in neuronal maturation. These defects are rescued by GDNF expression in muscle cells in the conditions where muscular APP has been previously silenced. Expression of GDNF in muscles of amyloid precursor protein null mice corrected the aberrant synaptic morphology of NMJs. Our findings highlight for the first time that APP-dependent GDNF expression drives the process of NMJ formation, providing new insights into the link between APP gene regulatory network and physiologic functions.-Stanga, S., Zanou, N., Audouard, E., Tasiaux, B., Contino, S., Vandermeulen, G., René, F., Loeffler, J.-P., Clotman, F., Gailly, P., Dewachter, I., Octave, J.-N., Kienlen-Campard, P. APP-dependent glial cell line-derived neurotrophic factor gene expression drives neuromuscular junction formation.

  18. Effects of erythropoietin on cell cycle and neurotrophic factor secretion of Schwann cells in vitro%红细胞生成素对体外培养许旺细胞增殖周期及其分泌神经营养因子的影响

    Institute of Scientific and Technical Information of China (English)

    曹能力; 吴学建; 朱旭; 王楷

    2009-01-01

    Objective To observe the effects of EPO on cell cycle and the secretion of NGF and CNTF of SCs in vitro and explore how EPO improve peripheral nerve regeneration. Methods The sciatic nerves of the adult New Zealand rabbits were ligated and pre - degenerated. Mter a week, the pre - degenerated nerves were fetched and their ectoblastics were removed. We adopted a method of trasplant piece and speed difference adhestion to cultivte and purify SCs. Different concentrations of EPO were added into cutures of puri-fied SCs respectively. The cell cycle distribution of SCs.was observed by flow cytometry, and the level of NGFand CNTF in the culture was detected by ELSIA. Results In the EPO supplemention group,the number of SCs in S phase(S%) was significantly increased,G1% obviously decreased and the PrI[(S + G2M)%] enhanced when the SCs were stimulated. Conclusions EPO enhance SCs proliferation activity and increase the secretion of neurotrophic factors, which may be explain how EPO improve peripheral nerve regeneration.%目的 观察促红细胞生成素(EPO)对体外培养许旺细胞(SCs)增殖周期及其分泌神经生长因子(NGF)和睫状神经生长因子(CNTF)的影响,探讨EPO促进周围神经再生的机制.方法 对成年新西兰兔的坐骨神经进行结扎顸变性,1周后取预变性的坐骨神经,剥净神经外膜,用植块法、差速贴壁法培养纯化许旺细胞,向培养基中加入不同剂量的EPO,通过流式细胞术检测细胞增殖周期分布,应用ELISA测定培养上清液中的NGF和CNTF水平.结果 与对照组相比,实验组许旺细胞处于S期数量(S%)和增殖指数PrI值[(S+G2M)%]都有明显升高,培养上清液中的NGF和CNTF水平明显增高.结论 EPO可提高体外培养许旺细胞的增殖活性,并且能提高许旺细胞分泌神经营养因子的水平,这可能是其促进周围神经再生的作用机制之一.

  19. Glial cell derived neurotrophic factor induces spermatogonial stem cell marker genes in chicken mesenchymal stem cells.

    Science.gov (United States)

    Boozarpour, Sohrab; Matin, Maryam M; Momeni-Moghaddam, Madjid; Dehghani, Hesam; Mahdavi-Shahri, Naser; Sisakhtnezhad, Sajjad; Heirani-Tabasi, Asieh; Irfan-Maqsood, Muhammad; Bahrami, Ahmad Reza

    2016-06-01

    Mesenchymal stem cells (MSCs) are known with the potential of multi-lineage differentiation. Advances in differentiation technology have also resulted in the conversion of MSCs to other kinds of stem cells. MSCs are considered as a suitable source of cells for biotechnology purposes because they are abundant, easily accessible and well characterized cells. Nowadays small molecules are introduced as novel and efficient factors to differentiate stem cells. In this work, we examined the potential of glial cell derived neurotrophic factor (GDNF) for differentiating chicken MSCs toward spermatogonial stem cells. MSCs were isolated and characterized from chicken and cultured under treatment with all-trans retinoic acid (RA) or glial cell derived neurotrophic factor. Expression analysis of specific genes after 7days of RA treatment, as examined by RT-PCR, proved positive for some germ cell markers such as CVH, STRA8, PLZF and some genes involved in spermatogonial stem cell maintenance like BCL6b and c-KIT. On the other hand, GDNF could additionally induce expression of POU5F1, and NANOG as well as other genes which were induced after RA treatment. These data illustrated that GDNF is relatively more effective in diverting chicken MSCs towards Spermatogonial stem cell -like cells in chickens and suggests GDNF as a new agent to obtain transgenic poultry, nevertheless, exploitability of these cells should be verified by more experiments.

  20. LncRNA analysis of mouse spermatogonial stem cells following glial cell-derived neurotrophic factor treatment

    Directory of Open Access Journals (Sweden)

    Lufan Li

    2015-09-01

    Full Text Available Spermatonial stem cells (SSCs are the foundation of spermatogenesis. Long non-coding RNAs (lncRNAs are a class of non-coding RNAs with at least 200 bp in length, which play important roles in various biological processes. Growth factor glial cell line-derived neurotrophic factor (GDNF, secreted from testis niches, is critical for self-renewal of SSCs in vitro and in vivo. Using Illumina HiSeq™ 2000 high throughput sequencing, we found 55924 lncRNAs which were regulated by GDNF in SSCs in vitro; these included 21,929 known lncRNAs from NONCODE library (version 3.0 and 33,975 predicted lncRNAs which were identified using Coding Potential Calculator. Analyses of these data should provide new insights into regulated mechanism in SSC self-renewal and proliferation. The data have been deposited in the Gene Expression Omnibus (series GSE66998.

  1. Use of RNAi silencing to target preconditioned glial cell line-derived neurotrophic factor in neuronal apoptosis

    Institute of Scientific and Technical Information of China (English)

    Hongliang Guo; Zhongxin Xu; Xinhua Li; Jing Mang; Ying Xing; Jinting He; Guihua Xu; Shijun Yan; Lifeng Liu; Chunli Mei

    2011-01-01

    Several studies have suggested that exogenous glial cell line-derived neurotrophic factor may protect neurons from cerebral ischemic injury. However, the mechanisms underlying the neuroprotective effects of endogenous glial cell line-derived neurotrophic factor remain unclear. The present experiments sought to elucidate the influence of various conditioned media on neuronal apoptosis, using a normal culture medium for astrocytes, an astrocyte medium highly expressing glial cell line-derived neurotrophic factor, and an astrocyte medium in which glial cell line-derived neurotrophic factor expression was silenced using RNAi technology. The results confirmed that the use of RNAi silencing to target pretreated glial cell line-derived neurotrophic factor expression promoted neuronal apoptosis. In addition, oxygen and glucose deprivation preconditioning was found to upregulate glial cell line-derived neurotrophic factor expression, and significantly reduce neuronal apoptosis.

  2. Study of brain-derived neurotrophic factor gene transgenic neural stem cells in the rat retina

    Institute of Scientific and Technical Information of China (English)

    ZHOU Xue-mei; YUAN Hui-ping; WU Dong-lai; ZHOU Xin-rong; SUN Da-wei; LI Hong-yi; SHAO Zheng-bo

    2009-01-01

    Background Neural stem cells (NSCs) transplantation and gene therapy have been widely investigated for treating the cerebullar and myelonic injuries, however, studies on the ophthalmology are rare. The aim of this study was to investigate the migration and differentiation of brain-derived neurotrophic factor (BDNF) gene transgenic NSCs transplanted into the normal rat retinas. Methods NSCs were cultured and purified in vitro and infected with recombinant retrovirus pLXSN-BDNF and pLXSN respectively, to obtain the BDNF overexpressed NSCs (BDNF-NSCs) and control cells (p-NSCs). The expression of BDNF genes in two transgenic NSCs and untreated NSCs were measured by fluorescent quantitative polymerase chain reaction (FQ-PCR) and enzyme-linked immunosorbent assay (ELISA). BDNF-NSCs and NSCs were infected with adeno-associated viruses-enhanced green fluorescent protein (AAV-EGFP) to track them in vivo and served as donor cells for transplantation into the subretinal space of normal rat retinas, phosphated buffer solution (PBS) served as pseudo transplantation for a negative control. Survival, migration, and differentiation of donor cells in host retinas were observed and analyzed with Heidelberg retina angiograph (HRA) and immunohistochemistry, respectively. Results NSCs were purified successfully by limiting dilution assay. The expression of BDNF gene in BDNF-NSCs was the highest among three groups both at mRNA level tested by FQ-PCR (P<0.05) and at protein level measured by ELISA (P<0.05), which showed that BDNF was overexpressed in BDNF-NSCs. The results of HRA demonstrated that graft cells could survive well and migrate into the host retinas, while the immunohistochemical analysis revealed that transplanted BDNF-NSCs differentiated into neuron more efficiently compared with the control NSCs 2 months after transplantation. Conclusions The seed cells of NSCs highly secreting BDNF were established. BDNF can promote NSCs to migrate and differentiate into neural cells in

  3. Secretion of invertase in mitotic yeast cells.

    OpenAIRE

    Makarow, M

    1988-01-01

    In mammalian cells intracellular transport is inhibited during mitosis. Here we show that in the yeast Saccharomyces cerevisiae secretion continues uninterrupted during mitosis. S. cerevisiae cells were arrested in mitosis by treating wild-type cells with the microtubule-inhibitor nocodazole, or by incubating a temperature-sensitive cell division cycle mutant (cdc16) at the restrictive temperature. Secretion of invertase into the periplasmic space was equally efficient in mitotic and in unsyn...

  4. Cell Secretion: Current Structural and Biochemical Insights

    Directory of Open Access Journals (Sweden)

    Saurabh Trikha

    2010-01-01

    Full Text Available Essential physiological functions in eukaryotic cells, such as release of hormones and digestive enzymes, neurotransmission, and intercellular signaling, are all achieved by cell secretion. In regulated (calcium-dependent secretion, membrane-bound secretory vesicles dock and transiently fuse with specialized, permanent, plasma membrane structures, called porosomes or fusion pores. Porosomes are supramolecular, cup-shaped lipoprotein structures at the cell plasma membrane that mediate and control the release of vesicle cargo to the outside of the cell. The sizes of porosomes range from 150nm in diameter in acinar cells of the exocrine pancreas to 12nm in neurons. In recent years, significant progress has been made in our understanding of the porosome and the cellular activities required for cell secretion, such as membrane fusion and swelling of secretory vesicles. The discovery of the porosome complex and the molecular mechanism of cell secretion are summarized in this article.

  5. Glial cell line-derived neurotrophic factor influences proliferation of osteoblastic cells.

    Science.gov (United States)

    Gale, Zoe; Cooper, Paul R; Scheven, Ben A

    2012-02-01

    Little is known about the role of neurotrophic growth factors in bone metabolism. This study investigated the short-term effects of glial cell line-derived neurotrophic factor (GDNF) on calvarial-derived MC3T3-E1 osteoblasts. MC3T3-E1 expressed GDNF as well as its canonical receptors, GFRα1 and RET. Addition of recombinant GDNF to cultures in serum-containing medium modestly inhibited cell growth at high concentrations; however, under serum-free culture conditions GDNF dose-dependently increased cell proliferation. GDNF effects on cell growth were inversely correlated with its effect on alkaline phosphatase (AlP) activity showing a significant dose-dependent inhibition of relative AlP activity with increasing concentrations of GDNF in serum-free culture medium. Live/dead and lactate dehydrogenase assays demonstrated that GDNF did not significantly affect cell death or survival under serum-containing and serum-free conditions. The effect of GDNF on cell growth was abolished in the presence of inhibitors to GFRα1 and RET indicating that GDNF stimulated calvarial osteoblasts via its canonical receptors. Finally, this study found that GDNF synergistically increased tumor necrosis factor-α (TNF-α)-stimulated MC3T3-E1 cell growth suggesting that GDNF interacted with TNF-α-induced signaling in osteoblastic cells. In conclusion, this study provides evidence for a direct, receptor-mediated effect of GDNF on osteoblasts highlighting a novel role for GDNF in bone physiology.

  6. Effect of platelet-rich plasma (PRP) concentration on proliferation, neurotrophic function and migration of Schwann cells in vitro.

    Science.gov (United States)

    Zheng, Canbin; Zhu, Qingtang; Liu, Xiaolin; Huang, Xijun; He, Caifeng; Jiang, Li; Quan, Daping; Zhou, Xiang; Zhu, Zhaowei

    2016-05-01

    Platelet-rich plasma (PRP) contains various growth factors and appears to have the potential to promote peripheral nerve regeneration, but evidence is lacking regarding its biological effect on Schwann cells (SCs). The present study was designed to investigate the effect of PRP concentration on SCs in order to determine the plausibility of using this plasma-derived therapy for peripheral nerve injury. PRP was obtained from rats by double-step centrifugation and was characterized by determining platelet numbers and growth factor concentrations. Primary cultures of rat SCs were exposed to various concentrations of PRP (40%, 20%, 10%, 5% and 2.5%). Cell proliferation assays and flow cytometry were performed to study to assess SC proliferation. Quantitative real-time PCR and ELISA analysis were performed to determine the ability of PRP to induce SCs to produce nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF). Microchemotaxis assay was used to analyse the cell migration capacity. The results obtained indicated that the platelet concentration and growth factors in our PRP preparations were significantly higher than in whole blood. Cell culture experiments showed that 2.5-20% PRP significantly stimulated SC proliferation and migration compared to untreated controls in a dose-dependent manner. In addition, the expression and secretion of NGF and GDNF were significantly increased. However, the above effects of SCs were suppressed by high PRP concentrations (40%). In conclusion, the appropriate concentration of PRP had the potency to stimulate cell proliferation, induced the synthesis of neurotrophic factors and significantly increased migration of SCs dose-dependently. Copyright © 2013 John Wiley & Sons, Ltd.

  7. S100B protein, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor in human milk.

    Directory of Open Access Journals (Sweden)

    Ruisong Li

    Full Text Available BACKGROUND: Human milk contains a wide variety of nutrients that contribute to the fulfillment of its functions, which include the regulation of newborn development. However, few studies have investigated the concentrations of S100B protein, brain-derived neurotrophic factor (BDNF, and glial cell line-derived neurotrophic factor (GDNF in human milk. The associations of the concentrations of S100B protein, BDNF, and GDNF with maternal factors are not well explored. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the concentrations of S100B protein, BDNF, and GDNF in human milk and characterize the maternal factors associated with their levels in human milk, human milk samples were collected at days 3, 10, 30, and 90 after parturition. Levels of S100B protein, BDNF, and GDNF, and their mRNAs in the samples were detected. Then, these concentrations were compared with lactation and other maternal factors. S100B protein levels in human milk samples collected at 3, 10, 30, and 90 d after parturition were 1249.79±398.10, 1345.05±539.16, 1481.83±573.30, and 1414.39±621.31 ng/L, respectively. On the other hand, the BDNF concentrations in human milk samples were 10.99±4.55, 13.01±5.88, 13.35±6.43, and 2.83±5.47 µg/L, while those of GDNF were 10.90±1.65, 11.38±1., 11.29±3.10, and 11.40±2.21 g/L for the same time periods. Maternal post-pregnancy body mass index was positively associated with S100B levels in human milk (r = 0.335, P = 0.030<0.05. In addition, there was a significant correlation between the levels of S100B protein and BDNF (z = 2.09, P = 0.037<0.05. Delivery modes were negatively associated with the concentration of GDNF in human milk. CONCLUSIONS: S100B protein, BDNF, and GDNF are present in all samples of human milk, and they may be responsible for the long term effects of breast feeding.

  8. Long-term survival of encapsulated GDNF secreting cells implanted within the striatum of parkinsonized rats.

    Science.gov (United States)

    Grandoso, Laura; Ponce, Sara; Manuel, Ivan; Arrúe, Aurora; Ruiz-Ortega, Jose A; Ulibarri, Isabel; Orive, Gorka; Hernández, Rosa M; Rodríguez, Alicia; Rodríguez-Puertas, Rafael; Zumárraga, Mercedes; Linazasoro, Gurutz; Pedraz, Jose Luis; Ugedo, Luisa

    2007-10-01

    Several findings suggest that glial cell line-derived neurotrophic factor (GDNF) may be a useful tool to treat parkinsonism by acting as a neuroprotective and neurotrophic factor for dopaminergic neurotransmission systems. In the present study, we implanted alginate-poly-L-lysine-alginate microcapsules containing immobilized Fischer rat 3T3 fibroblasts transfected to produce GDNF in vitro into the striatum of 6-hydroxydopamine (6-OHDA) lesioned rats. Microencapsulated GDNF secreting cells were stable for at least 3 weeks in vitro. Intrastriatal implantation of microencapsulated GDNF secreting cells into 6-OHDA lesioned rats resulted in a decrease in apomorphine-induced rotations by 84%, 64%, 84%, 60% and 52% (2, 5, 8, 16 and 24 weeks, respectively) with respect to the value before implantation and with respect to the value obtained from the empty microcapsule implanted-group at each time point. Six months after transplantation, immunohistochemical detection of GDNF revealed strong immunoreactivity in the striatal tissue surrounding the microcapsules in the absence of tissue damage due to microcapsule implantation. No changes in the levels of dopamine and its metabolites or of tyrosine hydroxylase immunoreactivity were detected in the striatum. In summary, the implantation of microencapsulated GDNF secreting cells allows the delivery of this molecule into the rat striatum for at least 6 months and results in substantial behavioral improvement.

  9. Efficient Transduction of Feline Neural Progenitor Cells for Delivery of Glial Cell Line-Derived Neurotrophic Factor Using a Feline Immunodeficiency Virus-Based Lentiviral Construct

    Directory of Open Access Journals (Sweden)

    X. Joann You

    2011-01-01

    Full Text Available Work has shown that stem cell transplantation can rescue or replace neurons in models of retinal degenerative disease. Neural progenitor cells (NPCs modified to overexpress neurotrophic factors are one means of providing sustained delivery of therapeutic gene products in vivo. To develop a nonrodent animal model of this therapeutic strategy, we previously derived NPCs from the fetal cat brain (cNPCs. Here we use bicistronic feline lentiviral vectors to transduce cNPCs with glial cell-derived neurotrophic factor (GDNF together with a GFP reporter gene. Transduction efficacy is assessed, together with transgene expression level and stability during induction of cellular differentiation, together with the influence of GDNF transduction on growth and gene expression profile. We show that GDNF overexpressing cNPCs expand in vitro, coexpress GFP, and secrete high levels of GDNF protein—before and after differentiation—all qualities advantageous for use as a cell-based approach in feline models of neural degenerative disease.

  10. Effect of glial cell line-derived neurotrophic factor on retinal function after experimental branch retinal vein occlusion

    DEFF Research Database (Denmark)

    Ejstrup, Rasmus; Dornonville de la Cour, Morten; Kyhn, Maria Voss;

    2012-01-01

    The objective of the study was to investigate the effect of glial cell line-derived neurotrophic factor (GDNF) on the multifocal electroretinogram (mfERG) following an induced branch retinal vein occlusion (BRVO) in pigs.......The objective of the study was to investigate the effect of glial cell line-derived neurotrophic factor (GDNF) on the multifocal electroretinogram (mfERG) following an induced branch retinal vein occlusion (BRVO) in pigs....

  11. The SNARE machinery in mast cell secretion

    Directory of Open Access Journals (Sweden)

    Axel eLorentz

    2012-06-01

    Full Text Available Mast cells are known as inflammatory cells which exert their functions in allergic and anaphylactic reactions by secretion of numerous inflammatory mediators. During an allergic response, the high-affinity IgE receptor, FcεRI, becomes cross-linked by receptor-bound IgE and antigen resulting in immediate release of pre-synthesized mediators – stored in granules – as well as in de novo synthesis of various mediators like cytokines and chemokines. Soluble N-ethylmaleimide-Sensitive Factor (NSF Attachment Protein (SNAP Receptors (SNARE proteins were found to play a central role in regulating membrane fusion events during exocytosis. In addition, several accessory regulators like Munc13, Munc18, Rab GTPases, SCAMPs, complexins or synaptotagmins were found to be involved in membrane fusion. In this review we summarize our current knowledge about the SNARE machinery and its mechanism of action in mast cell secretion.

  12. 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.

  13. Axonal localization of Ca2+-dependent activator protein for secretion 2 is critical for subcellular locality of brain-derived neurotrophic factor and neurotrophin-3 release affecting proper development of postnatal mouse cerebellum.

    Directory of Open Access Journals (Sweden)

    Tetsushi Sadakata

    Full Text Available Ca2+-dependent activator protein for secretion 2 (CAPS2 is a protein that is essential for enhanced release of brain-derived neurotrophic factor (BDNF and neurotrophin-3 (NT-3 from cerebellar granule cells. We previously identified dex3, a rare alternative splice variant of CAPS2, which is overrepresented in patients with autism and is missing an exon 3 critical for axonal localization. We recently reported that a mouse model CAPS2Δex3/Δex3 expressing dex3 showed autistic-like behavioral phenotypes including impaired social interaction and cognition and increased anxiety in an unfamiliar environment. Here, we verified impairment in axonal, but not somato-dendritic, localization of dex3 protein in cerebellar granule cells and demonstrated cellular and physiological phenotypes in postnatal cerebellum of CAPS2Δex3/Δex3 mice. Interestingly, both BDNF and NT-3 were markedly reduced in axons of cerebellar granule cells, resulting in a significant decrease in their release. As a result, dex3 mice showed developmental deficits in dendritic arborization of Purkinje cells, vermian lobulation and fissurization, and granule cell precursor proliferation. Paired-pulse facilitation at parallel fiber-Purkinje cell synapses was also impaired. Together, our results indicate that CAPS2 plays an important role in subcellular locality (axonal vs. somato-dendritic of enhanced BDNF and NT-3 release, which is indispensable for proper development of postnatal cerebellum.

  14. Stem cell-based delivery of brain-derived neurotrophic factor gene in the rat retina.

    Science.gov (United States)

    Park, Hae-Young Lopilly; Kim, Jie Hyun; Sun Kim, Hwa; Park, Chan Kee

    2012-08-21

    As an alternative to a viral vector, the application of stem cells to transfer specific genes is under investigation in various organs. Using this strategy may provide more effective method to supply neurotrophic factor to the neurodegenerative diseases caused by neurotrophic factor deprivation. This study investigated the possibility and efficacy of stem cell-based delivery of the brain-derived neurotrophic factor (BDNF) gene to rat retina. Rat BDNF cDNA was transduced into rat bone marrow mesenchymal stem cells (rMSCs) using a retroviral vector. Its incorporation into the experimental rat retina and the expression of BDNF after intravitreal injection or subretinal injection were detected by real-time PCR, western blot analysis, and immunohistochemical staining. For the incorporated rMSCs, retinal-specific marker staining was performed to investigate the changes in morphology and the characteristics of the stem cells. Transduction of the rMSCs by retrovirus was effective, and the transduced rMSCs expressed high levels of the BDNF gene and protein. The subretinal injection of rMSCs produced rMSC migration and incorporation into the rat retina (about 15.7% incorporation rate), and retinal BDNF mRNA and protein expression was increased at 4 weeks after transplantation. When subretinal injection of rMSCs was applied to axotomized rat retina, it significantly increased the expression of BDNF until 4 weeks after transplantation. Some of the transplanted rMSCs exhibited morphological changes, but the retinal-specific marker stain was not sufficient to indicate whether neuronal differentiation had occurred. Using mesenchymal stem cells to deliver the BDNF gene to the retina may provide new treatment for glaucoma.

  15. Stem cells modified by brain-derived neurotrophic fac-tor to promote stem cells differentiation into neurons and enhance neuromotor function after brain injury

    Institute of Scientific and Technical Information of China (English)

    ZHANG Sai; LIU Xiao-zhi; LIU Zhen-lin; WANG Yan-min; HU Qun-liang; MA Tie-zhu; SUN Shi-zhong

    2009-01-01

    Objective: To promote stem cells differentiation into neurons and enhance neuromotor function after brain in-jury through brain-derived neurotrophic factor (BDNF) induction.Methods: Recombinant adenovirus vector was ap-plied to the transfection of BDNF into human-derived um-bilical cord mesenchymal stem cells (UCMSCs). Enzyme linked immunosorbent assay (ELISA) was used to deter-mine the secretion phase of BDNF. The brain injury model of athymic mice induced by hydraulic pressure percussion was established for transplantation of stem cells into the edge of injury site. Nerve function scores were obtained, and the expression level of transfected and non-transfected BDNF, proportion of neuron specific enolase (NSE) andglial fibrillary acidic protein (GFAP), and the number of apoptosis cells were compared respectively. Results: The BDNF expression achieved its stabiliza-tion at a high level 72 hours after gene transfection. The mouse obtained a better score of nerve function, and the proportion of the NSE-positive cells increased significantly (P<0.05), but GFAP-positive cells decreased in BDNF-UCMSCs group compared with the other two groups (P<0.05). At the site of high expression of BDNF, the number of apoptosis cells decreased markedly.Conclusion: BDNF gene can promote the differentia-tion of the stem cells into neurons rather than gliai cells, and enhance neuromotor function after brain injury.

  16. Adenovirus-mediated human brain-derived neurotrophic factor gene-modified bone marrow mesenchymal stem cell transplantation for spinal cord injury

    Institute of Scientific and Technical Information of China (English)

    Changsheng Wang; Jianhua Lin; Chaoyang Wu; Rongsheng Chen

    2011-01-01

    Rat bone marrow mesenchymal stem cells expressing brain-derived neurotrophic factor were successfully obtained using a gene transfection method, then intravenously transplanted into rats with spinal cord injury. At 1, 3, and 5 weeks after transplantation, the expression of ??brain-derived neurotrophic factor and neurofilament-200 was upregulated in the injured spinal cord, spinal cord injury was alleviated, and Basso-Beattie-Bresnahan scores of hindlimb motor function were significantly increased. This evidence suggested that intravenous transplantation of adenovirus- mediated brain-derived neurotrophic factor gene-modified rat bone marrow mesenchymal stem cells could play a dual role, simultaneously providing neural stem cells and neurotrophic factors.

  17. Glial cell line-derived neurotrophic factor induces cell proliferation in the mouse urogenital sinus.

    Science.gov (United States)

    Park, Hyun-Jung; Bolton, Eric C

    2015-02-01

    Glial cell line-derived neurotrophic factor (GDNF) is a TGFβ family member, and GDNF signals through a glycosyl-phosphatidylinositol-linked cell surface receptor (GFRα1) and RET receptor tyrosine kinase. GDNF signaling plays crucial roles in urogenital processes, ranging from cell fate decisions in germline progenitors to ureteric bud outgrowth and renal branching morphogenesis. Gene ablation studies in mice have revealed essential roles for GDNF signaling in urogenital development, although its role in prostate development is unclear. We investigated the functional role of GDNF signaling in the urogenital sinus (UGS) and the developing prostate of mice. GDNF, GFRα1, and RET show time-specific and cell-specific expression during prostate development in vivo. In the UGS, GDNF and GFRα1 are expressed in the urethral mesenchyme (UrM) and epithelium (UrE), whereas RET is restricted to the UrM. In each lobe of the developing prostate, GDNF and GFRα1 expression declines in the epithelium and becomes restricted to the stroma. Using a well-established organ culture system, we determined that exogenous GDNF increases proliferation of UrM and UrE cells, altering UGS morphology. With regard to mechanism, GDNF signaling in the UrM increased RET expression and phosphorylation of ERK1/2. Furthermore, inhibition of RET kinase activity or ERK kinases suppressed GDNF-induced proliferation of UrM cells but not UrE cells. We therefore propose that GDNF signaling in the UGS increases proliferation of UrM and UrE cells by different mechanisms, which are distinguished by the role of RET receptor tyrosine kinase and ERK kinase signaling, thus implicating GDNF signaling in prostate development and growth.

  18. Involvement of stathmin 1 in the neurotrophic effects of PACAP in PC12 cells.

    Science.gov (United States)

    Dejda, Agnieszka; Chan, Philippe; Seaborn, Tommy; Coquet, Laurent; Jouenne, Thierry; Fournier, Alain; Vaudry, Hubert; Vaudry, David

    2010-09-01

    Rat pheochromocytoma PC12 cells have been widely used to investigate the neurotrophic activities of pituitary adenylate cyclase-activating polypeptide (PACAP). In particular, PACAP has been shown to promote differentiation and to inhibit apoptosis of PC12 cells. In order to identify the mechanisms mediating these effects, we sought for proteins that are phosphorylated upon PACAP treatment. High-performance liquid chromatography and 2D gel electrophoresis analysis, coupled with mass spectrometry, revealed that stathmin 1 is strongly phosphorylated within only 5 min of exposure to PACAP. Western blot experiments confirmed that PACAP induced a robust phosphorylation of stathmin 1 in a time-dependent manner. On the other hand, PACAP decreased stathmin 1 gene expression. Investigations of the signaling mechanisms known to be activated by PACAP revealed that phosphorylation of stathmin 1 was mainly mediated through the protein kinase A and mitogen-activated protein kinase pathways. Blockage of stathmin 1 expression with small interfering RNA did not affect PC12 cell differentiation induced by PACAP but reduced the ability of the peptide to inhibit caspase 3 activity and significantly decreased its neuroprotective action. Taken together, these data demonstrate that stathmin 1 is involved in the neurotrophic effect of PACAP in PC12 cells.

  19. Repair of peripheral nerve defects with chemically extracted acellular nerve allografts loaded with neurotrophic factors-transfected bone marrow mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Yan-ru Zhang; Ka Ka; Ge-chen Zhang; Hui Zhang; Yan Shang; Guo-qiang Zhao; Wen-hua Huang

    2015-01-01

    Chemically extracted acellular nerve allografts loaded with brain-derived neurotrophic fac-tor-transfected or ciliary neurotrophic factor-transfected bone marrow mesenchymal stem cells have been shown to repair sciatic nerve injury better than chemically extracted acellular nerve allografts alone, or chemically extracted acellular nerve allografts loaded with bone marrow mesenchymal stem cells. We hypothesized that these allografts compounded with both brain-derived neurotrophic factor- and ciliary neurotrophic factor-transfected bone marrow mesenchymal stem cells may demonstrate even better effects in the repair of peripheral nerve injury. We cultured bone marrow mesenchymal stem cells expressing brain-derived neuro-trophic factor and/or ciliary neurotrophic factor and used them to treat sciatic nerve injury in rats. We observed an increase in sciatic functional index, triceps wet weight recovery rate, myelin thickness, number of myelinated nerve ifbers, amplitude of motor-evoked potentials and nerve conduction velocity, and a shortened latency of motor-evoked potentials when al-lografts loaded with both neurotrophic factors were used, compared with allografts loaded with just one factor. Thus, the combination of both brain-derived neurotrophic factor and cili-ary neurotrophic factor-transfected bone marrow mesenchymal stem cells can greatly improve nerve injury.

  20. The intricacies of neurotrophic factor therapy for retinal ganglion cell rescue in glaucoma:a case for gene therapy

    Institute of Scientific and Technical Information of China (English)

    Marianna Foldvari; Ding Wen Chen

    2016-01-01

    Regeneration of damaged retinal ganglion cells (RGC) and their axons is an important aspect of reversing vision loss in glaucoma patients. While current therapies can effectively lower intraocular pressure, they do not provide extrinsic support to RGCs to actively aid in their protection and regeneration. The unmet need could be addressed by neurotrophic factor gene therapy, where plasmid DNA, encoding neurotrophic factors, is delivered to retinal cells to maintain sufifcient levels of neurotrophins in the retina. In this review, we aim to describe the intricacies in the design of the therapy including: the choice of neurotrophic factor, the site and route of administration and target cell populations for gene delivery. Furthermore, we also dis-cuss the challenges currently being faced in RGC-related therapy development with special considerations to the existence of multiple RGC subtypes and the lack of efifcient and representativein vitro models for rapid and reliable screening in the drug development process.

  1. The intricacies of neurotrophic factor therapy for retinal ganglion cell rescue in glaucoma: a case for gene therapy

    Directory of Open Access Journals (Sweden)

    Marianna Foldvari

    2016-01-01

    Full Text Available Regeneration of damaged retinal ganglion cells (RGC and their axons is an important aspect of reversing vision loss in glaucoma patients. While current therapies can effectively lower intraocular pressure, they do not provide extrinsic support to RGCs to actively aid in their protection and regeneration. The unmet need could be addressed by neurotrophic factor gene therapy, where plasmid DNA, encoding neurotrophic factors, is delivered to retinal cells to maintain sufficient levels of neurotrophins in the retina. In this review, we aim to describe the intricacies in the design of the therapy including: the choice of neurotrophic factor, the site and route of administration and target cell populations for gene delivery. Furthermore, we also discuss the challenges currently being faced in RGC-related therapy development with special considerations to the existence of multiple RGC subtypes and the lack of efficient and representative in vitro models for rapid and reliable screening in the drug development process.

  2. Glial cell line-derived neurotrophic factor protects against high-fat diet-induced obesity.

    Science.gov (United States)

    Mwangi, Simon Musyoka; Nezami, Behtash Ghazi; Obukwelu, Blessing; Anitha, Mallappa; Marri, Smitha; Fu, Ping; Epperson, Monica F; Le, Ngoc-Anh; Shanmugam, Malathy; Sitaraman, Shanthi V; Tseng, Yu-Hua; Anania, Frank A; Srinivasan, Shanthi

    2014-03-01

    Obesity is a growing epidemic with limited effective treatments. The neurotrophic factor glial cell line-derived neurotrophic factor (GDNF) was recently shown to enhance β-cell mass and improve glucose control in rodents. Its role in obesity is, however, not well characterized. In this study, we investigated the ability of GDNF to protect against high-fat diet (HFD)-induced obesity. GDNF transgenic (Tg) mice that overexpress GDNF under the control of the glial fibrillary acidic protein promoter and wild-type (WT) littermates were maintained on a HFD or regular rodent diet for 11 wk, and weight gain, energy expenditure, and insulin sensitivity were monitored. Differentiated mouse brown adipocytes and 3T3-L1 white adipocytes were used to study the effects of GDNF in vitro. Tg mice resisted the HFD-induced weight gain, insulin resistance, dyslipidemia, hyperleptinemia, and hepatic steatosis seen in WT mice despite similar food intake and activity levels. They exhibited significantly (PGDNF enhanced β-adrenergic-mediated cAMP release in brown adipocytes and suppressed lipid accumulation in differentiated 3T3L-1 cells through a p38MAPK signaling pathway. Our studies demonstrate a novel role for GDNF in the regulation of high-fat diet-induced obesity through increased energy expenditure. They show that GDNF and its receptor agonists may be potential targets for the treatment or prevention of obesity.

  3. In vivo induction of glial cell proliferation and axonal outgrowth and myelination by brain-derived neurotrophic factor.

    NARCIS (Netherlands)

    Groot, D.M. de; Coenen, A.J.M.; Verhofstad, A.A.J.; Herp, F. van; Martens, G.J.M.

    2006-01-01

    Brain-derived neurotrophic factor (BDNF) belongs to the neurotrophin family of neuronal cell survival and differentiation factors but is thought to be involved in neuronal cell proliferation and myelination as well. To explore the role of BDNF in vivo, we employed the intermediate pituitary melanotr

  4. Cell-based delivery of brain-derived neurotrophic factor in experimental allergic encephalomyelitis.

    Science.gov (United States)

    Makar, Tapas K; Nimmagadda, Vamshi K C; Trisler, David; Bever, Christopher T

    2014-08-01

    Brain-derived neurotrophic factor (BDNF) is a pleiotropic cytokine with neuroprotective properties that has been identified as a potential therapeutic agent for diseases of the central nervous system (CNS). The use of BDNF has been limited by a short serum half-life and poor penetration of the blood-brain barrier. To address this limitation we have explored cell-based approaches to delivery. We have used experimental allergic encephalomyelitis (EAE), an inflammatory disease of the CNS, as a model system. We engineered hematopoietic stem cells to produce BDNF to determine the feasibility and effectiveness of cell-based delivery of BDNF into the CNS in EAE. We review those studies here.

  5. Glial cell line-derived neurotrophic factor (GDNF) enhances sympathetic neurite growth in rat hearts at early developmental stages.

    Science.gov (United States)

    Miwa, Keiko; Lee, Jong-Kook; Takagishi, Yoshiko; Opthof, Tobias; Fu, Xianming; Kodama, Itsuo

    2010-12-01

    Molecular signaling of sympathetic innervation of myocardium is an unresolved issue. The purpose of this study was to investigate the effect of neurotrophic factors on sympathetic neurite growth towards cardiomyocytes. Cardiomyocytes (CMs) and sympathetic neurons (SNs) were isolated from neonatal rat hearts and superior cervical ganglia, and were co-cultured, either in a random or localized way. Neurite growth from SNs toward CMs was assessed by immunohistochemistry for neurofilament M and α-actinin in response to neurotrophic factors-nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF) and a chemical repellent, semaphorin 3A. As a result, GDNF as well as NGF and BDNF stimulated neurite growth. GDNF enhanced neurite outgrowth even under the NGF-depleted culture condition, excluding an indirect effect of GDNF via NGF. Quantification of mRNA and protein by real-time PCR and immunohistochemistry at different developmental stages revealed that GDNF is abundantly expressed in the hearts of embryos and neonates, but not in adult hearts. GDNF plays an important role in inducing cardiac sympathetic innervation at the early developmental stages. A possible role in (re)innervation of injured or transplanted or cultured and transplanted myocardium may deserve investigation.

  6. Axon guidance of sympathetic neurons to cardiomyocytes by glial cell line-derived neurotrophic factor (GDNF).

    Science.gov (United States)

    Miwa, Keiko; Lee, Jong-Kook; Takagishi, Yoshiko; Opthof, Tobias; Fu, Xianming; Hirabayashi, Masumi; Watabe, Kazuhiko; Jimbo, Yasuhiko; Kodama, Itsuo; Komuro, Issei

    2013-01-01

    Molecular signaling of cardiac autonomic innervation is an unresolved issue. Here, we show that glial cell line-derived neurotrophic factor (GDNF) promotes cardiac sympathetic innervation in vitro and in vivo. In vitro, ventricular myocytes (VMs) and sympathetic neurons (SNs) isolated from neonatal rat ventricles and superior cervical ganglia were cultured at a close distance. Then, morphological and functional coupling between SNs and VMs was assessed in response to GDNF (10 ng/ml) or nerve growth factor (50 ng/ml). As a result, fractions of neurofilament-M-positive axons and synapsin-I-positive area over the surface of VMs were markedly increased with GDNF by 9-fold and 25-fold, respectively, compared to control without neurotrophic factors. Pre- and post-synaptic stimulation of β1-adrenergic receptors (BAR) with nicotine and noradrenaline, respectively, resulted in an increase of the spontaneous beating rate of VMs co-cultured with SNs in the presence of GDNF. GDNF overexpressing VMs by adenovirus vector (AdGDNF-VMs) attracted more axons from SNs compared with mock-transfected VMs. In vivo, axon outgrowth toward the denervated myocardium in adult rat hearts after cryoinjury was also enhanced significantly by adenovirus-mediated GDNF overexpression. GDNF acts as a potent chemoattractant for sympathetic innervation of ventricular myocytes, and is a promising molecular target for regulation of cardiac function in diseased hearts.

  7. Neurogenic and neurotrophic effects of BDNF peptides in mouse hippocampal primary neuronal cell cultures.

    Directory of Open Access Journals (Sweden)

    Maria del Carmen Cardenas-Aguayo

    Full Text Available The level of brain-derived neurotrophic factor (BDNF, a member of the neurotrophin family, is down regulated in Alzheimer's disease (AD, Parkinson's disease (PD, depression, stress, and anxiety; conversely the level of this neurotrophin is increased in autism spectrum disorders. Thus, modulating the level of BDNF can be a potential therapeutic approach for nervous system pathologies. In the present study, we designed five different tetra peptides (peptides B-1 to B-5 corresponding to different active regions of BDNF. These tetra peptides were found to be non-toxic, and they induced the expression of neuronal markers in mouse embryonic day 18 (E18 primary hippocampal neuronal cultures. Additionally, peptide B-5 induced the expression of BDNF and its receptor, TrkB, suggesting a positive feedback mechanism. The BDNF peptides induced only a moderate activation (phosphorylation at Tyr 706 of the TrkB receptor, which could be blocked by the Trk's inhibitor, K252a. Peptide B-3, when combined with BDNF, potentiated the survival effect of this neurotrophin on H(2O(2-treated E18 hippocampal cells. Peptides B-3 and B-5 were found to work as partial agonists and as partial antagonists competing with BDNF to activate the TrkB receptor in a dose-dependent manner. Taken together, these results suggest that the described BDNF tetra peptides are neurotrophic, can modulate BDNF signaling in a partial agonist/antagonist way, and offer a novel therapeutic approach to neural pathologies where BDNF levels are dysregulated.

  8. Axon guidance of sympathetic neurons to cardiomyocytes by glial cell line-derived neurotrophic factor (GDNF.

    Directory of Open Access Journals (Sweden)

    Keiko Miwa

    Full Text Available Molecular signaling of cardiac autonomic innervation is an unresolved issue. Here, we show that glial cell line-derived neurotrophic factor (GDNF promotes cardiac sympathetic innervation in vitro and in vivo. In vitro, ventricular myocytes (VMs and sympathetic neurons (SNs isolated from neonatal rat ventricles and superior cervical ganglia were cultured at a close distance. Then, morphological and functional coupling between SNs and VMs was assessed in response to GDNF (10 ng/ml or nerve growth factor (50 ng/ml. As a result, fractions of neurofilament-M-positive axons and synapsin-I-positive area over the surface of VMs were markedly increased with GDNF by 9-fold and 25-fold, respectively, compared to control without neurotrophic factors. Pre- and post-synaptic stimulation of β1-adrenergic receptors (BAR with nicotine and noradrenaline, respectively, resulted in an increase of the spontaneous beating rate of VMs co-cultured with SNs in the presence of GDNF. GDNF overexpressing VMs by adenovirus vector (AdGDNF-VMs attracted more axons from SNs compared with mock-transfected VMs. In vivo, axon outgrowth toward the denervated myocardium in adult rat hearts after cryoinjury was also enhanced significantly by adenovirus-mediated GDNF overexpression. GDNF acts as a potent chemoattractant for sympathetic innervation of ventricular myocytes, and is a promising molecular target for regulation of cardiac function in diseased hearts.

  9. Intraspinal transplantation of motoneuron-like cell combined with delivery of polymer-based glial cell line-derived neurotrophic factor for repair of spinal cord contusion injury

    Institute of Scientific and Technical Information of China (English)

    Alireza Abdanipour; Taki Tiraihi; Taher Taheri

    2014-01-01

    To evaluate the effects of glial cell line-derived neurotrophic factor transplantation combined with adipose-derived stem cells-transdifferentiated motoneuron delivery on spinal cord con-tusion injury, we developed rat models of spinal cord contusion injury, 7 days later, injected adipose-derived stem cells-transdifferentiated motoneurons into the epicenter, rostral and caudal regions of the impact site and simultaneously transplanted glial cell line-derived neuro-trophic factor-gelfoam complex into the myelin sheath. Motoneuron-like cell transplantation combined with glial cell line-derived neurotrophic factor delivery reduced cavity formations and increased cell density in the transplantation site. The combined therapy exhibited superior promoting effects on recovery of motor function to transplantation of glial cell line-derived neurotrophic factor, adipose-derived stem cells or motoneurons alone. These ifndings suggest that motoneuron-like cell transplantation combined with glial cell line-derived neurotrophic factor delivery holds a great promise for repair of spinal cord injury.

  10. Pharmacokinetics of intravitreal glial cell line-derived neurotrophic factor: experimental studies in pigs

    DEFF Research Database (Denmark)

    Ejstrup, Rasmus; Kiilgaard, J F; Tucker, B A;

    2010-01-01

    The purpose of this study was to establish the intravitreal (ITV) pharmacokinetics of glial cell line-derived neurotrophic factor (GDNF) and observe possible complications after ITV injection. Twenty Danish landrace pigs and 34 eyes were included in the study; 30 were injected with 100 ng of GDNF......, two controls were injected without GDNF, and two received no injection. At post-injection time points of 1, 2, 3, 6 hours (h), 1, 2, 4 or 7 days (d) eyes were enucleated and the ITV concentration of GDNF (cGDNF) was determined by enzyme-linked immunosorbent assay, and activity was tested using...... a retinal ganglion cell line (RGC5) bioassay. Indirect ophthalmoscopy, intraocular pressure assessment, and fundus photography were performed before enucleation. There was initial variability in the cGDNF, but after 24h GDNF was cleared in a monoexponential fashion with a half-life of 37 h (CL 33-43 h...

  11. Human umbilical cord blood stem cells and brain-derived neurotrophic factor for optic nerve injury:a biomechanical evaluation

    Institute of Scientific and Technical Information of China (English)

    Zhong-jun Zhang; Ya-jun Li; Xiao-guang Liu; Feng-xiao Huang; Tie-jun Liu; Dong-mei Jiang; Xue-man Lv; Min Luo

    2015-01-01

    Treatment for optic nerve injury by brain-derived neurotrophic factor or the transplantation of human umbilical cord blood stem cells has gained progress, but analysis by biomechanical indicators is rare. Rabbit models of optic nerve injury were established by a clamp. At 7 days after injury, the vitreous body received a one-time injection of 50 μg brain-derived neurotrophic factor or 1 × 106 human umbilical cord blood stem cells. After 30 days, the maximum load, max-imum stress, maximum strain, elastic limit load, elastic limit stress, and elastic limit strain had clearly improved in rabbit models of optical nerve injury after treatment with brain-derived neu-rotrophic factor or human umbilical cord blood stem cells. The damage to the ultrastructure of the optic nerve had also been reduced. These ifndings suggest that human umbilical cord blood stem cells and brain-derived neurotrophic factor effectively repair the injured optical nerve, im-prove biomechanical properties, and contribute to the recovery after injury.

  12. Brain-derived neurotrophic factor produced by human umbilical tissue-derived cells is required for its effect on hippocampal dendritic differentiation.

    Science.gov (United States)

    Alder, Janet; Kramer, Brian C; Hoskin, Casey; Thakker-Varia, Smita

    2012-06-01

    The potential for nonembryonic cells to promote differentiation of neuronal cells has therapeutic implications for regeneration of neurons damaged by stroke or injury and avoids many ethical and safety concerns. The authors have assessed the capacity of human umbilical tissue-derived cells (hUTC) and human mesenchymal stromal cells (hMSC) to enhance differentiation of rodent hippocampal neurons. Co-culture of hippocampal cells with hUTC or hMSC in transwell inserts for 3 days resulted in increase of several dendritic parameters including the number and length of primary dendrites. The effect of hUTC or hMSC on dendritic maturation was only apparent on neurons grown for 2 weeks in vitro prior to co-culture. Changes in dendritic morphology in the presence of hUTC were also accompanied by increased expression of the presynaptic marker synaptotagmin and the postsynaptic marker postsynaptic density protein 95 kDa (PSD95) suggesting that there may also be an increase in the number of synapses formed in the presence of hUTC. The effect of hUTC and hMSC on hippocampal cells in co-culture was comparable to those induced by treatment with recombinant human brain-derived neurotrophic factor (BDNF) implying that a similar factor may be released from hUTC or hMSC. Analysis of hUTC-conditioned medium by ELISA demonstrated that BDNF was indeed secreted. An antibody that blocks the actions of BDNF partially inhibited the actions of hUTC on dendritic morphology suggesting that BDNF is at least one of the factors secreted from the cells to promote dendritic maturation. These results indicate that hUTC secrete biologically active BDNF, which can affect dendritic morphology.

  13. Effect of glial cell line-derived neurotrophic factor on peripheral nerve regeneration in adult rat

    Institute of Scientific and Technical Information of China (English)

    CHEN Zhe-yu; LI Jian-hong; ZHENG Xing-dong; LU Chang-lin; HE Cheng

    2001-01-01

    Objective: To study the effect of glial cell line-derived neurotrophic (GDNF) on adult peripheral nerve regeneration. Methods: Transectioned sciatic nerve in adult rats was sutured into silicone channel. GDNF or SAL solution was injected into the silicone channels during operation. Four weeks later, the effect of GDNF on axonal regeneration was evaluated by degenerative neurofiber staining and HRP retrograde tracing. Results: Compared with SAL group, the percentage of degenerative neurofiber areas decreased from 17.3% to 1.9% ( P<0.01 ) and the ratio of labeled spinal somas number was significantly increased from 43.5% to 68.3% ( P<0.01 ) in GDNF group. Conclusion: The results suggest that exogenous GDNF can obviously enhance adult peripheral nerve regeneration.

  14. Glial cell line-derived neurotrophic factor (GDNF therapy for Parkinson's disease.

    Directory of Open Access Journals (Sweden)

    Shingo,Tetsuro

    2007-04-01

    Full Text Available Many studies using animals clarify that glial cell line-derived neurotrophic factor (GDNF has strong neuroprotective and neurorestorative effects on dopaminergic neurons. Several pilot studies clarified the validity of continuous intraputaminal GDNF infusion to patients with Parkinson's disease (PD, although a randomized controlled trial of GDNF therapy published in 2006 resulted in negative outcomes, and controversy remains about the efficacy and safety of the treatment. For a decade, our laboratory has investigated the efficacy and the most appropriate method of GDNF administration using animals, and consequently we have obtained some solid data that correspond to the results of clinical trials. In this review, we present an outline of our studies and other key studies related to GDNF, the current state of the research, problems to be overcome, and predictions regarding the use of GDNF therapy for PD in the future.

  15. Postnatal roles of glial cell line-derived neurotrophic factor family members in nociceptors plasticity

    Institute of Scientific and Technical Information of China (English)

    Sacha A. Malin; Brian M. Davis

    2008-01-01

    The neurotrophin and glial cell line-derived neurotrophic factor (GDNF) family of growth factors have been extensively studied because of their proven ability to regulate development of the peripheral nervous system. The neurotrophin family,which includes nerve growth factor (NGF), NT-3, NT4/5 and BDNF, is also known for its ability to regulate the function of adult sensory neurons. Until recently, little was known concerning the role of the GNDF-family (that includes GDNF, artemin, neurturin and persephin) in adult sensory neuron function. Here we describe recent data that indicates that the GDNF family can regulate sensory neuron function, that some of its members are elevated in inflammatory pain models and that application of these growth factors produces pain in vivo. Finally we discuss how these two families of growth factors may converge on a single membrane receptor, TRPV 1, to produce long-lasting hyperalgesia.

  16. Glial cell line-derived neurotrophic factor (GDNF) as a novel candidate gene of anxiety.

    Science.gov (United States)

    Kotyuk, Eszter; Keszler, Gergely; Nemeth, Nora; Ronai, Zsolt; Sasvari-Szekely, Maria; Szekely, Anna

    2013-01-01

    Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor for dopaminergic neurons with promising therapeutic potential in Parkinson's disease. A few association analyses between GDNF gene polymorphisms and psychiatric disorders such as schizophrenia, attention deficit hyperactivity disorder and drug abuse have also been published but little is known about any effects of these polymorphisms on mood characteristics such as anxiety and depression. Here we present an association study between eight (rs1981844, rs3812047, rs3096140, rs2973041, rs2910702, rs1549250, rs2973050 and rs11111) GDNF single nucleotide polymorphisms (SNPs) and anxiety and depression scores measured by the Hospital Anxiety and Depression Scale (HADS) on 708 Caucasian young adults with no psychiatric history. Results of the allele-wise single marker association analyses provided significant effects of two single nucleotide polymorphisms on anxiety scores following the Bonferroni correction for multiple testing (p = 0.00070 and p = 0.00138 for rs3812047 and rs3096140, respectively), while no such result was obtained on depression scores. Haplotype analysis confirmed the role of these SNPs; mean anxiety scores raised according to the number of risk alleles present in the haplotypes (p = 0.00029). A significant sex-gene interaction was also observed since the effect of the rs3812047 A allele as a risk factor of anxiety was more pronounced in males. In conclusion, this is the first demonstration of a significant association between the GDNF gene and mood characteristics demonstrated by the association of two SNPs of the GDNF gene (rs3812047 and rs3096140) and individual variability of anxiety using self-report data from a non-clinical sample.

  17. Glial cell line-derived neurotrophic factor (GDNF as a novel candidate gene of anxiety.

    Directory of Open Access Journals (Sweden)

    Eszter Kotyuk

    Full Text Available Glial cell line-derived neurotrophic factor (GDNF is a neurotrophic factor for dopaminergic neurons with promising therapeutic potential in Parkinson's disease. A few association analyses between GDNF gene polymorphisms and psychiatric disorders such as schizophrenia, attention deficit hyperactivity disorder and drug abuse have also been published but little is known about any effects of these polymorphisms on mood characteristics such as anxiety and depression. Here we present an association study between eight (rs1981844, rs3812047, rs3096140, rs2973041, rs2910702, rs1549250, rs2973050 and rs11111 GDNF single nucleotide polymorphisms (SNPs and anxiety and depression scores measured by the Hospital Anxiety and Depression Scale (HADS on 708 Caucasian young adults with no psychiatric history. Results of the allele-wise single marker association analyses provided significant effects of two single nucleotide polymorphisms on anxiety scores following the Bonferroni correction for multiple testing (p = 0.00070 and p = 0.00138 for rs3812047 and rs3096140, respectively, while no such result was obtained on depression scores. Haplotype analysis confirmed the role of these SNPs; mean anxiety scores raised according to the number of risk alleles present in the haplotypes (p = 0.00029. A significant sex-gene interaction was also observed since the effect of the rs3812047 A allele as a risk factor of anxiety was more pronounced in males. In conclusion, this is the first demonstration of a significant association between the GDNF gene and mood characteristics demonstrated by the association of two SNPs of the GDNF gene (rs3812047 and rs3096140 and individual variability of anxiety using self-report data from a non-clinical sample.

  18. Somatomammotrophic cells in GH-secreting and PRL-secreting human pituitary adenomas.

    Science.gov (United States)

    Bassetti, M; Brina, M; Spada, A; Giannattasio, G

    1989-11-01

    A morphological study has been carried out on 20 GH-secreting adenomas removed from acromegalic normoprolactinemic patients, on 29 PRL-secreting adenomas removed from hyperprolactinemic patients without signs of acromegaly and on one normal human anterior pituitary gland collected at autopsy. The protein A-gold immunoelectron microscopic technique has been utilized in order to verify the presence of mixed cells producing both GH and PRL (somatomammotrophs) in these pituitary tissues. In the normal pituitary a considerable number of somatomammotrophs (15-20%) was found, thus supporting the idea that these cells are normal components of the human anterior pituitary gland. In 10 GH-secreting adenomas and in 10 PRL-secreting adenomas somatomammotrophs were present in a variable number (from 4 to 20% of the whole cell population in GH adenomas and from 1 to 47% in PRL tumors). It can be concluded therefore that these cells, largely present in all GH/PRL-secreting adenomas, can also be found in GH-secreting and PRL-secreting tumors without clinical evidence of a mixed secretion. Adenomatous somatomammotrophs displayed ultrastructural features of adenomatous somatotrophs and mammotrophs (prominent Golgi complexes, abundant rough endoplasmic reticulum, irregular nuclei). The size and the number of granules were variable. In some cells GH and PRL were stored in distinct secretory granules, in others in mixed granules or both in mixed and distinct granules, thus suggesting that in adenomatous somatomammotrophs the efficiency of the mechanisms of sorting of the two hormones varies from one cell to another.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Brain-derived neurotrophic factor genes transfect rat bone marrow mesenchymal stem cells based on cationic polymer vector

    Institute of Scientific and Technical Information of China (English)

    Zunsheng Zhang; Kun Zan; Yonghai Liu; Xia Shen

    2009-01-01

    BACKGROUND: Gene therapy is an effective expression of genes within target cells after transferring exogenous target genes. Both vector selection and transfection method are important factors for gene transfection. An ideal gene vector is required for a high transfusion of target gene and an exact introduction of target gene into specific target cells so as to express gene products. OBJECTIVE: To study the expression of mRNA and protein after transfecting rat bone marrow mesenchymal stem cells (BMSCs) with brain-derived neurotrophic factor (BDNF) genes based on cationic polymer vector. DESIGN, TIME AND SETTING: A randomized, controlled in vitro study using gene engineering, performed at the Neurobiology Laboratory, Xuzhou Medical College between October 2007 and April 2008. MATERIALS: PcDNA3.1 BDNF was obtained from Youbiai Biotechnological Company, Beijing and cationic polymer vector used was the SofastTM gene transfection reagent that was made by Taiyangma Biotechnological Co., Ltd., Xiamen. METHODS: BMSCs extracted from six Sprague Dawley (SD) rats aged 1 month were isolated and cultured in vitro. Third passage BMSCs were inoculated on a 6-well culture plate at the density of 1×106 cells/L. At about 80% confluence, BMSCs were transfected with PcDNA3.1-BDNF (2 μg) combined with SofastTM gene transfection reagent (6 μg) (BDNF group) or with PcDNA3.1 (2 μg) combined with SofastTM gene transfection reagent (6 μg) (blank vector group). Cells that were not transfected with any reagents but still cultured under primary culture conditions were used as a non-transfection group.MAIN OUTCOME MEASURES: Enzyme linked immunosorbent assay was used to measure time efficiency of BMSC-secreted BDNF protein. Twenty-four hours after gene transfection, RT-PCR was used to detect expression of BDNF mRNA in the BMSCs. Immunohistochemistry was used to determine expression of BDNF protein in the BMSCs.RESULTS: BDNF protein expression was detected at day 1 after gene transfection

  20. Effects of fibroblast growth factor and glial-derived neurotrophic factor on akinesia, F-DOPA uptake and dopamine cells in parkinsonian primates

    NARCIS (Netherlands)

    Fontan, A; Rojo, A; Pernaute, RS; Hernandez, [No Value; Lopez, [No Value; Castilla, C; Albisua, JS; Higueras, AP; Al-Rashid, [No Value; Rabano, A; Gonzalo, [No Value; Mena, MA; Cools, A; Eshuis, S; Maguire, P; Pruim, J; Leenders, K; de Yebenes, JG

    2002-01-01

    Parkinson's disease (PD) is a common neurodegenerative disorder that produces progressive disability despite symptomatic treatment. Several strategies, including stereotaxic brain lesions, deep brain stimulation, transplants of dopamine cells and administration of neurotrophic factors, have been pro

  1. Curative effect of transplantation of mesenchymal stem cells transfected with recombinant lentiviral vectors carrying brain-derived neurotrophic factor gene on intracerebral hemorrhage in rats

    Institute of Scientific and Technical Information of China (English)

    任瑞芳

    2013-01-01

    Objective To observe the curative effect of transplantation of mesenchymal stem cells(MSCs) transfected with recombinant lentiviral vectors carrying brain-derived neurotrophic factor(BDNF) gene on intracerebral

  2. The roles of glial cell line-derived neurotrophic factor, brain-derived neurotrophic factor and nerve growth factor during the final stage of folliculogenesis: a focus on oocyte maturation.

    Science.gov (United States)

    Linher-Melville, Katja; Li, Julang

    2013-02-01

    Neurotrophic factors were first identified to promote the growth, survival or differentiation of neurons and have also been associated with the early stages of ovarian folliculogenesis. More recently, their effects on the final stage of follicular development, including oocyte maturation and early embryonic development, have been reported. Glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), which are expressed in numerous peripheral tissues outside of the CNS, most notably the ovary, are now known to stimulate oocyte maturation in various species, also enhancing developmental competence. The mechanisms that underlie their actions in antral follicles, as well as the targets ultimately controlled by these factors, are beginning to emerge. GDNF, BDNF and NGF, alone or in combination, could be added to the media currently utilized for in vitro oocyte maturation, thereby potentially increasing the production and/or quality of early embryos.

  3. Brain-derived Neurotrophic Factor Promotes the Migration of Olfactory Ensheathing Cells Through TRPC Channels.

    Science.gov (United States)

    Wang, Ying; Teng, Hong-Lin; Gao, Yuan; Zhang, Fan; Ding, Yu-Qiang; Huang, Zhi-Hui

    2016-12-01

    Olfactory ensheathing cells (OECs) are a unique type of glial cells with axonal growth-promoting properties in the olfactory system. Organized migration of OECs is essential for neural regeneration and olfactory development. However, the molecular mechanism of OEC migration remains unclear. In the present study, we examined the effects of brain-derived neurotrophic factor (BDNF) on OEC migration. Initially, the "scratch" migration assay, the inverted coverslip and Boyden chamber migration assays showed that BDNF could promote the migration of primary cultured OECs. Furthermore, BDNF gradient attracted the migration of OECs in single-cell migration assays. Mechanistically, TrkB receptor expressed in OECs mediated BDNF-induced OEC migration, and BDNF triggered calcium signals in OECs. Finally, transient receptor potential cation channels (TRPCs) highly expressed in OECs were responsible for BDNF-induced calcium signals, and required for BDNF-induced OEC migration. Taken together, these results demonstrate that BDNF promotes the migration of cultured OECs and an unexpected finding is that TRPCs are required for BDNF-induced OEC migration. GLIA 2016;64:2154-2165.

  4. A novel mechanism of hepatocellular carcinoma cell apoptosis induced by lupeol via Brain-Derived Neurotrophic Factor Inhibition and Glycogen Synthase Kinase 3 beta reactivation.

    Science.gov (United States)

    Zhang, Lingli; Tu, Yi; He, Wen; Peng, Yan; Qiu, Zhenpeng

    2015-09-05

    Lupeol is a naturally available triterpenoid with selective anticancerous potential on various human cancer cells. The present study shows that lupeol can inhibit cell proliferation of hepatocellular carcinoma (HCC) HCCLM3 cells in a time- and dose-dependent manner, through caspase-3 dependent activation and Poly ADP-Ribose Polymerase (PARP) cleavage. Lupeol-induced cell death is associated with a marked decrease in the protein expression of Brain-Derived Neurotrophic Factor (BDNF) and ser-9-phosphoryltion of Glycogen Synthase Kinase 3 Beta (GSK-3β), with concomitant suppression of Akt1, phosphatidyl inositol 3-kinase (PI3K), β-catenin, c-Myc and Cyclin D1 mRNA expression. Suppressing overexpression of BDNF by lupeol results in decreased protein expression of p-Akt and PI3K (p110α), as well as reactivation of GSK-3β function in HepG2 cells. Lupeol treatment also inhibits LiCl-induced activation of Wnt signaling pathway and exerts the in vitro anti-invasive activity in Huh-7 cells. LiCl-triggered high expression of β-catenin, c-Myc and Cyclin D1 protein is reduced followed by lupeol exposure. The findings suggest a mechanistic link between caspase dependent pathway, BDNF secretion and Akt/PI3K/GSK-3β in HCC cells. These results indicate that lupeol can suppress HCC cell proliferation by inhibiting BDNF secretion and phosphorylation of GSK-3β(Ser-9), cooperated with blockade of Akt/PI3K and Wnt signaling pathway.

  5. Glial cell line-derived neurotrophic factor promotes barrier maturation and wound healing in intestinal epithelial cells in vitro.

    Science.gov (United States)

    Meir, Michael; Flemming, Sven; Burkard, Natalie; Bergauer, Lisa; Metzger, Marco; Germer, Christoph-Thomas; Schlegel, Nicolas

    2015-10-15

    Recent data suggest that neurotrophic factors from the enteric nervous system are involved in intestinal epithelial barrier regulation. In this context the glial cell line-derived neurotrophic factor (GDNF) was shown to affect gut barrier properties in vivo directly or indirectly by largely undefined processes in a model of inflammatory bowel disease (IBD). We further investigated the potential role and mechanisms of GDNF in the regulation of intestinal barrier functions. Immunostaining of human gut specimen showed positive GDNF staining in enteric neuronal plexus and in enterocytes. In Western blots of the intestinal epithelial cell lines Caco2 and HT29B6, significant amounts of GDNF were detected, suggesting that enterocytes represent an additional source of GDNF. Application of recombinant GDNF on Caco2 and HT29B6 cells for 24 h resulted in significant epithelial barrier stabilization in monolayers with immature barrier functions. Wound-healing assays showed a significantly faster closure of the wounded areas after GDNF application. GDNF augmented cAMP levels and led to significant inactivation of p38 MAPK in immature cells. Activation of p38 MAPK signaling by SB-202190 mimicked GDNF-induced barrier maturation, whereas the p38 MAPK activator anisomycin blocked GDNF-induced effects. Increasing cAMP levels had adverse effects on barrier maturation, as revealed by permeability measurements. However, increased cAMP augmented the proliferation rate in Caco2 cells, and GDNF-induced proliferation of epithelial cells was abrogated by the PKA inhibitor H89. Our data show that enterocytes represent an additional source of GDNF synthesis. GDNF contributes to wound healing in a cAMP/PKA-dependent manner and promotes barrier maturation in immature enterocytes cells by inactivation of p38 MAPK signaling.

  6. Neuronal cell death, nerve growth factor and neurotrophic models: 50 years on.

    Science.gov (United States)

    Bennet, M R; Gibson, W G; Lemon, G

    2002-01-10

    Viktor Hamburger has just died at the age of 100. It is 50 years since he and Rita Levi-Montalcini laid the foundations for the study of naturally occurring cell death and of neurotrophic factors in the nervous system. In a period of less than 10 years, from 1949 to 1958, Hamburger and Levi-Montalcini made the following seminal discoveries: that neuron cell death occurs in dorsal root ganglia, sympathetic ganglia and the cervical column of motoneurons; that the predictions arising from this observation, namely that survival is dependent on the supply of a trophic factor, could be substantiated by studying the effects of a sarcoma on the proliferation of ganglionic processes both in vivo and in vitro; and that the proliferation of these processes could be used as an assay system to isolate the factor. This work provides a short review mostly of the early history of this subject in the context of the Hamburger/Levi-Montalcini paradigm. This acts as an introduction to a consideration of models that have been proposed to account for how the different sources of growth factors provide for the survival of neurons during development. It is suggested that what has been called the 'social-control' model provides the most parsimonious quantitative description of the contribution of trophic factors to neuronal survival, a concept for which we are in debt to Viktor Hamburger and Rita Levi-Montalcini.

  7. Influence of brain-derived neurotrophic factor on pathfinding of dentate granule cell axons, the hippocampal mossy fibers

    OpenAIRE

    Tamura Makoto; Tamura Naohiro; Ikeda Takamitsu; Koyama Ryuta; Ikegaya Yuji; Matsuki Norio; Yamada Maki K

    2009-01-01

    Abstract Mossy fibers, the dentate granule cell axons, are generated throughout an animal's lifetime. Mossy fiber paths and synapses are primarily restricted to the stratum lucidum within the CA3 region. Brain-derived neurotrophic factor (BDNF), a neurotrophin family protein that activates Trk neurotrophin receptors, is highly expressed in the stratum lucidum in an activity-dependent manner. The addition of a Trk neurotrophin receptor inhibitor, K252a, to cultured hippocampal slices induced a...

  8. ROCKing cytokine secretion balance in human T cells.

    Science.gov (United States)

    Zanin-Zhorov, Alexandra; Waksal, Samuel D

    2015-04-01

    Balanced regulation of cytokine secretion in T cells is critical for maintenance of immune homeostasis and prevention of autoimmunity. The Rho-associated kinase (ROCK) 2 signaling pathway was previously shown to be involved in controlling of cellular movement and shape. However, recent work from our group and others has demonstrated a new and important role of ROCK2 in regulating cytokine secretion in T cells. We found that ROCK2 promotes pro-inflammatory cytokines such as IL-17 and IL-21, whereas IL-2 and IL-10 secretion are negatively regulated by ROCK2 under Th17-skewing activation. Also, in disease, but not in steady state conditions, ROCK2 contributes to regulation of IFN-γ secretion in T cells from rheumatoid arthritis patients. Thus, ROCK2 signaling is a key pathway in modulation of T-cell mediated immune responses underscoring the therapeutic potential of targeted inhibition of ROCK2 in autoimmunity.

  9. Peroxicretion: a novel secretion pathway in the eukaryotic cell

    NARCIS (Netherlands)

    Sagt, C.M.J.; Ten Haaft, P.J.T; Minneboo, I.M.; Hartog, M.P.; Damveld, R.A.; Van der Laan, J.M.; Akeroyd, M; Wenzel, T.J.; Luesken, F.A.; Veenhuis, M.; Van der Klei, I.; De Winde, J.H.

    2009-01-01

    Background: Enzyme production in microbial cells has been limited to secreted enzymes or intracellular enzymes followed by expensive down stream processing. Extracellular enzymes consists mainly of hydrolases while intracellular enzymes exhibit a much broader diversity. If these intracellular enzyme

  10. Peroxicretion : a novel secretion pathway in the eukaryotic cell

    NARCIS (Netherlands)

    Sagt, Cees M.J.; Haaft, Peter J. ten; Minneboo, Ingeborg M.; Hartog, Miranda P.; Damveld, Robbert A.; Laan, Jan Metske van der; Akeroyd, Michiel; Wenzel, Thibaut J.; Luesken, Francisca A.; Veenhuis, Marten; Klei, Ida van der; Winde, Johannes H. de

    2009-01-01

    Background: Enzyme production in microbial cells has been limited to secreted enzymes or intracellular enzymes followed by expensive down stream processing. Extracellular enzymes consists mainly of hydrolases while intracellular enzymes exhibit a much broader diversity. If these intracellular enzyme

  11. EFFECTS OF SECRETABLE PLACENTAL FACTORS UPON SECRETION OF CYTOKINES BY THP-1 MONOCYTE-LIKE CELLS

    Directory of Open Access Journals (Sweden)

    Ya. S. Onokhina

    2013-01-01

    Full Text Available Abstract. Мonocytes in feto-placental circulation are exposed to factors secreted by placental tissue. These factors influence monocyte functions in pregnancy. In present study, an in vitro model (monocyte-like THP-1 cells was used for assessing effects of soluble placental factors obtained from women with physiological pregnancies, or preeclampsia cases. The following effects of placental factors were revealed: increased secretion of VEGF by THP-1 cells along with decreased secretion of IL-6, IL-8 and MCP-1 under the influence of placental factors from the I. trimester of pregnancy in comparison with III. trimester. Secretion of IL-6 and MCP-1 by THP-1 cells was increased, and secretion of soluble TNFRII was decreased upon co-cultivation with soluble placental factors from the women with preeclampsia, as compared with placental products from physiological pregnancies.The work is supported by grants ГК № 02.740.11.0711 from Ministry of Education and Science, and НШ-3594.2010.7 grant from the President of Russian Federation.

  12. TRPC3 Regulates Release of Brain-Derived Neurotrophic Factor From Human Airway Smooth Muscle

    OpenAIRE

    Vohra, Pawan K.; Thompson, Michael A.; Sathish, Venkatachalem; Kiel, Alexander; Jerde, Calvin; Pabelick, Christina M.; Singh, Brij B.; Prakash, Y. S.

    2013-01-01

    Exogenous brain-derived neurotrophic factor (BDNF) enhances Ca2+ signaling and cell proliferation in human airway smooth muscle (ASM), especially with inflammation. Human ASM also expresses BDNF, raising the potential for autocrine/paracrine effects. The mechanisms by which ASM BDNF secretion occurs are not known. Transient receptor potential channels (TRPCs) regulate a variety of intracellular processes including store-operated Ca2+ entry (SOCE; including in ASM) and secretion of factors suc...

  13. Neural stem cells express melatonin receptors and neurotrophic factors: colocalization of the MT1 receptor with neuronal and glial markers

    Directory of Open Access Journals (Sweden)

    McMillan Catherine R

    2004-10-01

    Full Text Available Abstract Background In order to optimize the potential benefits of neural stem cell (NSC transplantation for the treatment of neurodegenerative disorders, it is necessary to understand their biological characteristics. Although neurotrophin transduction strategies are promising, alternative approaches such as the modulation of intrinsic neurotrophin expression by NSCs, could also be beneficial. Therefore, utilizing the C17.2 neural stem cell line, we have examined the expression of selected neurotrophic factors under different in vitro conditions. In view of recent evidence suggesting a role for the pineal hormone melatonin in vertebrate development, it was also of interest to determine whether its G protein-coupled MT1 and MT2 receptors are expressed in NSCs. Results RT-PCR analysis revealed robust expression of glial cell-line derived neurotrophic factor (GDNF, brain-derived neurotrophic factor (BDNF and nerve growth factor (NGF in undifferentiated cells maintained for two days in culture. After one week, differentiating cells continued to exhibit high expression of BDNF and NGF, but GDNF expression was lower or absent, depending on the culture conditions utilized. Melatonin MT1 receptor mRNA was detected in NSCs maintained for two days in culture, but the MT2 receptor was not seen. An immature MT1 receptor of about 30 kDa was detected by western blotting in NSCs cultured for two days, whereas a mature receptor of about 40 – 45 kDa was present in cells maintained for longer periods. Immunocytochemical studies demonstrated that the MT1 receptor is expressed in both neural (β-tubulin III positive and glial (GFAP positive progenitor cells. An examination of the effects of melatonin on neurotrophin expression revealed that low physiological concentrations of this hormone caused a significant induction of GDNF mRNA expression in NSCs following treatment for 24 hours. Conclusions The phenotypic characteristics of C17.2 cells suggest that they are

  14. Brain-derived neurotrophic factor induces neuron-like cellular differentiation of mesenchymal stem cells derived from human umbilical cord blood cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Lei Chen; Guozhen Hui; Zhongguo Zhang; Bing Chen; Xiaozhi Liu; Zhenlin Liu; Hongliang Liu; Gang Li; Zhiguo Su; Junfei Wang

    2011-01-01

    Human umbilical cord blood was collected from full-term deliveries scheduled for cesarean section. Mononuclear cells were isolated, amplified and induced as mesenchymal stem cells. Isolated mesenchymal stem cells tested positive for the marker CD29, CD44 and CD105 and negative for typical hematopoietic and endothelial markers. Following treatment with neural induction medium containing brain-derived neurotrophic factor for 7 days, the adherent cells exhibited neuron-like cellular morphology. Immunohistochemical staining and reverse transcription-PCR revealed that the induced mesenchymal stem cells expressed the markers for neuron-specific enolase and neurofilament. The results demonstrated that human umbilical cord blood-derived mesenchymal stem cells can differentiate into neuron-like cells induced by brain-derived neurotrophic factor in vitro.

  15. Presynaptic modulation of spinal nociceptive transmission by glial cell line-derived neurotrophic factor (GDNF).

    Science.gov (United States)

    Salio, Chiara; Ferrini, Francesco; Muthuraju, Sangu; Merighi, Adalberto

    2014-10-01

    The role of glial cell line-derived neurotrophic factor (GDNF) in nociceptive pathways is still controversial, as both pronociceptive and antinociceptive actions have been reported. To elucidate this role in the mouse, we performed combined structural and functional studies in vivo and in acute spinal cord slices where C-fiber activation was mimicked by capsaicin challenge. Nociceptors and their terminals in superficial dorsal horn (SDH; laminae I-II) constitute two separate subpopulations: the peptidergic CGRP/somatostatin+ cells expressing GDNF and the nonpeptidergic IB4+ neurons expressing the GFRα1-RET GDNF receptor complex. Ultrastructurally the dorsal part of inner lamina II (LIIid) harbors a mix of glomeruli that either display GDNF/somatostatin (GIb)-IR or GFRα1/IB4 labeling (GIa). LIIid thus represents the preferential site for ligand-receptor interactions. Functionally, endogenous GDNF released from peptidergic CGRP/somatostatin+ nociceptors upon capsaicin stimulation exert a tonic inhibitory control on the glutamate excitatory drive of SDH neurons as measured after ERK1/2 phosphorylation assay. Real-time Ca(2+) imaging and patch-clamp experiments with bath-applied GDNF (100 nM) confirm the presynaptic inhibition of SDH neurons after stimulation of capsaicin-sensitive, nociceptive primary afferent fibers. Accordingly, the reduction of the capsaicin-evoked [Ca(2+)]i rise and of the frequency of mEPSCs in SDH neurons is specifically abolished after enzymatic ablation of GFRα1. Therefore, GDNF released from peptidergic CGRP/somatostatin+ nociceptors acutely depresses neuronal transmission in SDH signaling to nonpeptidergic IB4+ nociceptors at glomeruli in LIIid. These observations are of potential pharmacological interest as they highlight a novel modality of cross talk between nociceptors that may be relevant for discrimination of pain modalities.

  16. Extracellular poly(ADP-ribose) is a neurotrophic signal that upregulates glial cell line-derived neurotrophic factor (GDNF) levels in vitro and in vivo.

    Science.gov (United States)

    Nakajima, Hidemitsu; Itakura, Masanori; Sato, Keishi; Nakamura, Sunao; Azuma, Yasu-Taka; Takeuchi, Tadayoshi

    2017-03-04

    Synthesis of poly(ADP-ribose) (PAR) is catalyzed by PAR polymerase-1 (PARP-1) in neurons. PARP1 plays a role in various types of brain damage in neurodegenerative disorders. In neurons, overactivation of PARP-1 during oxidative stress induces robust PAR formation, which depletes nicotinamide adenine dinucleotide levels and leads to cell death. However, the role of the newly-formed PAR in neurodegenerative disorders remains elusive. We hypothesized that the effects of PAR could occur in the extracellular space after it is leaked from damaged neurons. Here we report that extracellular PAR (EC-PAR) functions as a neuroprotective molecule by inducing the synthesis of glial cell line-derived neurotrophic factor (GDNF) in astrocytes during neuronal cell death, both in vitro and in vivo. In primary rat astrocytes, exogenous treatment with EC-PAR produced GDNF but not other neurotrophic factors. The effect was concentration-dependent and did not affect cell viability in rat C6 astrocytoma cells. Topical injection of EC-PAR into rat striatum upregulated GDNF levels in activated astrocytes and improved pathogenic rotation behavior in a unilateral 6-hydroxydopamine model of Parkinson disease in rats. These findings indicate that EC-PAR acts as a neurotrophic enhancer by upregulating GDNF levels. This effect protects the remaining neurons following oxidative stress-induced brain damage, such as that seen with Parkinson disease.

  17. Glial cell line-derived neurotrophic factor gene delivery via a polyethylene imine grafted chitosan carrier.

    Science.gov (United States)

    Peng, Yu-Shiang; Lai, Po-Liang; Peng, Sydney; Wu, His-Chin; Yu, Siang; Tseng, Tsan-Yun; Wang, Li-Fang; Chu, I-Ming

    2014-01-01

    Parkinson's disease is known to result from the loss of dopaminergic neurons. Direct intracerebral injections of high doses of recombinant glial cell line-derived neurotrophic factor (GDNF) have been shown to protect adult nigral dopaminergic neurons. Because GDNF does not cross the blood-brain barrier, intracerebral gene transfer is an ideal option. Chitosan (CHI) is a naturally derived material that has been used for gene transfer. However, the low water solubility often leads to decreased transfection efficiency. Grafting of highly water-soluble polyethylene imines (PEI) and polyethylene glycol onto polymers can increase their solubility. The purpose of this study was to design a non-viral gene carrier with improved water solubility as well as enhanced transfection efficiency for treating Parkinsonism. Two molecular weights (Mw =600 and 1,800 g/mol) of PEI were grafted onto CHI (PEI600-g-CHI and PEI1800-g-CHI, respectively) by opening the epoxide ring of ethylene glycol diglycidyl ether (EX-810). This modification resulted in a non-viral gene carrier with less cytotoxicity. The transfection efficiency of PEI600-g-CHI/deoxyribonucleic acid (DNA) polyplexes was significantly higher than either PEI1800-g-CHI/DNA or CHI/DNA polyplexes. The maximal GDNF expression of PEI600-g-CHI/DNA was at the polymer:DNA weight ratio of 10:1, which was 1.7-fold higher than the maximal GDNF expression of PEI1800-g-CHI/DNA. The low toxicity and high transfection efficiency of PEI600-g-CHI make it ideal for application to GDNF gene therapy, which has potential for the treatment of Parkinson's disease.

  18. 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.

  19. Association between smoking behaviour and genetic variants of glial cell line-derived neurotrophic factor

    Indian Academy of Sciences (India)

    ESZTER KOTYUK; NORA NEMETH; ZSOLT RONAI; ZSOLT DEMETROVICS; MARIA SASVARI-SZEKELY; ANNA SZEKELY

    2016-12-01

    Glial cell line-derived neurotrophic factor (GDNF) promotes development and differentiation of dopaminergic neurons, thus it has an important role in dopamine-related neuropsychiatric disorders. Since the role of dopamine system in smoking iswell established, we hypothesized that GDNF gene variants may affect smoking behaviour. Self-reported data on smoking behaviour (never smoked, quit, occasional, or regular smokers) and level of nicotine addiction (Hooked on Nicotine Checklist and Fagerstrom Nicotine Addiction Scale), anxiety, as well as buccal samples were obtained from 930 Hungarian young adults (18–35 years). Genetic analysis involved eight GDNF single-nucleotide polymorphisms (SNP) (rs1981844, rs3812047, rs3096140, rs2973041, rs2910702, rs1549250, rs2973050 and rs11111). Allele-wise association analyses of the eight GDNF SNPs provided a significant association between smoking behaviour and rs3096140 (P = 0.0039). The minor allele (C) was more frequent in those groups who smoked in some form (quit, occasional or regular smokers) as compared to those who neversmoked (P = 0.0046). This result remained significant after Bonferroni correction for multiple testing. In the ever smoking group, no significant differences were found in the level of nicotine addiction by the alleles of these polymorphisms. Also, nosignificant interaction of rs3096140 and smoking categories were observed on anxiety mean scores. Although previous data demonstrated an association between GDNF rs2910704 and severity of methamphetamine use to the best of our knowledge, this is the first study on the role of GDNF genetic variations in smoking behaviour. Our results suggest that GDNF rs3096140 might be involved in the genetic background of smoking, independent of anxiety characteristics.

  20. Peroxicretion: a novel secretion pathway in the eukaryotic cell

    Directory of Open Access Journals (Sweden)

    Luesken Francisca A

    2009-05-01

    Full Text Available Abstract Background Enzyme production in microbial cells has been limited to secreted enzymes or intracellular enzymes followed by expensive down stream processing. Extracellular enzymes consists mainly of hydrolases while intracellular enzymes exhibit a much broader diversity. If these intracellular enzymes could be secreted by the cell the potential of industrial applications of enzymes would be enlarged. Therefore a novel secretion pathway for intracellular proteins was developed, using peroxisomes as secretion vesicles. Results Peroxisomes were decorated with a Golgi derived v-SNARE using a peroxisomal membrane protein as an anchor. This allowed the peroxisomes to fuse with the plasma membrane. Intracellular proteins were transported into the peroxisomes by adding a peroxisomal import signal (SKL tag. The proteins which were imported in the peroxisomes, were released into the extra-cellular space through this artificial secretion pathway which was designated peroxicretion. This concept was supported by electron microscopy studies. Conclusion Our results demonstrate that it is possible to reroute the intracellular trafficking of vesicles by changing the localisation of SNARE molecules, this approach can be used in in vivo biological studies to clarify the different control mechanisms regulating intracellular membrane trafficking. In addition we demonstrate peroxicretion of a diverse set of intracellular proteins. Therefore, we anticipate that the concept of peroxicretion may revolutionize the production of intracellular proteins from fungi and other microbial cells, as well as from mammalian cells.

  1. Human Beta Cells Produce and Release Serotonin to Inhibit Glucagon Secretion from Alpha Cells

    OpenAIRE

    Joana Almaça; Judith Molina; Danusa Menegaz; Pronin, Alexey N.; Alejandro Tamayo; Vladlen Slepak; Per-Olof Berggren; Alejandro Caicedo

    2016-01-01

    In the pancreatic islet, serotonin is an autocrine signal increasing beta cell mass during metabolic challenges such as those associated with pregnancy or high-fat diet. It is still unclear whether serotonin is relevant for regular islet physiology and hormone secretion. Here, we show that human beta cells produce and secrete serotonin when stimulated with increases in glucose concentration. Serotonin secretion from beta cells decreases cyclic AMP (cAMP) levels in neighboring alpha cells via ...

  2. Nerve injury induces glial cell line-derived neurotrophic factor (GDNF) expression in Schwann cells through purinergic signaling and the PKC-PKD pathway.

    Science.gov (United States)

    Xu, Pin; Rosen, Kenneth M; Hedstrom, Kristian; Rey, Osvaldo; Guha, Sushovan; Hart, Courtney; Corfas, Gabriel

    2013-07-01

    Upon peripheral nerve injury, specific molecular events, including increases in the expression of selected neurotrophic factors, are initiated to prepare the tissue for regeneration. However, the mechanisms underlying these events and the nature of the cells involved are poorly understood. We used the injury-induced upregulation of glial cell-derived neurotrophic factor (GDNF) expression as a tool to gain insights into these processes. We found that both myelinating and nonmyelinating Schwann cells are responsible for the dramatic increase in GDNF expression after injury. We also demonstrate that the GDNF upregulation is mediated by a signaling cascade involving activation of Schwann cell purinergic receptors, followed by protein kinase C signaling which activates protein kinase D (PKD), which leads to increased GDNF transcription. Given the potent effects of GDNF on survival and repair of injured peripheral neurons, we propose that targeting these pathways may yield therapeutic tools to treat peripheral nerve injury and neuropathies.

  3. Expression of neurotrophic factors in injured spinal cord after transplantation of human-umbilical cord blood stem cells in rats.

    Science.gov (United States)

    Chung, Hyo-jin; Chung, Wook-hun; Lee, Jae-Hoon; Chung, Dai-Jung; Yang, Wo-Jong; Lee, A-Jin; Choi, Chi-Bong; Chang, Hwa-Seok; Kim, Dae-Hyun; Suh, Hyun Jung; Lee, Dong-Hun; Hwang, Soo-Han; Do, Sun Hee; Kim, Hwi-Yool

    2016-03-01

    We induced percutaneous spinal cord injuries (SCI) using a balloon catheter in 45 rats and transplanted human umbilical cord blood derived mesenchymal stem cells (hUCB-MSCs) at the injury site. Locomotor function was significantly improved in hUCB-MSCs transplanted groups. Quantitative ELISA of extract from entire injured spinal cord showed increased expression of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and neurotrophin-3 (NT-3). Our results show that treatment of SCI with hUCB-MSCs can improve locomotor functions, and suggest that increased levels of BDNF, NGF and NT-3 in the injured spinal cord were the main therapeutic effect.

  4. Glial cell line-derived neurotrophic factor gene delivery via a polyethylene imine grafted chitosan carrier

    Directory of Open Access Journals (Sweden)

    Peng YS

    2014-06-01

    Full Text Available Yu-Shiang Peng,1,* Po-Liang Lai,2,* Sydney Peng,1 His-Chin Wu,3 Siang Yu,1 Tsan-Yun Tseng,4 Li-Fang Wang,5 I-Ming Chu1 1Department of Chemical Engineering, National Tsing Hua University, Hsinchu, 2Department of Orthopedic Surgery, Chang Gung Memorial Hospital at Linkou, Chang Gung University College of Medicine, Taoyuan, 3Department of Materials Engineering, Tatung University, Taipei, 4Graduate School of Biotechnology and Bioengineering, College of Engineering, Yuan Ze University, Chung-Li, 5Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung, Taiwan *Yu-Shiang Peng and Po-Liang Lai contributed equally to this work Abstract: Parkinson’s disease is known to result from the loss of dopaminergic neurons. Direct intracerebral injections of high doses of recombinant glial cell line-derived neurotrophic factor (GDNF have been shown to protect adult nigral dopaminergic neurons. Because GDNF does not cross the blood–brain barrier, intracerebral gene transfer is an ideal option. Chitosan (CHI is a naturally derived material that has been used for gene transfer. However, the low water solubility often leads to decreased transfection efficiency. Grafting of highly water-soluble polyethylene imines (PEI and polyethylene glycol onto polymers can increase their solubility. The purpose of this study was to design a non-viral gene carrier with improved water solubility as well as enhanced transfection efficiency for treating Parkinsonism. Two molecular weights (Mw =600 and 1,800 g/mol of PEI were grafted onto CHI (PEI600-g-CHI and PEI1800-g-CHI, respectively by opening the epoxide ring of ethylene glycol diglycidyl ether (EX-810. This modification resulted in a non-viral gene carrier with less cytotoxicity. The transfection efficiency of PEI600-g-CHI/deoxyribonucleic acid (DNA polyplexes was significantly higher than either PEI1800-g-CHI/DNA or CHI/DNA polyplexes. The maximal GDNF expression of PEI600-g-CHI/DNA was at the

  5. Secretion of immunoregulatory cytokines by mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Dobroslav; Kyurkchiev; Ivan; Bochev; Ekaterina; Ivanova-Todorova; Milena; Mourdjeva; Tsvetelina; Oreshkova; Kalina; Belemezova; Stanimir; Kyurkchiev

    2014-01-01

    According to the minimal criteria of the International Society of Cellular Therapy, mesenchymal stem cells(MSCs) are a population of undifferentiated cells defined by their ability to adhere to plastic surfaces when cultured under standard conditions, express a certain panel of phenotypic markers and can differentiate into osteogenic, chondrogenic and adipogenic lineages when cultured in specific inducing media. In parallel with their major role as undifferentiated cell reserves, MSCs have immunomodulatory functions which are exerted by direct cell-to-cell contacts, secretion of cytokines and/or by a combination of both mechanisms. There are no convincing data about a principal difference in the profile of cytokines secreted by MSCs isolated from different tissue sources, although some papers report some quantitative but not qualitative differences in cytokine secretion. The present review focuses on the basic cytokines secreted by MSCs as described in the literature by which the MSCs exert immunodulatory effects. It should be pointed out that MSCs themselves are objects of cytokine regulation. Hypothetical mechanisms by which the MSCs exert their immunoregulatory effects are also discussed in this review. These mechanisms may either influence the target immune cells directly or indirectly by affecting the activities of predominantly dendritic cells. Chemokines are also discussed as participants in this process by recruiting cells of the immune systems and thus making them targets of immunosuppression. This review aims to present and discuss the published data and the personal experience of the authors regarding cytokines secreted by MSCs and their effects on the cells of the immune system.

  6. Are bone marrow regenerative cells ideal seed cells for the treatment of cerebral ischemia?★

    OpenAIRE

    Li, Yi; Hua, Xuming; Hua, Fang; Mao, Wenwei; Wan, Liang; Li, Shiting

    2013-01-01

    Bone marrow cells for the treatment of ischemic brain injury may depend on the secretion of a large number of neurotrophic factors. Bone marrow regenerative cells are capable of increasing the secretion of neurotrophic factors. In this study, after tail vein injection of 5-fluorouracil for 7 days, bone marrow cells and bone marrow regenerative cells were isolated from the tibias and femurs of rats, and then administered intravenously via the tail vein after focal cerebral ischemia. Immunohist...

  7. Control of renin secretion from kidneys with renin cell hyperplasia.

    Science.gov (United States)

    Kurt, Birgül; Karger, Christian; Wagner, Charlotte; Kurtz, Armin

    2014-02-01

    In states of loss-of-function mutations of the renin-angiotensin-aldosterone system, kidneys develop a strong hyperplasia of renin-producing cells. Those additional renin cells are located outside the classic juxtaglomerular areas, mainly in the walls of preglomerular vessels and most prominently in multilayers surrounding afferent arterioles. Since the functional behavior of those ectopic renin cells is yet unknown, we aimed to characterize the control of renin secretion from kidneys with renin cell hyperplasia. As a model, we used kidneys from mice lacking aldosterone synthase (AS⁻/⁻ mice), which displayed 10-fold elevations of renin mRNA and plasma renin concentrations. On the absolute level, renin secretion from isolated AS⁻/⁻ kidneys was more than 10-fold increased over wild-type kidneys. On the relative level, the stimulation of renin secretion by the β-adrenergic activator isoproterenol or by lowering of the concentration of extracellular Ca²⁺ was very similar between the two genotypes. In addition, the inhibitory effects of ANG II and of perfusion pressure were similar between the two genotypes. Deletion of connexin40 blunted the pressure dependency of renin secretion and the stimulatory effect of low extracellular Ca²⁺ on renin secretion in the same manner in kidneys of AS⁻/⁻ mice as in wild-type mice. Our findings suggest a high degree of functional similarity between renin cells originating during development and located at different positions in the adult kidney. They also suggest a high similarity in the expression of membrane proteins relevant for the control of renin secretion, such as β₁-adrenergic receptors, ANG II type 1 receptors, and connexin40.

  8. New developments in goblet cell mucus secretion and function.

    Science.gov (United States)

    Birchenough, G M H; Johansson, M E V; Gustafsson, J K; Bergström, J H; Hansson, G C

    2015-07-01

    Goblet cells and their main secretory product, mucus, have long been poorly appreciated; however, recent discoveries have changed this and placed these cells at the center stage of our understanding of mucosal biology and the immunology of the intestinal tract. The mucus system differs substantially between the small and large intestine, although it is built around MUC2 mucin polymers in both cases. Furthermore, that goblet cells and the regulation of their secretion also differ between these two parts of the intestine is of fundamental importance for a better understanding of mucosal immunology. There are several types of goblet cell that can be delineated based on their location and function. The surface colonic goblet cells secrete continuously to maintain the inner mucus layer, whereas goblet cells of the colonic and small intestinal crypts secrete upon stimulation, for example, after endocytosis or in response to acetyl choline. However, despite much progress in recent years, our understanding of goblet cell function and regulation is still in its infancy.

  9. Activation of AMPK Stimulates Neurotensin Secretion in Neuroendocrine Cells.

    Science.gov (United States)

    Li, Jing; Song, Jun; Weiss, Heidi L; Weiss, Todd; Townsend, Courtney M; Evers, B Mark

    2016-01-01

    AMP-activated protein kinase (AMPK), a critical fuel-sensing enzyme, regulates the metabolic effects of various hormones. Neurotensin (NT) is a 13-amino acid peptide predominantly localized in enteroendocrine cells of the small bowel and released by fat ingestion. Increased fasting plasma levels of pro-NT (a stable NT precursor fragment produced in equimolar amounts relative to NT) are associated with an increased risk of diabetes, cardiovascular disease, and mortality; however, the mechanisms regulating NT release are not fully defined. We previously reported that inhibition of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) increases NT secretion and gene expression through activation of the MEK/ERK pathway. Here, we show that activation of AMPK increases NT secretion from endocrine cell lines (BON and QGP-1) and isolated mouse crypt cells enriched for NT-positive cells. In addition, plasma levels of NT increase in mice treated with 5-aminoimidazole-4-carboxamide riboside, a pharmacologic AMPK activator. Small interfering RNA-mediated knockdown of AMPKα decrease, whereas overexpression of the subunit significantly enhances, NT secretion from BON cells treated with AMPK activators or oleic acid. Similarly, small interfering RNA knockdown of the upstream AMPK kinases, liver kinase B1 and Ca(2+) calmodulin-dependent protein kinase kinase 2, also attenuate NT release and AMPK phosphorylation. Moreover, AMPK activation increases NT secretion through inhibition of mTORC1 signaling. Together, our findings show that AMPK activation enhances NT release through inhibition of mTORC1 signaling, thus demonstrating an important cross talk regulation for NT secretion.

  10. Purification and cultivation of human pituitary growth hormone secreting cells

    Science.gov (United States)

    Hymer, W. C.

    1978-01-01

    The maintainance of actively secreting human pituitary growth hormone cells (somatotrophs) in vitro was studied. The primary approach was the testing of agents which may be expected to increase the release of the human growth hormone (hGH). A procedure for tissue procurement is described along with the methodologies used to dissociate human pituitary tissue (obtained either at autopsy or surgery) into single cell suspensions. The validity of the Biogel cell column perfusion system for studying the dynamics of GH release was developed and documented using a rat pituitary cell system.

  11. TRPM4 controls insulin secretion in pancreatic beta-cells.

    Science.gov (United States)

    Cheng, Henrique; Beck, Andreas; Launay, Pierre; Gross, Stefan A; Stokes, Alexander J; Kinet, Jean-Pierre; Fleig, Andrea; Penner, Reinhold

    2007-01-01

    TRPM4 is a calcium-activated non-selective cation channel that is widely expressed and proposed to be involved in cell depolarization. In excitable cells, TRPM4 may regulate calcium influx by causing the depolarization that drives the activation of voltage-dependent calcium channels. We here report that insulin-secreting cells of the rat pancreatic beta-cell line INS-1 natively express TRPM4 proteins and generate large depolarizing membrane currents in response to increased intracellular calcium. These currents exhibit the characteristics of TRPM4 and can be suppressed by expressing a dominant negative TRPM4 construct, resulting in significantly decreased insulin secretion in response to a glucose stimulus. Reduced insulin secretion was also observed with arginine vasopressin stimulation, a Gq-coupled receptor agonist in beta-cells. Moreover, the recruitment of TRPM4 currents was biphasic in both INS-1 cells as well as HEK-293 cells overexpressing TRPM4. The first phase is due to activation of TRPM4 channels localized within the plasma membrane followed by a slower secondary phase, which is caused by the recruitment of TRPM4-containing vesicles to the plasma membrane during exocytosis. The secondary phase can be observed during perfusion of cells with increasing [Ca(2+)](i), replicated with agonist stimulation, and coincides with an increase in cell capacitance, loss of FM1-43 dye, and vesicle fusion. Our data suggest that TRPM4 may play a key role in the control of membrane potential and electrical activity of electrically excitable secretory cells and the dynamic translocation of TRPM4 from a vesicular pool to the plasma membrane via Ca(2+)-dependent exocytosis may represent a key short- and midterm regulatory mechanism by which cells regulate electrical activity.

  12. Sequence analysis and functional study of the Han Nationality glial cell line-derived neurotrophic factor transcript

    Institute of Scientific and Technical Information of China (English)

    CHEN Zhe-yu; HUANG Ai-jun; LU Chang-lin; WU Xiang-fu; HE Cheng

    2001-01-01

    To study the sequence and function of the glial cell line-derived neurotrophic factor (GDNF) transcript in subjects of Han nationality. Methods: The Han nationality GDNF transcript was amplified by RT-PCR and expressed by baculovirus expression system. Biological activity of the expressed product was measured by the primary culture of midbrain dopaminergic neurons. Results: There only existed the shorter GDNF transcript of 555 bp in the Han nationality. The secretory expression product of the shorter transcript in insect cells promoted the survival and differentiation of dopaminergic neurons. Conclusion: It is found that there is a 78 bp deletion in the Han nationality GDNF transcript compared with the reported 633 bp GDNF transcript. The 78 bp deletion does not affect the secretory expression and biological activity of GDNF mature protein.

  13. Ghrelin directly stimulates glucagon secretion from pancreatic alpha-cells.

    Science.gov (United States)

    Chuang, Jen-Chieh; Sakata, Ichiro; Kohno, Daisuke; Perello, Mario; Osborne-Lawrence, Sherri; Repa, Joyce J; Zigman, Jeffrey M

    2011-09-01

    Previous work has demonstrated that the peptide hormone ghrelin raises blood glucose. Such has been attributed to ghrelin's ability to enhance GH secretion, restrict insulin release, and/or reduce insulin sensitivity. Ghrelin's reported effects on glucagon have been inconsistent. Here, both animal- and cell-based systems were used to determine the role of glucagon in mediating ghrelin's effects on blood glucose. The tissue and cell distribution of ghrelin receptors (GHSR) was evaluated by quantitative PCR and histochemistry. Plasma glucagon levels were determined following acute acyl-ghrelin injections and in pharmacological and/or transgenic mouse models of ghrelin overexpression and GHSR deletion. Isolated mouse islets and the α-cell lines αTC1 and InR1G9 were used to evaluate ghrelin's effects on glucagon secretion and the role of calcium and ERK in this activity. GHSR mRNA was abundantly expressed in mouse islets and colocalized with glucagon in α-cells. Elevation of acyl-ghrelin acutely (after sc administration, such that physiologically relevant plasma ghrelin levels were achieved) and chronically (by slow-releasing osmotic pumps and as observed in transgenic mice harboring ghrelinomas) led to higher plasma glucagon and increased blood glucose. Conversely, genetic GHSR deletion was associated with lower plasma glucagon and reduced fasting blood glucose. Acyl-ghrelin increased glucagon secretion in a dose-dependent manner from mouse islets and α-cell lines, in a manner requiring elevation of intracellular calcium and phosphorylation of ERK. Our study shows that ghrelin's regulation of blood glucose involves direct stimulation of glucagon secretion from α-cells and introduces the ghrelin-glucagon axis as an important mechanism controlling glycemia under fasting conditions.

  14. Hsp60 is actively secreted by human tumor cells.

    Directory of Open Access Journals (Sweden)

    Anna M Merendino

    Full Text Available BACKGROUND: Hsp60, a Group I mitochondrial chaperonin, is classically considered an intracellular chaperone with residence in the mitochondria; nonetheless, in the last few years it has been found extracellularly as well as in the cell membrane. Important questions remain pertaining to extracellular Hsp60 such as how generalized is its occurrence outside cells, what are its extracellular functions and the translocation mechanisms that transport the chaperone outside of the cell. These questions are particularly relevant for cancer biology since it is believed that extracellular chaperones, like Hsp70, may play an active role in tumor growth and dissemination. METHODOLOGY/PRINCIPAL FINDINGS: Since cancer cells may undergo necrosis and apoptosis, it could be possible that extracellular Hsps are chiefly the result of cell destruction but not the product of an active, physiological process. In this work, we studied three tumor cells lines and found that they all release Hsp60 into the culture media by an active mechanism independently of cell death. Biochemical analyses of one of the cell lines revealed that Hsp60 secretion was significantly reduced, by inhibitors of exosomes and lipid rafts. CONCLUSIONS/SIGNIFICANCE: Our data suggest that Hsp60 release is the result of an active secretion mechanism and, since extracellular release of the chaperone was demonstrated in all tumor cell lines investigated, our observations most likely reflect a general physiological phenomenon, occurring in many tumors.

  15. Dietary gluten increases natural killer cell cytotoxicity and cytokine secretion.

    Science.gov (United States)

    Larsen, Jesper; Dall, Morten; Antvorskov, Julie Christine; Weile, Christian; Engkilde, Kåre; Josefsen, Knud; Buschard, Karsten

    2014-10-01

    Dietary gluten influences the development of type 1 diabetes in nonobese diabetic (NOD) mice and biobreeding rats, and has been shown to influence a wide range of immunological factors in the pancreas and gut. In the present study, the effects of gluten on NK cells were studied in vitro and in vivo. We demonstrated that gliadin increased direct cytotoxicity and IFN-γ secretion from murine splenocytes and NK cells toward the pancreatic beta-cell line MIN6 cells. Additionally, stimulation of MIN6 cells led to a significantly increased proportion of degranulating C57BL/6 CD107a(+) NK cells. Stimulation of C57BL/6 pancreatic islets with gliadin significantly increased secretion of IL-6 more than ninefold. In vivo, the gluten-containing diet led to a higher expression of NKG2D and CD71 on NKp46(+) cells in all lymphoid organs in BALB/c and NOD mice compared with the gluten-free diet. Collectively, our data suggest that dietary gluten increases murine NK-cell activity against pancreatic beta cells. This mechanism may contribute to development of type 1 diabetes and explain the higher disease incidence associated with gluten intake in NOD mice.

  16. Histamine content and secretion in basophils and mast cells.

    Science.gov (United States)

    Dvorak, A M

    1998-01-01

    Biochemical determinations of the histamine content and secretion from basophils and mast cells have been available for some time, and much of the complex anatomy of these cellular populations and their release reactions has been documented using the electron microscope. The ultrastructural analyses led to the description of vesicular transport between secretory granules and the plasma membrane as a mechanism for secretion from basophils and mast cells--a process termed piecemeal degranulation. Proof of concepts incorporated in a general degranulation model put forth in 1975 (DVORAK, H.F. and DVORAK, A.M.) requires high magnification imaging of a granule constituent in trafficking vesicles in the process of a stimulated release reaction in which the constituent release is monitored biochemically. Development and application of a new enzyme-affinity method to detect histamine at high magnifications in well-preserved ultrastructural samples have provided the necessary means to establish proof that appropriate secretagogues can stimulate the vesicular transport of histamine in basophils and mast cells during release reactions monitored biochemically. The background information necessary to the understanding of this result is presented here, as well as the development and verification of the diamine oxidase-gold method to image histamine in human mast cell granules as the test system. Also presented are applications using this technology to examine histamine stores and secretion in vitro, in vivo, and ex vivo in human basophils and mast cells and in mouse mast cells. Specifically examined are histamine stores developing in maturing mast cells induced to develop de novo from cultured human cord blood cells, secretagogue-stimulated release and recovery of histamine stores from isolated, purified human lung mast cells ex vivo, cytokine-stimulated degranulation of human skin mast cells and their histamine stores in vivo, piecemeal degranulation of human gut mast cells and

  17. INCREASED EXPRESSION OF GLIAL CELL LINE-DERIVED NEUROTROPHIC FACTOR IN RAT BRAIN AFTER TRAUMATIC BRAIN INJURY

    Directory of Open Access Journals (Sweden)

    V. Rahimi-Movaghar

    2005-04-01

    Full Text Available Glial cell line-derived neurotrophic factor (GDNF plays important roles not only for the differentiation of neurons during normal development but also for the survival and recovery of many populations of mature neurons. The effect of traumatic brain injury (TBI on the expression of GDNF is currently unknown. To determine if there is alteration in GDNF after TBI we examined the effect of controlled cortical impact (CCI injury on GDNF protein levels at 6 hours, 1 day, 1 week, and 4 weeks following injury by utilizing a commercially available antibody specific to GDNF. Rats were anesthetized and surgically prepared for CCI injury (4 m/sec, 2.7 mm and sham surgery. Injured and sham animals (n=6 per group were sacrificed at 6 hours, 1 day, 1 week, and 4 weeks and perfused with 4% paraformaldehyde. Coronal sections (35 mm thick were cut through the hippocampus. An increased expression of GDNF protein was observed by immunohistochemistry in the dentate gyrus of hippocampus and the cortex in injured rats compared to sham controls. The increased expression of GDNF was more evidently observed in the ipsilateral dentate gyrus and the area around the contusion in the cortex. In the cortex, GDNF immunoreactivity appeared greatest in cells with glial morphology but in the hippocampus, GDNF immunoreactivity was greatest in neuronal-like cells. These changes were observed at 1 day, 1 and 4 weeks postinjury. We speculate that the up-regulation of the GDNF protein may reflect its neurotrophic and neuroprotective effect on dopaminergic system responding to the TBI insult.

  18. Plasma cell endocytosis: is it related to immunoglobulin secretion?

    Science.gov (United States)

    Tartakoff, A; Vassalli, P; Montesano, R

    1981-12-01

    Mouse plasma cells have been exposed to a wide range of soluble and adsorptive macromolecular tracers for 10 min to 4 h to explore the possibility of membrane recycling related to the high secretory rate of these nonregulated secretory cells. Electron microscopic examination showed in all cases labeling of multivesicular and multilamellar bodies and a lesser labeling of smooth-surfaced vesicles. Using cationized ferritin as tracer, an additional very restricted labeling of Golgi cisternae was observed. Comparable labeling patterns were observed when immunoglobulin (Ig) secretion was blocked with the Golgi-specific pertrurbant, monensin, and in the case of poorly differentiated B immunoblasts which secrete little or no Ig. Our observations therefore emphasize that available approaches cannot yet determine whether a mandatory circuit of vesicular traffic couples Ig exocytosis to endocytosis.

  19. Exosomes from B cells and Dendritic cells: mechanisms of formation, secretion and targeting

    OpenAIRE

    Buschow, S.I.

    2006-01-01

    Many cell types, including dendritic cells (DC) and B cells, secrete small vesicles called exosomes. Exosomes from immune cells are thought to have immuno-regulatory functions but their precise role remains unresolved. The aim of the studies presented in this thesis was to get more insight into the factors that determine exosome formation, composition and secretion as well as to learn more about their physiological relevance. Exosomes are equivalent to Luminal Vesicles (LV) of Multi Vesicular...

  20. Glial cell line-derived neurotrophic factor (GDNF) expression and NMJ plasticity in skeletal muscle following endurance exercise.

    Science.gov (United States)

    Gyorkos, A M; McCullough, M J; Spitsbergen, J M

    2014-01-17

    Glial cell line-derived neurotrophic factor (GDNF) supports and maintains the neuromuscular system during development and through adulthood by promoting neuroplasticity. The aim of this study was to determine if different modes of exercise can promote changes in GDNF expression and neuromuscular junction (NMJ) morphology in slow- and fast-twitch muscles. Rats were randomly assigned to a run training (run group), swim training (swim group), or sedentary control group. GDNF protein content was determined by enzyme-linked immunosorbant assay. GDNF protein content increased significantly in soleus (SOL) following both training protocols (PGDNF content and total end plate area were positively correlated. End plate area decreased in EDL of the run group and increased in SOL of the swim group. The results indicate that GDNF expression and NMJ morphological changes are activity dependent and that different changes may be observed by varying the exercise intensity in slow- and fast-twitch fibers.

  1. Lipid-mediated glial cell line-derived neurotrophic factor gene transfer to cultured porcine ventral mesencephalic tissue

    DEFF Research Database (Denmark)

    Bauer, Matthias; Meyer, Morten; Brevig, Thomas;

    2002-01-01

    -mediated transfer of the gene for human glial cell line-derived neurotrophic factor (GDNF) to embryonic (E27/28) porcine VM tissue kept as organotypic explant cultures. Treatment of the developing VM with two mitogens, basic fibroblast growth factor and epidermal growth factor, prior to transfection significantly...... numbers of tyrosine hydroxylase-positive neurons in the cultured VM tissue. We conclude that lipid-mediated gene transfer employed on embryonic pig VM explant cultures is a safe and effective method to improve survival of dopaminergic neurons and may become a valuable tool to improve allo......Transplantation of dopaminergic ventral mesencephalic (VM) tissue into the basal ganglia of patients with Parkinson's disease (PD) shows at best moderate symptomatic relief in some of the treated cases. Experimental animal studies and clinical trials with allogenic and xenogenic pig-derived VM...

  2. Mesenchymal Stem Cells Expressing Brain-Derived Neurotrophic Factor Enhance Endogenous Neurogenesis in an Ischemic Stroke Model

    Directory of Open Access Journals (Sweden)

    Chang Hyun Jeong

    2014-01-01

    Full Text Available Numerous studies have reported that mesenchymal stem cells (MSCs can ameliorate neurological deficits in ischemic stroke models. Among the various hypotheses that have been suggested to explain the therapeutic mechanism underlying these observations, neurogenesis is thought to be critical. To enhance the therapeutic benefits of human bone marrow-derived MSCs (hBM-MSCs, we efficiently modified hBM-MSCs by introduction of the brain-derived neurotrophic factor (BDNF gene via adenoviral transduction mediated by cell-permeable peptides and investigated whether BDNF-modified hBM-MSCs (MSCs-BDNF contributed to functional recovery and endogenous neurogenesis in a rat model of middle cerebral artery occlusion (MCAO. Transplantation of MSCs induced the proliferation of 5-bromo-2′-deoxyuridine (BrdU- positive cells in the subventricular zone. Transplantation of MSCs-BDNF enhanced the proliferation of endogenous neural stem cells more significantly, while suppressing cell death. Newborn cells differentiated into doublecortin (DCX- positive neuroblasts and Neuronal Nuclei (NeuN- positive mature neurons in the subventricular zone and ischemic boundary at higher rates in animals with MSCs-BDNF compared with treatment using solely phosphate buffered saline (PBS or MSCs. Triphenyltetrazolium chloride staining and behavioral analysis revealed greater functional recovery in animals with MSCs-BDNF compared with the other groups. MSCs-BDNF exhibited effective therapeutic potential by protecting cell from apoptotic death and enhancing endogenous neurogenesis.

  3. Mesenchymal stem cells expressing brain-derived neurotrophic factor enhance endogenous neurogenesis in an ischemic stroke model.

    Science.gov (United States)

    Jeong, Chang Hyun; Kim, Seong Muk; Lim, Jung Yeon; Ryu, Chung Heon; Jun, Jin Ae; Jeun, Sin-Soo

    2014-01-01

    Numerous studies have reported that mesenchymal stem cells (MSCs) can ameliorate neurological deficits in ischemic stroke models. Among the various hypotheses that have been suggested to explain the therapeutic mechanism underlying these observations, neurogenesis is thought to be critical. To enhance the therapeutic benefits of human bone marrow-derived MSCs (hBM-MSCs), we efficiently modified hBM-MSCs by introduction of the brain-derived neurotrophic factor (BDNF) gene via adenoviral transduction mediated by cell-permeable peptides and investigated whether BDNF-modified hBM-MSCs (MSCs-BDNF) contributed to functional recovery and endogenous neurogenesis in a rat model of middle cerebral artery occlusion (MCAO). Transplantation of MSCs induced the proliferation of 5-bromo-2'-deoxyuridine (BrdU-) positive cells in the subventricular zone. Transplantation of MSCs-BDNF enhanced the proliferation of endogenous neural stem cells more significantly, while suppressing cell death. Newborn cells differentiated into doublecortin (DCX-) positive neuroblasts and Neuronal Nuclei (NeuN-) positive mature neurons in the subventricular zone and ischemic boundary at higher rates in animals with MSCs-BDNF compared with treatment using solely phosphate buffered saline (PBS) or MSCs. Triphenyltetrazolium chloride staining and behavioral analysis revealed greater functional recovery in animals with MSCs-BDNF compared with the other groups. MSCs-BDNF exhibited effective therapeutic potential by protecting cell from apoptotic death and enhancing endogenous neurogenesis.

  4. Acetylcholine regulates pancreastatin secretion from the human pancreastatin-producing cell line (QGP-1N).

    Science.gov (United States)

    Funakoshi, A; Tateishi, K; Tsuru, M; Jimi, A; Wakasugi, H; Kono, A

    1991-07-01

    Studies were made of pancreastatin (PST) secretion from a human PST-producing cell line (QGP-1N) in response to various secretagogues. Cells with immunoreactivity for PST were observed in monolayer cultures of QGP-1N cells. Carbachol stimulated PST secretion and the intracellular Ca2+ mobilization concentration dependently in the range of 10(-6)-10(-4) M. The PST secretion and Ca2+ mobilization induced by carbachol were inhibited by atropine. The calcium ionophore (A23187) stimulated PST secretion. However, cholecystokinin and gastrin-releasing peptide did not stimulate either PST secretion or Ca2+ mobilization. Secretin also did not stimulate PST secretion. The glucose concentration in the culture medium had no effect on PST secretion. These results suggest that PST secretion is mainly regulated by acetylcholine through a muscarinic receptor, and that an increase in intracellular Ca2+ plays an important role in stimulus-secretion coupling in QGP-1N cells.

  5. Glial cell line-derived neurotrophic factor induced the differentiation of amniotic fluid-derived stem cells into vascular endothelial-like cells in vitro.

    Science.gov (United States)

    Zhang, Ruyu; Lu, Ying; Li, Ju; Wang, Jia; Liu, Caixia; Gao, Fang; Sun, Dong

    2016-02-01

    Amniotic fluid-derived stem cells (AFSCs) are a novel source of stem cells that are isolated and cultured from second trimester amniocentesis. Glial cell line-derived neurotrophic factor (GDNF) acts as a tissue morphogen and regulates stem cell proliferation and differentiation. This study investigated the effect of an adenovirus-mediated GDNF gene, which was engineered into AFSCs, on the cells' biological properties and whether GDNF in combination with AFSCs can be directionally differentiated into vascular endothelial-like cells in vitro. AFSCs were isolated and cultured using the plastic adherence method in vitro and identified by the transcription factor Oct-4, which is the primary marker of pluripotent stem cells. AFSCs were efficiently transfected by a GFP-labeled plasmid system of an adenovirus vector carrying the GDNF gene (Ad-GDNF-GFP). Transfected AFSCs stably expressed GDNF. Transfected AFSCs were cultured in endothelial growth medium-2 containing vascular endothelial growth factor. After 1 week, AFSCs were positive for von Willebrand factor (vWF) and CD31, which are markers of endothelial cells, and the recombinant GDNF group was significantly higher than undifferentiated controls and the GFP only group. These results demonstrated that AFSCs differentiated into vascular endothelial-like cells in vitro, and recombinant GDNF promoted differentiation. The differentiation-induced AFSCs may be used as seed cells to provide a new manner of cell and gene therapies for transplantation into the vascular injury site to promote angiogenesis.

  6. RNA interference-mediated knockdown of brain-derived neurotrophic factor (BDNF) promotes cell cycle arrest and apoptosis in B-cell lymphoma cells.

    Science.gov (United States)

    Xia, D; Li, W; Zhang, L; Qian, H; Yao, S; Qi, X

    2014-01-01

    Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin superfamily that has been reported to be involved in a number of neurological and psychological situations. Recently, high expression level of BDNF is observed in diverse human malignancies, delineating a role of BDNF in tumorigenesis. Nevertheless, its effect on B-cell lymphoma remains unclear. In this study, RNA interference technology mediated by short hairpin RNA (shRNA) was performed to inhibit endogenous BDNF expression in B-cell lymphoma cells. Results showed that knockdown of BDNF reduced cell growth and proliferation of Raji and Ramos cells. Furthermore, down-regulation of BDNF induced a cell cycle arrest at G0/G1 phase in Raji cells, and consequently led to cell apoptosis in vitro. Meanwhile, down-regulation of Bcl-2 and up-regulation of Bax, activated caspase-3 and caspase-9 and cleaved poly (ADP-ribose) polymerase (PARP) were observed in Raji cells when endogenous BDNF was inhibited. Besides, we also found that suppression of BDNF in Raji cells increased their sensitivity to chemotherapeutic drug, 5-Fluorouracil (5-FU). Our research provides a promising therapeutic strategy for human B-cell lymphoma by targeting BDNF.

  7. Brain-derived neurotrophic factor (BDNF) gene delivery into the CNS using bone marrow cells as vehicles in mice.

    Science.gov (United States)

    Makar, T K; Trisler, D; Eglitis, M A; Mouradian, M M; Dhib-Jalbut, S

    2004-02-19

    Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, is protective in animal models of neurodegenerative diseases. However, BDNF has a short half-life and its efficacy in the CNS when delivered peripherally is limited due to the blood-brain barrier. In the present study, bone marrow cells were used as vehicles to deliver the BDNF gene into the CNS. Marrow cells obtained from 6 to 8 week-old SJL/J mice were transduced with BDNF expressing pro-virus. RT-PCR analysis revealed that BDNF mRNA was expressed in transduced but not in non-transduced marrow cells. Additionally, virus transduced marrow cells expressed the BDNF protein (296+/-1.2 unit/ml). BDNF-transduced marrow cells were then transplanted into irradiated mice through the tail vein. Three months post-transplantation, significant increases in BDNF as well as glutamic acid decarboxylase (GAD(67)) mRNA were detected in the brains of BDNF transplanted mice compared to untransplanted animals, indicating biological activity of the BDNF transgene. Thus, bone marrow cells can be used as vehicles to deliver the BDNF gene into the brain with implications for the treatment of neurological diseases.

  8. Purification and cultivation of human pituitary growth hormone secreting cells

    Science.gov (United States)

    Hymer, W. C.

    1979-01-01

    Efforts were directed towards maintenance of actively secreting human pituitary growth hormone cells (somatotrophs) in vitro. The production of human growth hormone (hGH) by this means would be of benefit for the treatment of certain human hypopituitary diseases such as dwarfism. One of the primary approaches was the testing of agents which may logically be expected to increase hGH release. The progress towards this goal is summarized. Results from preliminary experiments dealing with electrophoresis of pituitary cell for the purpose of somatotroph separation are described.

  9. Brain derived neurotrophic factor contributes to the cardiogenic potential of adult resident progenitor cells in failing murine heart.

    Directory of Open Access Journals (Sweden)

    Rasmita Samal

    Full Text Available Resident cardiac progenitor cells show homing properties when injected into the injured but not to the healthy myocardium. The molecular background behind this difference in behavior needs to be studied to elucidate how adult progenitor cells can restore cardiac function of the damaged myocardium. Since the brain derived neurotrophic factor (BDNF moderates cardioprotection in injured hearts, we focused on delineating its regulatory role in the damaged myocardium.Comparative gene expression profiling of freshly isolated undifferentiated Sca-1 progenitor cells derived either from heart failure transgenic αMHC-CyclinT1/Gαq overexpressing mice or wildtype littermates revealed transcriptional variations. Bdnf expression was up regulated 5-fold during heart failure which was verified by qRT-PCR and confirmed at protein level. The migratory capacity of Sca-1 cells from transgenic hearts was improved by 15% in the presence of 25 ng/ml BDNF. Furthermore, BDNF-mediated effects on Sca-1 cells were studied via pulsed Stable Isotope Labeling of Amino acids in Cell Culture (pSILAC proteomics approach. After BDNF treatment significant differences between newly synthesized proteins in Sca-1 cells from control and transgenic hearts were observed for CDK1, SRRT, HDGF, and MAP2K3 which are known to regulate cell cycle, survival and differentiation. Moreover BDNF repressed the proliferation of Sca-1 cells from transgenic hearts.Comparative profiling of resident Sca-1 cells revealed elevated BDNF levels in the failing heart. Exogenous BDNF (i stimulated migration, which might improve the homing ability of Sca-1 cells derived from the failing heart and (ii repressed the cell cycle progression suggesting its potency to ameliorate heart failure.

  10. Glial cell line-derived neurotrophic factor attenuates behavioural deficits and regulates nigrostriatal dopaminergic and peptidergic markers in 6-hydroxydopamine-lesioned adult rats: comparison of intraventricular and intranigral delivery.

    Science.gov (United States)

    Lapchak, P A; Miller, P J; Collins, F; Jiao, S

    1997-05-01

    The effects of intranigrally- or intraventricularly-administered glial cell line-derived neurotrophic factor were tested on low dose (0.05 mg/kg) apomorphine-induced rotations and tyrosine hydroxylase activity in the substantia nigra and striatum of stable 6-hydroxydopamine-lesioned rats. In addition, we determined if 6-hydroxydopamine lesions in the absence or presence of treatment affected neuropeptide (substance P, met-enkephalin, dynorphin) content in the striatum. Glial cell line-derived neurotrophic factor, when administered intranigrally, prevented apomorphine-induced rotational behaviour for 11 weeks following a single injection. In comparison, intraventricularly-administered glial cell line-derived neurotrophic factor produced a transient reduction in rotational behaviour that lasted for two to three weeks following a single injection. We also show that rotational behaviour is reduced following each subsequent intraventricular injection of glial cell line-derived neurotrophic factor given every six weeks, a time-point when baseline rotation deficits were re-established. Intranigrally- or intraventricularly-administered glial cell line-derived neurotrophic factor significantly reduced weight gain in all 6-hydroxydopamine-lesioned rats in this study. Following behavioural analysis where a confirmed improvement of behaviour was established, tissues were dissected for neurochemical analysis. In lesioned rats with intranigral injections of administered glial cell line-derived neurotrophic factor, significant increases of nigral, but not striatal tyrosine hydroxylase activity were measured. Additionally, 6-hydroxydopamine lesions significantly increased striatal dynorphin (61-139%) and met-enkephalin (81-139%), but not substance P levels. In these rats, intranigrally-administered glial cell line-derived neurotrophic factor injections reversed lesion-induced increases in nigral dynorphin A levels and increased nigral dopamine levels, but did not alter nigral met

  11. Upregulation of glutathione peroxidase-1 expression and activity by glial cell line-derived neurotrophic factor promotes high-level protection of PC12 cells against 6-hydroxydopamine and hydrogen peroxide toxicities.

    Science.gov (United States)

    Gharib, Ehsan; Gardaneh, Mossa; Shojaei, Sahar

    2013-06-01

    We examined the impact of strong co-presence and function of glutathione peroxidase-1 (GPX-1) and glial cell line-derived neurotrophic factor (GDNF) on protecting the rat dopaminergic pheochromocytoma cell line PC12 against 6-hydroxydopamine (6-OHDA) and hydrogen peroxide (H₂O₂) toxicities. Primarily, GPX-1 over-expression by PC12 cells infected with pLV-GPX1 lentivirus vectors significantly increased cell survival against 6-OHDA toxicity (pcells with astro-CM of GDNF-over-secreting astrocytes (Test astro-CM) significantly induced GPX-1 expression, peroxidase enzymatic activity, and intra-cellular glutathione (GSH) levels. These changes paralleled with protection of 90% of GDNF⁺/GPX1⁺ PC12 cells against toxicity, a rate that was 37% up from their un-infected un-treated (GDNF⁻/GPX1⁻) controls (pcells that received only Control astro-CM (GPX⁺/GDNF⁻) (pcell groups, increased cell survival against either compound was further confirmed by increased live cell counts measured by double staining. Following depletion of intra-cellular GSH, only 46% of pLV-GPX1 cells survived 6-OHDA toxicity, whereas over 70% of them were saved upon GDNF treatment (pcells and maximized by addition of GDNF. Comparison analyses established correlations between GPX-1-GDNF co-presence and both enhanced cell protection and diminished levels of activated caspase-3. Our data collectively indicate that GDNF is capable of inducing anti-oxidant activities of intra-cellular GPX-1 and that growth-promoting potential of GDNF and anti-oxidant properties of GPX-1 can, in concert, maximize survival of dopaminergic neurons.

  12. Localization of Brain-Derived Neurotrophic Factor to Distinct Terminals of Mossy Fiber Axons Implies Regulation of Both Excitation and Feedforward Inhibition of CA3 Pyramidal Cells

    OpenAIRE

    Danzer, Steve C.; McNamara, James O.

    2004-01-01

    Hippocampal dentate granule cells directly excite and indirectly inhibit CA3 pyramidal cells via distinct presynaptic terminal specializations of their mossy fiber axons. This mossy fiber pathway contains the highest concentration of brain-derived neurotrophic factor (BDNF) in the CNS, yet whether BDNF is positioned to regulate the excitatory and/or inhibitory pathways is unknown. To localize BDNF, confocal microscopy of green fluorescent protein transgenic mice was combined with BDNF immunoh...

  13. Secretion of neurotensin from a human pancreatic islet cell carcinoma cell line (QGP-1N).

    Science.gov (United States)

    Tateishi, K; Funakoshi, A; Kitayama, N; Matsuoka, Y

    1993-12-10

    Effects of various secretagogues on secretion of neurotensin from a pancreatic islet cell carcinoma cell line (QGP-1N) were examined. Carbachol stimulated secretion of neurotensin concentration-dependently in the range of 10(-6) - 10(-4) M. The neurotensin secretion stimulated with 10(-5) M carbachol was completely inhibited by atropine at 10(-5) M. Phorbol ester and calcium ionophore (A23187) stimulated secretion of neurotensin. The removal of extracellular Ca2+ suppressed the secretion through the stimulation with 10(-5) M carbachol. Fluoride, an activator of guanine nucleotide-binding (G) protein, stimulated secretion of neurotensin. Neurotensin released into culture medium through stimulation with carbachol coeluted with neurotensin 1-13 on a gel-chromatography. Our results suggest that secretion of neurotensin from QGP-1N cells is mainly regulated by acetylcholine through muscarinic receptors coupled to G protein and that an increase in intracellular Ca2+ and protein kinase C play an important role in stimulus-secretion coupling.

  14. Preservation of biological activity of glial cell line-derived neurotrophic factor (GDNF) after microencapsulation and sterilization by gamma irradiation.

    Science.gov (United States)

    Checa-Casalengua, P; Jiang, C; Bravo-Osuna, I; Tucker, B A; Molina-Martínez, I T; Young, M J; Herrero-Vanrell, R

    2012-10-15

    A main issue in controlled delivery of biotechnological products from injectable biodegradable microspheres is to preserve their integrity and functional activity after the microencapsulation process and final sterilization. The present experimental work tested different technological approaches to maintain the biological activity of an encapsulated biotechnological product within PLGA [poly (lactic-co-glycolic acid)] microspheres (MS) after their sterilization by gamma irradiation. GDNF (glial cell line-derived neurotrophic factor), useful in the treatment of several neurodegenerative diseases, was chosen as a labile model protein. In the particular case of optic nerve degeneration, GDNF has been demonstrated to improve the damaged retinal ganglion cells (RGC) survival. GDNF was encapsulated in its molecular state by the water-in-oil-in-water (W/O/W) technique or as solid according to the solid-in-oil-in-water (S/O/W) method. Based on the S/O/W technique, GDNF was included in the PLGA microspheres alone (S/O/W 1) or in combination with an antioxidant (vitamin E, Vit E) (S/O/W 2). Microspheres were sterilized by gamma-irradiation (dose of 25 kGy) at room and low (-78 °C) temperatures. Functional activity of GDNF released from the different microspheres was evaluated both before and after sterilization in their potential target cells (retinal cells). Although none of the systems proposed achieved with the goal of totally retain the structural stability of the GDNF-dimer, the protein released from the S/O/W 2 microspheres was clearly the most biologically active, showing significantly less retinal cell death than that released from either W/O/W or S/O/W 1 particles, even in low amounts of the neurotrophic factor. According to the results presented in this work, the biological activity of biotechnological products after microencapsulation and sterilization can be further preserved by the inclusion of the active molecule in its solid state in combination with

  15. Riluzole enhances expression of brain-derived neurotrophic factor with consequent proliferation of granule precursor cells in the rat hippocampus.

    Science.gov (United States)

    Katoh-Semba, Ritsuko; Asano, Tomiko; Ueda, Hiroshi; Morishita, Rika; Takeuchi, Ikuo K; Inaguma, Yutaka; Kato, Kanefusa

    2002-08-01

    The dentate gyrus of the hippocampus, generating new cells throughout life, is essential for normal recognition memory performance. Reduction of brain-derived neurotrophic factor (BDNF) in this structure impairs its functions. To elucidate the association between BDNF levels and hippocampal neurogenesis, we first conducted a search for compounds that stimulate endogenous BDNF production in hippocampal granule neurons. Among ion channel modulators tested, riluzole, a neuroprotective agent with anticonvulsant properties that is approved for treatment of amyotrophic lateral sclerosis, was highly effective as a single dose by an intraperitoneal injection, causing a rise in BDNF localized in dentate granule neurons, the hilus, and the stratum radiatum of the CA3 region. Repeated, but not single, injections resulted in prolonged elevation of hippocampal BDNF and were associated with increased numbers of newly generated cells in the granule cell layer. This appeared due to promoted proliferation rather than survival of precursor cells, many of which differentiated into neurons. Intraventricular administration of BDNF-specific antibodies blocked such riluzole effects, suggesting that BDNF increase is necessary for the promotion of precursor proliferation. Our results suggest the basis for a new strategy for treatment of memory dysfunction.

  16. Influence of brain-derived neurotrophic factor on pathfinding of dentate granule cell axons, the hippocampal mossy fibers.

    Science.gov (United States)

    Tamura, Makoto; Tamura, Naohiro; Ikeda, Takamitsu; Koyama, Ryuta; Ikegaya, Yuji; Matsuki, Norio; Yamada, Maki K

    2009-01-31

    Mossy fibers, the dentate granule cell axons, are generated throughout an animal's lifetime. Mossy fiber paths and synapses are primarily restricted to the stratum lucidum within the CA3 region. Brain-derived neurotrophic factor (BDNF), a neurotrophin family protein that activates Trk neurotrophin receptors, is highly expressed in the stratum lucidum in an activity-dependent manner. The addition of a Trk neurotrophin receptor inhibitor, K252a, to cultured hippocampal slices induced aberrant extension of mossy fibers into ectopic regions. BDNF overexpression in granule cells ameliorated the mossy fiber pathway abnormalities caused by a submaximal dose of K252a. A similar rescue was observed when BDNF was expressed in CA3 pyramidal cells, most notably in mossy fibers distal to the expression site. These findings are the first to clarify the role of BDNF in mossy fiber pathfinding, not as an attractant cue but as a regulator, possibly acting in a paracrine manner. This effect of BDNF may be as a signal for new fibers to fasciculate and extend further to form synapses with neurons that are far from active BDNF-expressing synapses. This mechanism would ensure the emergence of new independent dentate gyrus-CA3 circuits by the axons of new-born granule cells.

  17. Influence of brain-derived neurotrophic factor on pathfinding of dentate granule cell axons, the hippocampal mossy fibers

    Directory of Open Access Journals (Sweden)

    Tamura Makoto

    2009-01-01

    Full Text Available Abstract Mossy fibers, the dentate granule cell axons, are generated throughout an animal's lifetime. Mossy fiber paths and synapses are primarily restricted to the stratum lucidum within the CA3 region. Brain-derived neurotrophic factor (BDNF, a neurotrophin family protein that activates Trk neurotrophin receptors, is highly expressed in the stratum lucidum in an activity-dependent manner. The addition of a Trk neurotrophin receptor inhibitor, K252a, to cultured hippocampal slices induced aberrant extension of mossy fibers into ectopic regions. BDNF overexpression in granule cells ameliorated the mossy fiber pathway abnormalities caused by a submaximal dose of K252a. A similar rescue was observed when BDNF was expressed in CA3 pyramidal cells, most notably in mossy fibers distal to the expression site. These findings are the first to clarify the role of BDNF in mossy fiber pathfinding, not as an attractant cue but as a regulator, possibly acting in a paracrine manner. This effect of BDNF may be as a signal for new fibers to fasciculate and extend further to form synapses with neurons that are far from active BDNF-expressing synapses. This mechanism would ensure the emergence of new independent dentate gyrus-CA3 circuits by the axons of new-born granule cells.

  18. Exosomes from B cells and Dendritic cells: mechanisms of formation, secretion and targeting

    NARCIS (Netherlands)

    Buschow, S.I.

    2006-01-01

    Many cell types, including dendritic cells (DC) and B cells, secrete small vesicles called exosomes. Exosomes from immune cells are thought to have immuno-regulatory functions but their precise role remains unresolved. The aim of the studies presented in this thesis was to get more insight into the

  19. Effect of herpesvirus infection on pancreatic duct cell secretion

    Institute of Scientific and Technical Information of China (English)

    Péter Hegyi; András Varró; Mária K Kovács; Mike A Gray; Barry E Argent; Zsolt Boldogk(o)i; Balázs (O)rd(o)g; Zoltán Rakonczai Jr; Tamás Takács; János Lonovics; Annamária Szabolcs; Réka Sári; András Tóth; Julius G Papp

    2005-01-01

    AIM: To examine the effect of acute infection caused by herpesvirus (pseudorabies virus, PRV) on pancreatic ductal secretion.METHODS: The virulent Ba-DupGreen (BDG) and nonvirulent Ka-RREpOlacgfp (KEG) genetically modified strains of PRV were used in this study and both of them contain the gene for green fluorescent protein (GFP). Small intra/interlobular ducts were infected with BDG virus (107 PFU/mL for 6 h) or with KEG virus (1010 PFU/mL for 6 h), while non-infected ducts were incubated only with the culture media. The ducts were then cultured for a further 18 h.The rate of HCO3- secretion [base efflux -J(B-)] was determined from the buffering capacity of the cells and the initial rate of intracellular acidification (1) after sudden blockage of basolateral base loaders with dihydro-4,4,-diisothiocyanatostilbene-2,2,-disulfonic acid (500 μmol/L)and amiloride (200 μmol/L), and (2) after alkali loading the ducts by exposure to NH4Cl. All the experiments were performed in HCO3--buffered Ringer solution at 37 ℃ (n = 5ducts for each experimental condition). Viral structural proteins were visualized by immunohistochemistry. Virallyencoded GFP and immunofluorescence signals were recorded by a confocal laser scanning microscope.RESULTS: The BDG virus infected the majority of accessible cells of the duct as judged by the appearance of GFP and viral antigens in the ductal cells. KEG virus caused a similarly high efficiency of infection. After blockage of basolateral base loaders, BDG infection significantly elevated -J(B-) 24 h after the infection, compared to the non-infected group. However, KEG infection did not modify -J(B-). After alkali loading the ducts, -J(B-) was significantly elevated in the BDG group compared to the control group 24 h after the infection. As we found with the inhibitor stop method, no change was observed in the group KEG compared to the non-infected group.CONCLUSION: Incubation with the BDG or KEG strains of PRV results in an effective

  20. Evolution of brain-derived neurotrophic factor levels after autologous hematopietic stem cell transplantation in multiple sclerosis.

    Science.gov (United States)

    Blanco, Y; Saiz, A; Costa, M; Torres-Peraza, J F; Carreras, E; Alberch, J; Jaraquemada, D; Graus, F

    A neuroprotective role of inflammation has been suggested based on that immune cells are the main source of brain-derived neurotrophic factor (BDNF). We investigated the 3-year evolution of BDNF levels in serum, CSF and culture supernatant of peripheral blood mononuclear cells (PBMC), unstimulated and stimulated with anti-CD3 and soluble anti-CD28 antibodies, in 14 multiple sclerosis patients who underwent an autologous hematopoietic stem cell transplantation (AHSCT). BDNF levels were correlated with previously reported MRI measures that showed a reduction of T2 lesion load and increased brain atrophy, mainly at first year post-transplant. A significant decrease of serum BDNF levels was seen at 12 months post-transplant. BDNF values were found significantly lower in stimulated but not in unstimulated PBMC supernatants during the follow-up, supporting that AHSCT may induce a down-regulation of BDNF production. The only significant correlation was found between CSF BDNF levels and T2 lesion load before and 1 year after AHSCT, suggesting that BDNF reflects the past and ongoing inflammatory activity and demyelination of these highly active patients. Our study suggests that AHSCT can reduce BDNF levels to values associated with lower activity. This decrease does not seem to correlate with the brain atrophy measures observed in the MRI.

  1. Nanowell-based immunoassays for measuring single-cell secretion: characterization of transport and surface binding.

    Science.gov (United States)

    Torres, Alexis J; Hill, Abby S; Love, J Christopher

    2014-12-01

    Arrays of subnanoliter wells (nanowells) provide a useful system to isolate single cells and analyze their secreted proteins. Two general approaches have emerged: one that uses open arrays and local capture of secreted proteins, and a second (called microengraving) that relies on closed arrays to capture secreted proteins on a solid substrate, which is subsequently removed from the array. However, the design and operating parameters for efficient capture from these two approaches to analyze single-cell secretion have not been extensively considered. Using numerical simulations, we analyzed the operational envelope for both open and closed formats, as a function of the spatial distribution of capture ligands, their affinities for the protein, and the rates of single-cell secretion. Based on these analyses, we present a modified approach to capture secreted proteins in-well for highly active secreting cells. This simple method for in-well detection should facilitate rapid identification of cell lines with high specific productivities.

  2. The role of brain-derived neurotrophic factor in the regulation of cell growth and gene expression in melanotrope cells of Xenopus laevis.

    Science.gov (United States)

    Jenks, Bruce G; Kuribara, Miyuki; Kidane, Adhanet H; Kramer, Bianca M R; Roubos, Eric W; Scheenen, Wim J J M

    2012-07-01

    Brain-derived neurotrophic factor (BDNF) is, despite its name, also found outside the central nervous system (CNS), but the functional significance of this observation is largely unknown. This review concerns the expression of BDNF in the pituitary gland. While the presence of the neurotrophin in the mammalian pituitary gland is well documented its functional significance remains obscure. Studies on the pars intermedia of the pituitary of the amphibian Xenopus laevis have shown that BDNF is produced by the neuroendocrine melanotrope cells, its expression is physiologically regulated, and the melanotrope cells themselves express receptors for the neurotrophin. The neurotrophin has been shown to act as an autocrine factor on the melanotrope to promote cell growth and regulate gene expression. In doing so BDNF supports the physiological function of the cell to produce and release α-melanophore-stimulating hormone for the purpose of adjusting the animal's skin color to that of its background.

  3. Impact of Mesenchymal Stem Cell secreted PAI-1 on colon cancer cell migration and proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Hogan, Niamh M. [Discipline of Surgery, School of Medicine, National University of Ireland, Galway (Ireland); Joyce, Myles R. [Department of Colorectal Surgery, University College Hospital, Galway (Ireland); Murphy, J. Mary; Barry, Frank P.; O’Brien, Timothy [Regenerative Medicine Institute, National University of Ireland, Galway (Ireland); Kerin, Michael J. [Discipline of Surgery, School of Medicine, National University of Ireland, Galway (Ireland); Dwyer, Roisin M., E-mail: roisin.dwyer@nuigalway.ie [Discipline of Surgery, School of Medicine, National University of Ireland, Galway (Ireland)

    2013-06-14

    Highlights: •MSCs were directly co-cultured with colorectal cancer (CRC) cells on 3D scaffolds. •MSCs influence CRC protein/gene expression, proliferation and migration. •We report a significant functional role of MSC-secreted PAI-1 in colon cancer. -- Abstract: Mesenchymal Stem Cells are known to engraft and integrate into the architecture of colorectal tumours, with little known regarding their fate following engraftment. This study aimed to investigate mediators of Mesenchymal Stem Cell (MSC) and colon cancer cell (CCC) interactions. Mesenchymal Stem Cells and colon cancer cells (HT29 and HCT-116) were cultured individually or in co-culture on 3-dimensional scaffolds. Conditioned media containing all secreted factors was harvested at day 1, 3 and 7. Chemokine secretion and expression were analyzed by Chemi-array, ELISA (Macrophage migration inhibitory factor (MIF), plasminogen activator inhibitor type 1 (PAI-1)) and RQ-PCR. Colon cancer cell migration and proliferation in response to recombinant PAI-1, MSCs and MSCs + antibody to PAI-1 was analyzed using Transwell inserts and an MTS proliferation assay respectively. Chemi-array revealed secretion of a wide range of factors by each cell population, including PAI-1and MIF. ELISA analysis revealed Mesenchymal Stem Cells to secrete the highest levels of PAI-1 (MSC mean 10.6 ng/mL, CCC mean 1.01 ng/mL), while colon cancer cells were the principal source of MIF. MSC-secreted PAI-1 stimulated significant migration of both CCC lines, with an antibody to the chemokine shown to block this effect (67–88% blocking,). A cell-line dependant effect on CCC proliferation was shown for Mesenchymal Stem Cell-secreted PAI-1 with HCT-116 cells showing decreased proliferation at all concentrations, and HT29 cells showing increased proliferation in the presence of higher PAI-1 levels. This is the first study to identify PAI-1 as an important mediator of Mesenchymal Stem Cell/colon cancer cell interactions and highlights the

  4. Effects of lateral ventricular transplantation of bone marrow-derived mesenchymal stem cells modified with brain-derived neurotrophic factor gene on cognition in a rat model of Alzheimer's disease

    Institute of Scientific and Technical Information of China (English)

    Ping Zhang; Gangyong Zhao; Xianjiang Kang; Likai Su

    2012-01-01

    In the present study, transplantation of bone marrow-derived mesenchymal stem cells modified with brain-derived neurotrophic factor gene into the lateral ventricle of a rat model of Alzheimer's disease, resulted in significant attenuation of nerve cell damage in the hippocampal CA1 region. Furthermore, brain-derived neurotrophic factor and tyrosine kinase B mRNA and protein levels were significantly increased, and learning and memory were significantly improved. Results indicate that transplantation of bone marrow-derived mesenchymal stem cells modified with brain-derived neurotrophic factor gene can significantly improve cognitive function in a rat model of Alzheimer's disease, possibly by increasing the levels of brain-derived neurotrophic factor and tyrosine kinase B in the hippocampus.

  5. Brain-derived neurotrophic factor increases vascular endothelial growth factor expression and enhances angiogenesis in human chondrosarcoma cells.

    Science.gov (United States)

    Lin, Chih-Yang; Hung, Shih-Ya; Chen, Hsien-Te; Tsou, Hsi-Kai; Fong, Yi-Chin; Wang, Shih-Wei; Tang, Chih-Hsin

    2014-10-15

    Chondrosarcomas are a type of primary malignant bone cancer, with a potent capacity for local invasion and distant metastasis. Brain-derived neurotrophic factor (BDNF) is commonly upregulated during neurogenesis. The aim of the present study was to examine the mechanism involved in BDNF-mediated vascular endothelial growth factor (VEGF) expression and angiogenesis in human chondrosarcoma cells. Here, we knocked down BDNF expression in chondrosarcoma cells and assessed their capacity to control VEGF expression and angiogenesis in vitro and in vivo. We found knockdown of BDNF decreased VEGF expression and abolished chondrosarcoma conditional medium-mediated angiogenesis in vitro as well as angiogenesis effects in vivo in the chick chorioallantoic membrane and Matrigel plug nude mouse models. In addition, in the xenograft tumor angiogenesis model, the knockdown of BDNF significantly reduced tumor growth and tumor-associated angiogenesis. BDNF increased VEGF expression and angiogenesis through the TrkB receptor, PLCγ, PKCα, and the HIF-1α signaling pathway. Finally, we analyzed samples from chondrosarcoma patients by immunohistochemical staining. The expression of BDNF and VEGF protein in 56 chondrosarcoma patients was significantly higher than in normal cartilage. In addition, the high level of BDNF expression correlated strongly with VEGF expression and tumor stage. Taken together, our results indicate that BDNF increases VEGF expression and enhances angiogenesis through a signal transduction pathway that involves the TrkB receptor, PLCγ, PKCα, and the HIF-1α. Therefore, BDNF may represent a novel target for anti-angiogenic therapy for human chondrosarcoma.

  6. Effect of controlled release of brain-derived neurotrophic factor and neurotrophin-3 from collagen gel on neural stem cells.

    Science.gov (United States)

    Huang, Fei; Wu, Yunfeng; Wang, Hao; Chang, Jun; Ma, Guangwen; Yin, Zongsheng

    2016-01-20

    This study aimed to examine the effect of controlled release of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) from collagen gel on rat neural stem cells (NSCs). With three groups of collagen gel, BDNF/collagen gel, and NT-3/collagen gel as controls, BDNF and NT-3 were tested in the BDNF-NT-3/collagen gel group at different time points. The enzyme-linked immunosorbent assay results showed that BDNF and NT-3 were steadily released from collagen gels for 10 days. The cell viability test and the bromodeoxyuridine incorporation assay showed that BDNF-NT-3/collagen gel supported the survival and proliferation of NSCs. The results also showed that the length of processes was markedly longer and differentiation percentage from NSCs into neurons was much higher in the BDNF-NT-3/collagen gel group than those in the collagen gel, BDNF/collagen gel, and NT-3/collagen gel groups. These findings suggest that BDNF-NT-3/collagen gel could significantly improve the ability of NSCs proliferation and differentiation.

  7. The gene coding for glial cell line derived neurotrophic factor (GDNF) maps to chromosome 5p12-p13.1

    Energy Technology Data Exchange (ETDEWEB)

    Schindelhauer, D.; Schuffenhauer, S.; Meitinger, T. [Maximiland-Universitaet, Munich (Germany)] [and others

    1995-08-10

    The gene coding for glial cell line derived neurotrophic factor (GDNF) has biological properties that may have potential as a treatment for Parkinson`s and motoneuron diseases. Using the NIGMS Mapping Panel 2, we have localized the GDNF gene to human chromosome 5p12-p13.1. Large NruI and NotI fragments on chromosome 5 will facilitate the construction of a long-range map of the region. 26 refs., 1 fig., 1 tab.

  8. Brain-derived neurotrophic factor, but not neurotrophin-3, prevents ischaemia-induced neuronal cell death in organotypic rat hippocampal slice cultures.

    Science.gov (United States)

    Pringle, A K; Sundstrom, L E; Wilde, G J; Williams, L R; Iannotti, F

    1996-06-28

    We have investigated the neuroprotective actions of neurotrophins in a model of ischaemia using slice cultures. Ischaemia was induced in organotypic hippocampal cultures by simultaneous oxygen and glucose deprivation. Cell death was assessed 24 h later by propidium iodide fluorescence. Pre- but not post-ischaemic addition of brain-derived neurotrophic factor (BDNF) produced a concentration-dependent reduction in neuronal damage. Neurotrophin-3 was not neuroprotective. These data suggest that BDNF may form part of an endogenous neuroprotective mechanism.

  9. Human umbilical cord blood-derived stem cells and brain-derived neurotrophic factor protect injured optic nerve:viscoelasticity characterization

    Institute of Scientific and Technical Information of China (English)

    Xue-man Lv; Yan Liu; Fei Wu; Yi Yuan; Min Luo

    2016-01-01

    The optic nerve is a viscoelastic solid-like biomaterial. Its normal stress relaxation and creep properties enable the nerve to resist constant strain and protect it from injury. We hypothesized that stress relaxation and creep properties of the optic nerve change after injury. More-over, human brain-derived neurotrophic factor or umbilical cord blood-derived stem cells may restore these changes to normal. To validate this hypothesis, a rabbit model of optic nerve injury was established using a clamp approach. At 7 days after injury, the vitreous body re-ceived a one-time injection of 50 μg human brain-derived neurotrophic factor or 1 × 106 human umbilical cord blood-derived stem cells. At 30 days after injury, stress relaxation and creep properties of the optic nerve that received treatment had recovered greatly, with patho-logical changes in the injured optic nerve also noticeably improved. These results suggest that human brain-derived neurotrophic factor or umbilical cord blood-derived stem cell intervention promotes viscoelasticity recovery of injured optic nerves, and thereby contributes to nerve recovery.

  10. Brain-derived neurotrophic factor as an indicator of chemical neurotoxicity: an animal-free CNS cell culture model.

    Science.gov (United States)

    Woehrling, Elizabeth K; Hill, Eric J; Nagel, David; Coleman, Michael D

    2013-12-01

    Recent changes to the legislation on chemicals and cosmetics testing call for a change in the paradigm regarding the current 'whole animal' approach for identifying chemical hazards, including the assessment of potential neurotoxins. Accordingly, since 2004, we have worked on the development of the integrated co-culture of post-mitotic, human-derived neurons and astrocytes (NT2.N/A), for use as an in vitro functional central nervous system (CNS) model. We have used it successfully to investigate indicators of neurotoxicity. For this purpose, we used NT2.N/A cells to examine the effects of acute exposure to a range of test chemicals on the cellular release of brain-derived neurotrophic factor (BDNF). It was demonstrated that the release of this protective neurotrophin into the culture medium (above that of control levels) occurred consistently in response to sub-cytotoxic levels of known neurotoxic, but not non-neurotoxic, chemicals. These increases in BDNF release were quantifiable, statistically significant, and occurred at concentrations below those at which cell death was measureable, which potentially indicates specific neurotoxicity, as opposed to general cytotoxicity. The fact that the BDNF immunoassay is non-invasive, and that NT2.N/A cells retain their functionality for a period of months, may make this system useful for repeated-dose toxicity testing, which is of particular relevance to cosmetics testing without the use of laboratory animals. In addition, the production of NT2.N/A cells without the use of animal products, such as fetal bovine serum, is being explored, to produce a fully-humanised cellular model.

  11. A thermoreversible polymer mediates controlled release of glial cell line-derived neurotrophic factor to enhance kidney regeneration.

    Science.gov (United States)

    Gheisari, Yousof; Yokoo, Takashi; Matsumoto, Kei; Fukui, Akira; Sugimoto, Naomi; Ohashi, Toya; Kawamura, Tetsuya; Hosoya, Tatsuo; Kobayashi, Eiji

    2010-08-01

    Previously, we reported that human mesenchymal stem cells (hMSCs) that were cultivated in growing embryos differentiated in an appropriate developmental milieu, thereby facilitating the development of a functional renal unit. However, this approach required transfection with an adenovirus that expressed glial cell line-derived neurotrophic factor (GDNF) to enhance the development of hMSC-derived renal tissue, and safety issues restrict the clinical use of such viral vectors. To circumvent this problem, we tested an artificial polymer as a means to diffuse GDNF. This GDNF-polymer, which exists in liquid form at 4 degrees C but becomes a hydrogel upon heating to 37 degrees C, was used as a thermoreversible switch, allowing the injection of hMSCs at low viscosity using a mouth pipette, with subsequent slow diffusion of GDNF as it solidified. The polymer, which was dissolved in a solution of GDNF at 4 degrees C and then maintained at 37 degrees C, acted as a diffuser of GDNF for more than 48 h. LacZ-transfected hMSCs and the GDNF-polymer (at 4 degrees C) were placed in the nephrogenic sites of growing rat embryos that were maintained at 37 degrees C. Forty-eight hours later, the resultant kidney anlagen were dissected out and allowed to continue developing for 6 days in vitro. Whole-organ X-Gal staining and fluorescence activated cell sorter analysis showed that the number of hMSC-derived cells was significantly increased in developed anlagen that have been generated from hMSCs plus GDNF-polymer compared with those from hMSCs plus GDNF-containing medium and was comparable to those from adenovirus-transfected hMSCs. These findings suggest that the GDNF-polymer can be used as a diffuser of GDNF for kidney organogenesis.

  12. Origin of the angiotensin II secreted by cells.

    Science.gov (United States)

    Ganong, W F

    1994-03-01

    Circulating angiotensin II is unique in that it is formed in the blood by the interaction of circulating proteins. There are in addition many local renin-angiotensin systems in tissues in which angiotensin II is apparently secreted by various types of cells. This brief review considers the possible pathways for synthesis of locally produced angiotensin II in the brain, the anterior pituitary, the testes, the ovaries, the adrenal cortex, the kidneys, the heart, blood vessel walls, and brown and white fat. Synthesis by cells in culture is also reviewed. The possibility that certain cells contain a complete intracellular renin-angiotensin system is not ruled out, but there are problems with this hypothesis. Proteases other than renin may be involved, and there may be different pathways in different tissues. However, it appears that at least in some tissues, angiotensinogen is produced in one population of cells and transported in a paracrine fashion to other renin-containing cells, where it serves as the substrate for production of angiotensin II.

  13. Decreased glial cell line-derived neurotrophic factor levels in patients with depression: a meta-analytic study.

    Science.gov (United States)

    Lin, Pao-Yen; Tseng, Ping-Tao

    2015-04-01

    Glial cell-line derived neurotrophic factor (GDNF) has been shown to promote development, differentiation, and protection of CNS neurons and was thought to play an important role in various neuropsychiatric disorders. Several studies have examined the GDNF levels in patients with depression but shown inconsistent results. In this study, we compared blood GDNF levels between depressive patients and control subjects through meta-analytic method. The effect sizes (ESs) from all eligible studies were synthesized by using a random effect model. In this meta-analysis, we included 526 patients and 502 control subjects from 12 original articles. Compared to control subjects, blood GDNF levels are significantly decreased in patients with depression (ES = -0.62, p = 0.0011). However, significant heterogeneity was found among included studies. Through subgroup analysis, we found that GDNF was still decreased in studies with major depressive disorder (ES = -0.73, p = 0.0001); in studies with non-old-age depression (ES = -1.25, p = 0.0001), but not with old-age depression; and in studies using serum samples (ES = -0.86, p GDNF levels as a biomarker of depression as a whole, but the results were modulated by psychiatric diagnosis, age of included subjects, and sampling sources. With these results, future studies are required to examine whether effective antidepressant treatment is associated with an increase in serum GDNF levels.

  14. Association between serum levels of glial cell-line derived neurotrophic factor and attention deficits in schizophrenia.

    Science.gov (United States)

    Niitsu, Tomihisa; Shirayama, Yukihiko; Matsuzawa, Daisuke; Shimizu, Eiji; Hashimoto, Kenji; Iyo, Masaomi

    2014-07-11

    Several lines of evidence suggest that glial cell-line derived neurotrophic factor (GDNF) plays an important role in the pathophysiology of neuropsychiatric and neurodegenerative disorders. In this study, we investigated the association between GDNF serum levels and the clinical status of medicated patients with schizophrenia. Sixty-three medicated patients with schizophrenia and 52 age- and sex-matched healthy controls were recruited. Patients were evaluated using the brief psychiatry rating scale, the scale for the assessment of negative symptoms (SANS) and neuropsychological tests. Serum levels of GDNF were determined using an ELISA method. Serum levels of GDNF did not differ between schizophrenia patients and controls. Higher GDNF serum levels were associated with better performances on the Digit Span in healthy controls but not in schizophrenics. At the same time, higher GDNF serum levels were associated with severe attention deficits on the SANS subscale, in schizophrenics. Our preliminary study suggests that serum levels of GDNF may be an unsuitable biomarker for schizophrenia, although it may be associated with working memory in healthy controls and the pathophysiology of attention deficits in schizophrenia.

  15. Sympathetic Innervation Induced in Engrafted Engineered Cardiomyocyte Sheets by Glial Cell Line Derived Neurotrophic Factor In Vivo

    Directory of Open Access Journals (Sweden)

    Xian-ming Fu

    2013-01-01

    Full Text Available The aim of myocardial tissue engineering is to repair or regenerate damaged myocardium with engineered cardiac tissue. However, this strategy has been hampered by lack of functional integration of grafts with native myocardium. Autonomic innervation may be crucial for grafts to function properly with host myocardium. In this study, we explored the feasibility of in vivo induction of autonomic innervation to engineered myocardial tissue using genetic modulation by adenovirus encoding glial cell line derived neurotrophic factor (GDNF. GFP-transgene (control group or GDNF overexpressing (GDNF group engineered cardiomyocyte sheets were transplanted on cryoinjured hearts in rats. Nerve fibers in the grafts were examined by immunohistochemistry at 1, 2, and 4 weeks postoperatively. Growth associated protein-43 positive growing nerves and tyrosine hydroxylase positive sympathetic nerves were first detected in the grafts at 2 weeks postoperatively in control group and 1 week in GDNF group. The densities of growing nerve and sympathetic nerve in grafts were significantly increased in GDNF group. No choline acetyltransferase immunopositive parasympathetic nerves were observed in grafts. In conclusion, sympathetic innervation could be effectively induced into engrafted engineered cardiomyocyte sheets using GDNF.

  16. Rapamycin modulated brain-derived neurotrophic factor and B-cell lymphoma 2 to mitigate autism spectrum disorder in rats

    Science.gov (United States)

    Zhang, Jie; Liu, Li-Ming; Ni, Jin-Feng

    2017-01-01

    The number of children suffered from autism spectrum disorder (ASD) is increasing dramatically. However, the etiology of ASD is not well known. This study employed mammalian target of rapamycin inhibitor rapamycin to explore its effect on ASD and provided new therapeutic strategies for ASD. ASD rat model was constructed and valproic acid (VPA) was injected intraperitoneally into rats on pregnancy day 12.5. Offspring from VPA group were divided into ASD group and ASD + rapamycin (ASD + RAPA) group. Compared with normal group, the frequency and duration of social behavior and straight times of ASD group were shortened, but the grooming times were extended. Meanwhile, in ASD group, the average escape latency and the frequency of crossing plates were decreased, the apoptotic index (AI) detected by TUNEL assay was increased, and the expression of brain-derived neurotrophic factor (BDNF) and B-cell lymphoma 2 (Bcl-2) analyzed was decreased with great difference compared with normal group (P<0.01). However, rapamycin treatment in ASD rats mitigated the ASD-like social behavior, such as the frequencies of straight and grooming. Furthermore, rapamycin shortened the average escape latency, but increased the frequency of crossing plates of ASD rats. In hippocampus, rapamycin decreased the AI, but increased the levels of BDNF and Bcl-2 (P<0.01) of ASD rats. These findings revealed that rapamycin significantly mitigated the social behavior by enhancing the expression of BDNF and Bcl-2 to suppress the hippocampus apoptosis in VPA-induced ASD rats.

  17. Over-expression of brain-derived neurotrophic factor in mesenchymal stem cells transfected with recombinant lentivirus BDNF gene.

    Science.gov (United States)

    Zhang, X; Zhu, J; Zhang, K; Liu, T; Zhang, Z

    2016-12-30

    This study was aimed at investigating the expression of brain-derived neurotrophic factor (BDNF) in mesenchymal stem cells (MSCs) modified with recombinant lentivirus bearing BDNF gene. Lentivirus vectors bearing BDNF gene were constructed. MSCs were isolated from rats and cultured. The lentiviral vectors containing BDNF gene were transfected into the MSCs, and BDNF gene and protein expressions were monitored with enhanced green fluorescent protein (EGFP). RT-PCR and Western blot were used to measure gene and protein expressions, respectibvely in MSCs, MSCs-EGFP and MSCs-EGFP-BDNF groups. Green fluorescence assay confirmed successful transfection of BDNF gene recombinant lentivirus into MSCs. RT-PCR and Western blot revealed that BDNF gene and protein expressions in the MSCs-EGFP-BDNF group were significantly higher than that in MSCs group and MSCs-EGFP group. There were no statistically significant differences in gene expression between MSCs and MSCs-EGFP groups. MSCs can over-express BDNF when transfected with recombinant lentivirus bearing BDNF gene.

  18. Functional recovery after transplantation of neural stem cells modified by brain-derived neurotrophic factor in rats with cerebral ischaemia.

    Science.gov (United States)

    Zhu, J M; Zhao, Y Y; Chen, S D; Zhang, W H; Lou, L; Jin, X

    2011-01-01

    Functional recovery after transplantation of brain-derived neurotrophic factor (BDNF)-modified neural stem cells (NSCs) was evaluated in a rat model of cerebral ischaemia damage induced by temporary middle cerebral artery occlusion (tMCAO). Western blotting and enzyme-linked immunosorbent assay demonstrated upregulated BDNF protein expression by rat embryonic NSCs transfected with the human BDNF gene (BDNF-NSCs). BDNF-NSCs stimulated neurite outgrowth in cocultured dorsal root ganglion neurons, suggesting that BDNF increased neurogenesis in vitro. In vivo, BDNF promoted recovery of tMCAO. Phosphate-buffered saline, untransformed NSCs or BDNF-NSCs were introduced into the penumbra zone of the right striatum of tMCAO rats and neurological function deficit was assessed for up to 12 weeks using the neurological severity score (NSS). The NSS was significantly lower in the BDNF-NSC transfected transplant group than in all the other groups from week 10. BDNF-NSCs recovered 1 week after transplantation expressed BDNF protein. Transplanted NSCs had differentiated into mature neurons 12 weeks after transplantation. Transgenic NSCs have potential as a therapeutic agent for brain ischaemia.

  19. Cognitive disorder and changes in cholinergic receptors, N-methyl-D aspartate receptors, neural cell adhesion molecule, and brain-derived neurotrophic factor following brain injury

    Institute of Scientific and Technical Information of China (English)

    Weiliang Zhao; Dezhi Kang; Yuanxiang Lin

    2008-01-01

    BACKGROUND: Learning and memory damage is one of the most permanent and the severest symptoms of traumatic brain injury; it can seriously influence the normal life and work of patients. Some research has demonstrated that cognitive disorder is closely related to nicotine cholinergic receptors, N-methyl-D aspartate receptors, neural cell adhesion molecule, and brain-derived neurotrophic factor. OBJECTIVE: To summarize the cognitive disorder and changes in nicotine cholinergic receptors, N-methyl-D aspartate receptors, neural cell adhesion molecule, and brain-derived neurotrophic factor following brain injury. RETRIEVAL STRATEGY: A computer-based online search was conducted in PUBMED for English language publications containing the key words "brain injured, cognitive handicap, acetylcholine, N-methyl-D aspartate receptors, neural cell adhesion molecule, brain-derived neurotrophic factor" from January 2000 to December 2007. There were 44 papers in total. Inclusion criteria: ① articles about changes in nicotine cholinergic receptors, N-methyl-D aspartate receptors, neural cell adhesion molecule, and brain-derived neurotrophic factor following brain injury; ② articles in the same researching circle published in authoritative journals or recently published. Exclusion criteria: duplicated articles.LITERATURE EVALUATION: References were mainly derived from research on changes in these four factors following brain injury. The 20 included papers were clinical or basic experimental studies. DATA SYNTHESIS: After craniocerebral injury, changes in these four factors in brain were similar to those during recovery from cognitive disorder, to a certain degree. Some data have indicated that activation of nicotine cholinergic receptors, N-methyl-D aspartate receptors, neural cell adhesion molecule, and brain-derived neurotrophic factor could greatly improve cognitive disorder following brain injury. However, there are still a lot of questions remaining; for example, how do these

  20. MicroRNA regulation of central glial cell line-derived neurotrophic factor (GDNF) signalling in depression.

    Science.gov (United States)

    Maheu, M; Lopez, J P; Crapper, L; Davoli, M A; Turecki, G; Mechawar, N

    2015-02-17

    Although multiple studies have reported that peripheral glial cell line-derived neurotrophic factor (GDNF) is reduced in depression, cerebral GDNF signalling has yet to be examined in this condition. Here, we report an isoform-specific decrease in GDNF family receptor alpha 1 (GFRA1) mRNA expression, resulting in lowered GFRα1a protein levels in basolateral amygdala (BLA) samples from depressed subjects. Downregulation of GFRα1a was associated with increased expression of microRNAs, including miR-511, predicted to bind to long 3' untranslated region (3'-UTR)-containing transcripts (GFRA1-L) coding for GFRα1a. Transfection of human neural progenitor cells (NPCs) with a miR-511 mimic was sufficient to repress GFRA1-L/GFRα1a without altering GFRα1b, and resulted in pathway-specific changes in immediate early gene activity. Unexpectedly, GFRα1a knockdown did not reduce NPC responses to GDNF. Rather, it greatly enhanced mitogen-activated protein kinase signalling. This effect appeared to be mediated by GDNF/soluble GFRα1/neural cell adhesion molecule binding, and substituting the soluble GFRα1a/GFRα1b content of miR-511-transfected NPCs with that of controls rescued signalling. In light of previous reports suggesting that GFRα1b can inhibit GFRα1a-induced neuroplasticity, we also assessed the association between GFRα1 and doublecortin (DCX; a hyperplastic marker) in human BLA. Although controls displayed coordinated expression of GFRα1a and b isoforms and these correlated positively with DCX, the only significant association observed among depressed subjects was a strongly negative correlation between GFRα1b and DCX. Taken together, these results suggest that microRNA-mediated reductions of GFRα1a in depression change the quality, rather than the quantity, of GDNF signalling. They also suggest that central GDNF signalling may represent a novel target for antidepressant treatment.

  1. Expression and cell localization of brain-derived neurotrophic factor and TrkB during zebrafish retinal development.

    Science.gov (United States)

    Germanà, A; Sánchez-Ramos, C; Guerrera, M C; Calavia, M G; Navarro, M; Zichichi, R; García-Suárez, O; Pérez-Piñera, P; Vega, Jose A

    2010-09-01

    Brain-derived neurotrophic factor (BDNF) signaling through TrkB regulates different aspects of neuronal development, including survival, axonal and dendritic growth, and synapse formation. Despite recent advances in our understanding of the functional significance of BDNF and TrkB in the retina, the cell types in the retina that express BDNF and TrkB, and the variations in their levels of expression during development, remain poorly defined. The goal of the present study is to determine the age-dependent changes in the levels of expression and localization of BDNF and TrkB in the zebrafish retina. Zebrafish retinas from 10 days post-fertilization (dpf) to 180 dpf were used to perform PCR, Western blot and immunohistochemistry. Both BDNF and TrkB mRNAs, and BDNF and full-length TrkB proteins were detected at all ages sampled. The localization of these proteins in the retina was very similar at all time points studied. BDNF immunoreactivity was found in the outer nuclear layer, the outer plexiform layer and the inner plexiform layer, whereas TrkB immunoreactivity was observed in the inner plexiform layer and, to a lesser extent, in the ganglion cell layer. These results demonstrate that the pattern of expression of BDNF and TrkB in the retina of zebrafish remains unchanged during postembryonic development and adult life. Because TrkB expression in retina did not change with age, cells expressing TrkB may potentially be able to respond during the entire lifespan of zebrafish to BDNF either exogenously administered or endogenously produced, acting through paracrine mechanisms.

  2. Activated T cells recruit exosomes secreted by dendritic cells via LFA-1.

    NARCIS (Netherlands)

    Nolte-'t Hoen, E.N.; Buschow, S.I.; Anderton, S.M.; Stoorvogel, W.; Wauben, M.H.M.

    2009-01-01

    Dendritic cells (DCs) are known to secrete exosomes that transfer membrane proteins, like major histocompatibility complex class II, to other DCs. Intercellular transfer of membrane proteins is also observed during cognate interactions between DCs and CD4(+) T cells. The acquired proteins are functi

  3. Stimulation of mucin secretion from human bronchial epithelial cells by mast cell chymase

    Institute of Scientific and Technical Information of China (English)

    Shao-heng HE; Jian ZHENG

    2004-01-01

    AIM: To investigate the effect ofchymase on the mucin secretion from human bronchial epithelial cells. METHODS:Primarily-cultured human bronchial epithelial (PCHBE) cells and normal human bronchial epithelial (NHBE) cells were cultured with chymase or other stimulus in a mixture of bronchial epithelial growth medium (BEGM) and Dulbecco's modified Eagle's medium (DMEM), and the quantities of stimulatory mucin release were recorded.MUC5AC mucin was measured with an ELISA and dolichos biflorus agglutinin (DBA) mucin was determined with an enzyme linked DBA assay. RESULTS: A dose-dependent secretion of DBA mucin from PCHBE cells was observed with chymase with a maximum secretion of 98 % above baseline being achieved following 3 h incubation.The action of chymase started from 1 h, peaked at 3 h and dramatically decreased at 20 h following incubation.Chymase was able to also stimulate approximately 38 % increase in MUC5AC mucin release from PCHBE cells, and about 121% increase in DBA mucin release from NHBE cells. A chymase inhibitor soybean trypsin inhibitor (SBTI)was able to inhibit up to 85 % chymase induced mucin release, indicating that the enzymatic activity was essential for the actions of chymase on bronchial epithelial cells. CONCLUSION: Chymase is a potent stimulus of mucin secretion from human bronchial epithelial cells. It can contribute to mucus hypersecretion process in the patients with chronic obstructive pulmonary disease or asthma.

  4. Effect of Cytokines Secreted by Human Adipose Stromal Cells on Endothelial Cells

    Institute of Scientific and Technical Information of China (English)

    LI Bingong; ZENG Qiutang; WANG Hongxiang; MAO Xiaobo

    2006-01-01

    To isolate and culture adipose stromal cells (ASCs), and study the effect of cytokines secreted by ASCs on endothelial cells, human adipose tissue was digested with collagenase type Ⅰ solution and ASCs were derived by culture. The cells surface phenotype was examined by flow cytometry. ELISA was used to detect the secretion of VEGF, HGF, SDF-1 α and RT-PCR was employed to detect the expression of their mRNA. Then the ASC medium was utilized to culture human umbilical vein endothelial cells ECV304. Cells were counted by hemacytometer to determine the proliferation and Annexin V/PI was employed for the examination of the apoptosis rate of ECV304. ASCs were derived by culture and expressed CD34, CD105 while they did not express CD31 or CD45. ASCs secreted cytokines such as VEGF, HGF and SDF-1 α so the ASC medium could stimulate proliferation and counteract apoptosis of endothelial cells (P<0.05). Bcl-2 mRNA was also found to be up-regulated in the endothelial cells. It is concluded that ASCs can secrete cytokines and has significant effect on the proliferation of endothelial cells and apoptosis.

  5. Cancer-secreted AGR2 induces programmed cell death in normal cells

    Science.gov (United States)

    Vitello, Elizabeth A.; Quek, Sue-Ing; Kincaid, Heather; Fuchs, Thomas; Crichton, Daniel J.; Troisch, Pamela; Liu, Alvin Y.

    2016-01-01

    Anterior Gradient 2 (AGR2) is a protein expressed in many solid tumor types including prostate, pancreatic, breast and lung. AGR2 functions as a protein disulfide isomerase in the endoplasmic reticulum. However, AGR2 is secreted by cancer cells that overexpress this molecule. Secretion of AGR2 was also found in salamander limb regeneration. Due to its ubiquity, tumor secretion of AGR2 must serve an important role in cancer, yet its molecular function is largely unknown. This study examined the effect of cancer-secreted AGR2 on normal cells. Prostate stromal cells were cultured, and tissue digestion media containing AGR2 prepared from prostate primary cancer 10-076 CP and adenocarcinoma LuCaP 70CR xenograft were added. The control were tissue digestion media containing no AGR2 prepared from benign prostate 10-076 NP and small cell carcinoma LuCaP 145.1 xenograft. In the presence of tumor-secreted AGR2, the stromal cells were found to undergo programmed cell death (PCD) characterized by formation of cellular blebs, cell shrinkage, and DNA fragmentation as seen when the stromal cells were UV irradiated or treated by a pro-apoptotic drug. PCD could be prevented with the addition of the monoclonal AGR2-neutralizing antibody P3A5. DNA microarray analysis of LuCaP 70CR media-treated vs. LuCaP 145.1 media-treated cells showed downregulation of the gene SAT1 as a major change in cells exposed to AGR2. RT-PCR analysis confirmed the array result. SAT1 encodes spermidine/spermine N1-acetyltransferase, which maintains intracellular polyamine levels. Abnormal polyamine metabolism as a result of altered SAT1 activity has an adverse effect on cells through the induction of PCD. PMID:27283903

  6. Automated in situ measurement of cell-specific antibody secretion and laser-mediated purification for rapid cloning of highly-secreting producers.

    Science.gov (United States)

    Hanania, Elie G; Fieck, Annabeth; Stevens, Janine; Bodzin, Leon J; Palsson, Bernhard Ø; Koller, Manfred R

    2005-09-30

    Cloning of highly-secreting recombinant cells is critical for biopharmaceutical manufacturing, but faces numerous challenges including the fact that secreted protein does not remain associated with the producing cell. A fundamentally new approach was developed combining in situ capture and measurement of individual cell protein secretion followed by laser-mediated elimination of all non- and poorly-secreting cells, leaving only the highest-secreting cell in a well. Recombinant cells producing humanized antibody were cultured serum-free on a capture matrix, followed by staining with fluorescently-labeled anti-human antibody fragment. A novel, automated, high-throughput instrument (called LEAP) was used to image and locate every cell, quantify the cell-associated and secreted antibody (surrounding each cell), eliminate all undesired cells from a well via targeted laser irradiation, and then track clone outgrowth and stability. Temporarily sparing an island of helper cells around the clone of interest improved cloning efficiency (particularly when using serum-free medium), and helper cells were easily eliminated with the laser after several days. The in situ nature of this process allowed several serial sub-cloning steps to be performed within days of one another, resulting in rapid generation of clonal populations with significantly increased and more stable, homogeneous antibody secretion. Cell lines with specific antibody secretion rates of > 50 pg/cell per day (in static batch culture) were routinely obtained as a result of this cloning approach, often times representing up to 20% of the clones screened.

  7. The niche-derived glial cell line-derived neurotrophic factor (GDNF) induces migration of mouse spermatogonial stem/progenitor cells.

    Science.gov (United States)

    Dovere, Lisa; Fera, Stefania; Grasso, Margherita; Lamberti, Dante; Gargioli, Cesare; Muciaccia, Barbara; Lustri, Anna Maria; Stefanini, Mario; Vicini, Elena

    2013-01-01

    In mammals, the biological activity of the stem/progenitor compartment sustains production of mature gametes through spermatogenesis. Spermatogonial stem cells and their progeny belong to the class of undifferentiated spermatogonia, a germ cell population found on the basal membrane of the seminiferous tubules. A large body of evidence has demonstrated that glial cell line-derived neurotrophic factor (GDNF), a Sertoli-derived factor, is essential for in vivo and in vitro stem cell self-renewal. However, the mechanisms underlying this activity are not completely understood. In this study, we show that GDNF induces dose-dependent directional migration of freshly selected undifferentiated spermatogonia, as well as germline stem cells in culture, using a Boyden chamber assay. GDNF-induced migration is dependent on the expression of the GDNF co-receptor GFRA1, as shown by migration assays performed on parental and GFRA1-transduced GC-1 spermatogonial cell lines. We found that the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP) is specifically expressed in undifferentiated spermatogonia. VASP belongs to the ENA/VASP family of proteins implicated in actin-dependent processes, such as fibroblast migration, axon guidance, and cell adhesion. In intact seminiferous tubules and germline stem cell cultures, GDNF treatment up-regulates VASP in a dose-dependent fashion. These data identify a novel role for the niche-derived factor GDNF, and they suggest that GDNF may impinge on the stem/progenitor compartment, affecting the actin cytoskeleton and cell migration.

  8. Expression of glial cell line-derived neurotrophic factor and its receptors in cultured retinal Müller cells under high glucose circumstance.

    Science.gov (United States)

    Zhu, Xinping; Sun, Yan; Wang, Zhongping; Cui, Weigang; Peng, Yuwen; Li, Ruixi

    2012-03-01

    This study aimed to explore the effect of high glucose concentration on the expression of glial cell line-derived neurotrophic factor (GDNF) and its family ligand receptors (GFRs) GFRα1 and GFRα2 in Müller cells and the protective role of GDNF in cultured Müller cells under high glucose circumstance. Cultured Müller cells (untreated or treated with 200 ng/mL of GDNF) were exposed to high glucose conditions (20 mmol/L glucose). We found that the expression levels of GDNF and GFRα1 mRNA and protein increased gradually over time under high glucose and exogenous GDNF-treated conditions, whereas the upregulation in GFRα2 expression was observed only in the early stage of high glucose conditions. Exogenous GDNF not only decreased apoptosis in cultured Müller cells under high glucose circumstance, but also accelerated the levels and speed of synthesis of GDNF and GFRα1 proteins in Müller cells. These results suggest that Müller cells can synthesize GDNF and GFRs under high glucose conditions, and GDNF may play important role in protecting Müller cells during the early stage of diabetic retinopathy. The difference in GFRs expression indicated that GDNF and neurturin may exert different effects on Müller cells under high glucose circumstance.

  9. The niche-derived glial cell line-derived neurotrophic factor (GDNF induces migration of mouse spermatogonial stem/progenitor cells.

    Directory of Open Access Journals (Sweden)

    Lisa Dovere

    Full Text Available In mammals, the biological activity of the stem/progenitor compartment sustains production of mature gametes through spermatogenesis. Spermatogonial stem cells and their progeny belong to the class of undifferentiated spermatogonia, a germ cell population found on the basal membrane of the seminiferous tubules. A large body of evidence has demonstrated that glial cell line-derived neurotrophic factor (GDNF, a Sertoli-derived factor, is essential for in vivo and in vitro stem cell self-renewal. However, the mechanisms underlying this activity are not completely understood. In this study, we show that GDNF induces dose-dependent directional migration of freshly selected undifferentiated spermatogonia, as well as germline stem cells in culture, using a Boyden chamber assay. GDNF-induced migration is dependent on the expression of the GDNF co-receptor GFRA1, as shown by migration assays performed on parental and GFRA1-transduced GC-1 spermatogonial cell lines. We found that the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP is specifically expressed in undifferentiated spermatogonia. VASP belongs to the ENA/VASP family of proteins implicated in actin-dependent processes, such as fibroblast migration, axon guidance, and cell adhesion. In intact seminiferous tubules and germline stem cell cultures, GDNF treatment up-regulates VASP in a dose-dependent fashion. These data identify a novel role for the niche-derived factor GDNF, and they suggest that GDNF may impinge on the stem/progenitor compartment, affecting the actin cytoskeleton and cell migration.

  10. Co-culture of clonal beta cells with GLP-1 and glucagon-secreting cell line impacts on beta cell insulin secretion, proliferation and susceptibility to cytotoxins.

    Science.gov (United States)

    Green, Alastair D; Vasu, Srividya; Moffett, R Charlotte; Flatt, Peter R

    2016-06-01

    We investigated the direct effects on insulin releasing MIN6 cells of chronic exposure to GLP-1, glucagon or a combination of both peptides secreted from GLUTag L-cell and αTC1.9 alpha-cell lines in co-culture. MIN6, GLUTag and αTC1.9 cell lines exhibited high cellular hormone content and release of insulin, GLP-1 and glucagon, respectively. Co-culture of MIN6 cells with GLUTag cells significantly increased cellular insulin content, beta-cell proliferation, insulin secretory responses to a range of established secretogogues and afforded protection against exposure cytotoxic concentrations of glucose, lipid, streptozotocin or cytokines. Benefits of co-culture of MIN6 cells with αTC1.9 alphacells were limited to enhanced beta-cell proliferation with marginal positive actions on both insulin secretion and cellular protection. In contrast, co-culture of MIN6 with GLUTag cells plus αTC1.9 cells, markedly enhanced both insulin secretory responses and protection against beta-cell toxins compared with co-culture with GLUTag cells alone. These data indicate important long-term effects of conjoint GLP-1 and glucagon exposure on beta-cell function. This illustrates the possible functional significance of alpha-cell GLP-1 production as well as direct beneficial effects of dual agonism at beta-cell GLP-1 and glucagon receptors.

  11. Imipramine activates glial cell line-derived neurotrophic factor via early growth response gene 1 in astrocytes.

    Science.gov (United States)

    Kim, Yeni; Kim, Se Hyun; Kim, Yong Sik; Lee, Young Han; Ha, Kyooseob; Shin, Soon Young

    2011-06-01

    Recent evidence has suggested that deficits in glial plasticity contribute to the pathophysiology of depressive disorders. The present study explored early growth response 1 (EGR-1) transcriptional regulation of imipramine-induced glial cell line-derived neurotrophic factor (GDNF) expression in astrocytes. After we observed the induction of GDNF mRNA expression in rat astrocytes in response to imipramine, deletion mutant studies showed that the proximal region between -493 and -114 of the GDNF promoter, which contains three binding sites for EGR-1, was essential for maximal imipramine-induced activation of GDNF promoter. The dose-dependent upregulation of EGR-1 by imipramine, the activation of GDNF by the over-expression of EGR-1 without imipramine and the reduction in the imipramine-induced GDNF mRNA expression after silencing of endogenous EGR-1 demonstrated that EGR-1 is upregulated by imipramine to activate the GDNF promoter. Furthermore, imipramine-induced GDNF mRNA expression was strongly attenuated in primary astrocytes from Egr-1(-/-) mice, and the immunoreactivity to an anti-GDNF antibody in glial fibrillary acidic protein-positive cells was lower in imipramine-treated astrocytes from Egr-1(-/-) mice than in those from Egr-1(+/-) mice. To determine whether mitogen-activated protein kinases (MAPKs) were associated with imipramine-induced EGR-1 expression, we examined the induction of MAPK phosphorylation in response to imipramine. Pretreatment of rat primary astrocytes with the MAPK kinase inhibitor U0126 or the JNK inhibitor SP600125 strongly inhibited imipramine-stimulated EGR-1 expression. In conclusion, we found that imipramine induction of EGR-1 upregulated GDNF in astrocytes in a dose-dependent manner. This upregulation may occur through the MEK/ERK and JNK MAPK pathways, which suggests a new therapeutic mechanism of action for depressive disorders.

  12. Brain-derived neurotrophic factor from bone marrow-derived cells promotes post-injury repair of peripheral nerve.

    Directory of Open Access Journals (Sweden)

    Yoshinori Takemura

    Full Text Available Brain-derived neurotrophic factor (BDNF stimulates peripheral nerve regeneration. However, the origin of BNDF and its precise effect on nerve repair have not been clarified. In this study, we examined the role of BDNF from bone marrow-derived cells (BMDCs in post-injury nerve repair. Control and heterozygote BDNF knockout mice (BDNF+/- received a left sciatic nerve crush using a cerebral blood clip. Especially, for the evaluation of BDNF from BMDCs, studies with bone marrow transplantation (BMT were performed before the injury. We evaluated nerve function using a rotarod test, sciatic function index (SFI, and motor nerve conduction velocity (MNCV simultaneously with histological nerve analyses by immunohistochemistry before and after the nerve injury until 8 weeks. BDNF production was examined by immunohistochemistry and mRNA analyses. After the nerve crush, the controls showed severe nerve dysfunction evaluated at 1 week. However, nerve function was gradually restored and reached normal levels by 8 weeks. By immunohistochemistry, BDNF expression was very faint before injury, but was dramatically increased after injury at 1 week in the distal segment from the crush site. BDNF expression was mainly co-localized with CD45 in BMDCs, which was further confirmed by the appearance of GFP-positive cells in the BMT study. Variant analysis of BDNF mRNA also confirmed this finding. BDNF+/- mice showed a loss of function with delayed histological recovery and BDNF+/+→BDNF+/- BMT mice showed complete recovery both functionally and histologically. These results suggested that the attenuated recovery of the BDNF+/- mice was rescued by the transplantation of BMCs and that BDNF from BMDCs has an essential role in nerve repair.

  13. CFTR is involved in the regulation of glucagon secretion in human and rodent alpha cells.

    Science.gov (United States)

    Edlund, Anna; Pedersen, Morten Gram; Lindqvist, Andreas; Wierup, Nils; Flodström-Tullberg, Malin; Eliasson, Lena

    2017-12-01

    Glucagon is the main counterregulatory hormone in the body. Still, the mechanism involved in the regulation of glucagon secretion from pancreatic alpha cells remains elusive. Dysregulated glucagon secretion is common in patients with Cystic Fibrosis (CF) that develop CF related diabetes (CFRD). CF is caused by a mutation in the Cl(-) channel Cystic fibrosis transmembrane conductance regulator (CFTR), but whether CFTR is present in human alpha cells and regulate glucagon secretion has not been investigated in detail. Here, both human and mouse alpha cells showed CFTR protein expression, whereas CFTR was absent in somatostatin secreting delta cells. CFTR-current activity induced by cAMP was measured in single alpha cells. Glucagon secretion at different glucose levels and in the presence of forskolin was increased by CFTR-inhibition in human islets, whereas depolarization-induced glucagon secretion was unaffected. CFTR is suggested to mainly regulate the membrane potential through an intrinsic alpha cell effect, as supported by a mathematical model of alpha cell electrophysiology. In conclusion, CFTR channels are present in alpha cells and act as important negative regulators of cAMP-enhanced glucagon secretion through effects on alpha cell membrane potential. Our data support that loss-of-function mutations in CFTR contributes to dysregulated glucagon secretion in CFRD.

  14. Glial cell line-derived neurotrophic factor alters the growth characteristics and genomic imprinting of mouse multipotent adult germline stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Jung, Yoon Hee [Department of Bioscience and Biotechnology, Bio-Organ Research Center/Animal Resources Research Center, Konkuk University, Hwayang-dong, Gwangjin-Gu, Seoul 143 701 (Korea, Republic of); Gupta, Mukesh Kumar, E-mail: goops@konkuk.ac.kr [Department of Animal Biotechnology, Bio-Organ Research Center/Animal Resources Research Center, Konkuk University, Hwayang-dong, Gwangjin-Gu, Seoul 143 701 (Korea, Republic of); Oh, Shin Hye [Department of Bioscience and Biotechnology, Bio-Organ Research Center/Animal Resources Research Center, Konkuk University, Hwayang-dong, Gwangjin-Gu, Seoul 143 701 (Korea, Republic of); Uhm, Sang Jun [Department of Animal Biotechnology, Bio-Organ Research Center/Animal Resources Research Center, Konkuk University, Hwayang-dong, Gwangjin-Gu, Seoul 143 701 (Korea, Republic of); Lee, Hoon Taek, E-mail: htl3675@konkuk.ac.kr [Department of Bioscience and Biotechnology, Bio-Organ Research Center/Animal Resources Research Center, Konkuk University, Hwayang-dong, Gwangjin-Gu, Seoul 143 701 (Korea, Republic of); Department of Animal Biotechnology, Bio-Organ Research Center/Animal Resources Research Center, Konkuk University, Hwayang-dong, Gwangjin-Gu, Seoul 143 701 (Korea, Republic of)

    2010-03-10

    This study evaluated the essentiality of glial cell line-derived neurotrophic factor (GDNF) for in vitro culture of established mouse multipotent adult germline stem (maGS) cell lines by culturing them in the presence of GDNF, leukemia inhibitory factor (LIF) or both. We show that, in the absence of LIF, GDNF slows the proliferation of maGS cells and result in smaller sized colonies without any change in distribution of cells to different cell-cycle stages, expression of pluripotency genes and in vitro differentiation potential. Furthermore, in the absence of LIF, GDNF increased the expression of male germ-line genes and repopulated the empty seminiferous tubule of W/W{sup v} mutant mouse without the formation of teratoma. GDNF also altered the genomic imprinting of Igf2, Peg1, and H19 genes but had no effect on DNA methylation of Oct4, Nanog and Stra8 genes. However, these effects of GDNF were masked in the presence of LIF. GDNF also did not interfere with the multipotency of maGS cells if they are cultured in the presence of LIF. In conclusion, our results suggest that, in the absence of LIF, GDNF alters the growth characteristics of maGS cells and partially impart them some of the germline stem (GS) cell-like characteristics.

  15. Apoptosis signal-regulating kinase 1 is involved in brain-derived neurotrophic factor (BDNF)-enhanced cell motility and matrix metalloproteinase 1 expression in human chondrosarcoma cells.

    Science.gov (United States)

    Lin, Chih-Yang; Chang, Sunny Li-Yun; Fong, Yi-Chin; Hsu, Chin-Jung; Tang, Chih-Hsin

    2013-07-25

    Chondrosarcoma is the primary malignancy of bone that is characterized by a potent capacity to invade locally and cause distant metastasis, and is therefore associated with poor prognoses. Chondrosarcoma further shows a predilection for metastasis to the lungs. The brain-derived neurotrophic factor (BDNF) is a small molecule in the neurotrophin family of growth factors that is associated with the disease status and outcome of cancers. However, the effect of BDNF on cell motility in human chondrosarcoma cells is mostly unknown. Here, we found that human chondrosarcoma cell lines had significantly higher cell motility and BDNF expression compared to normal chondrocytes. We also found that BDNF increased cell motility and expression of matrix metalloproteinase-1 (MMP-1) in human chondrosarcoma cells. BDNF-mediated cell motility and MMP-1 up-regulation were attenuated by Trk inhibitor (K252a), ASK1 inhibitor (thioredoxin), JNK inhibitor (SP600125), and p38 inhibitor (SB203580). Furthermore, BDNF also promoted Sp1 activation. Our results indicate that BDNF enhances the migration and invasion activity of chondrosarcoma cells by increasing MMP-1 expression through a signal transduction pathway that involves the TrkB receptor, ASK1, JNK/p38, and Sp1. BDNF thus represents a promising new target for treating chondrosarcoma metastasis.

  16. Modulation of secreted proteins of mouse mammary epithelial cells by the collagenous substrata

    Energy Technology Data Exchange (ETDEWEB)

    Lee, E.Y.H.; Parry, G.; Bissell, M.J.

    1984-01-01

    It has been shown previously that cultures of mouse mammary epithelial cells retain their characteristic morphology and their ability to produce ..gamma..-casein, a member of the casein gene family, only if they are maintained on floating collagen gels. In this paper we show: (a) Cells on floating collagen gels secrete not only ..gamma..-casein but also ..cap alpha../sub 1/-, ..cap alpha../sub 2/-, and ..beta..-caseins. These are not secreted by cells on plastic and are secreted to only a very limited extent by cells on attached collagen gels. (b) The floating collagen gel regulates at the level of synthesis and/or stabilization of the caseins rather than at the level of secretion alone. Contraction of the floating gel is important in that cells cultured on floating glutaraldehyde cross-linked gels do not secrete any of the caseins. (c) The secretion of an 80,000-mol-wt protein, most probably transferrin, and a 67,000-mol-wt protein, probably butyrophilin, a major protein of the milk fat globule membrane, are partially modulated by substrata. However, in contrast to the caseins, these are always detectable in media from cells cultured on plastic and attached gels. (d) Whey acidic protein, a major whey protein, is actively secreted by freshly isolated cells but is secreted in extremely limited quantities in cultured cells regardless of the nature of the substratum used. Lactalbumin secretion is also decreased significantly in cultured cells. (e) A previously unreported set of proteins, which may be minor milk proteins, are prominently secreted by the mammary cells on all substrata tested. We conclude that while the substratum profoundly influences the secretion of the caseins, it does not regulate the expression of every milk-specific protein in the same way. The mechanistic implications of these findings are discussed.

  17. Upregulated gene expression of local brain-derived neurotrophic factor and nerve growth factor after intracisternal administration of marrow stromal cells in rats with traumatic brain injury

    Institute of Scientific and Technical Information of China (English)

    胡德志; 周良辅; 朱剑虹; 毛颖; 吴雪海

    2005-01-01

    Objective: To examine the effects of rat marrow stromal cells (rMSCs) on gene expression of local brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) after injection of rMSCs into Cistern Magnum of adult rats subjected to traumatic brain injury(TBI).Results: Group cell transplantation had higher BDNF and NGF gene expressions than Group saline control during a period of less than 3 weeks (P<0.05).Conclusions: rMSCs transplantation via Cistern Magnum in rats subjected to traumatic brain injury can enhance expressions of local brain NGF and BDNF to a certain extent.

  18. Regulation of IGFBP secretion and modulation of cell growth in MDBK cells.

    Science.gov (United States)

    Cohick, W S; Clemmons, D R

    1993-03-01

    The ability of IGF binding proteins (IGFBP) to modulate cell growth and IGF-I responsiveness of epithelial cells was examined using the Madin-Darby bovine kidney (MDBK) cell line. The predominant IGFBP present in conditioned media (CM) of untreated cells was found to be IGFBP-2. Following exposure to forskolin, the abundance of IGFBP-2 in CM was decreased, while IGFBP-3 and -4 were induced. These changes corresponded with alterations in mRNA abundance. Growth of MDBK cells in serum-free media was stimulated by addition of 2.5 to 50 ng/ml of IGF-I in a dose responsive manner. Coincubation with equimolar amounts of IGF-I and exogenous bovine IGFBP-3 potentiated the growth response observed with IGF-I alone. In order to alter endogenous IGFBP-3 secretion, cells were exposed to transfection with an expression vector containing sense IGFBP-3 cDNA. Following selection and amplification with methotrexate, cells underwent a permanent alteration in cell morphology, exhibiting characteristics of transporting epithelia. This was associated with secretion of IGFBP-3 under basal conditions. Secretion of IGFBP-3 was due to expression of endogenous IGFBP-3 and not to expression of the transgene. Cells expressing IGFBP-3 under basal conditions grew slower in serum, but were more responsive to 100 ng/ml of IGF-1 in serum-free media compared to wild-type MDBK cells. The role of IGFBP-3 in mediating these responses requires further study.

  19. Role of PI3-K/Akt pathway and its effect on glial cell line-derived neurotrophic factor in midbrain dopamine cells

    Institute of Scientific and Technical Information of China (English)

    Hong-jun WANG; Jun-ping CAO; Jing-kao YU; Dian-shuai GAO

    2007-01-01

    Aim: To explore the intracellular mechanisms underlying the survival/differentia-don effect of the glial cell line-derived neurotrophic factor (GDNF) on dopamine(DA) cells. Methods: Midbrain slice culture and primary cell culture were established, and the cultures were divided into 3 groups: control group, GDNF group, and the phosphatidylinositol 3-kinase/Akt (PI3-K/Akt) pathway-inhibited group. Then the expression of tyrosine hydroxylase (TH) was detected by immunostaining as well as Western blotting. Results: GDNF treatment induced an increase in the number of TH-immunoreactive (ir) cells and the neurite number of TH-ir cells, as well as in the level of TH expression in cultures (Number of TH-ir cells in the slice culture: control group, 8.76±0.75; GDNF group, 18.63±0.95.Number of TH-ir cells and neurite number of TH-ir cells in cell culture: controlgroup, 3.65±0.88 and 2.49±0.42; GDNF group, 6.01±0.43 and 4.89±0.46). Meanwhile, the stimulation of cultured cells with GDNF increased the phosphorylation of Akt, which is a downstream effector of PI3-K/Akt. The effects of GDNF were specifically blocked by the inhibitor of the PI3-K/Akt pathway, wortmannin (Number of TH-ir cells in slice culture: PI3-K/Akt pathway-inhibited group, 6.98±0.58. Num-ber of TH-ir cells and neurite number of TH-ir cells in cell culture: PI3-K/Aktpathway-inhibited group, 3.79±0.62 and 2.50±0.25, respectively). Conclusion: The PI3-K/Akt pathway mediates the survival/differentiation effect of GDNF on DA cells.8±0.58.

  20. Distribution of glial cell line-derived neurotrophic factor receptor alpha-1 in the brain of adult zebrafish.

    Science.gov (United States)

    Lucini, Carla; Carla, Lucini; Facello, Bruna; Bruna, Facello; Maruccio, Lucianna; Lucianna, Maruccio; Langellotto, Fernanda; Fernanda, Langellotto; Sordino, Paolo; Paolo, Sordino; Castaldo, Luciana; Luciana, Castaldo

    2010-08-01

    Glial cell line-derived neurotrophic factor (GDNF) is a potent trophic factor for several types of neurons in the central and peripheral nervous systems. The biological activity of GDNF is mediated by a multicomponent receptor complex that includes a common transmembrane signaling component (the rearranged during transfection (RET) proto-oncogene product, a tyrosine kinase receptor) as well as a GDNF family receptor alpha (GFRalpha) subunit, a high-affinity glycosyl phosphatidylinositol (GPI)-linked binding element. Among the four known GFRalpha subunits, GFRalpha1 preferentially binds to GDNF. In zebrafish (Danio rerio) embryos, the expression of the GFRalpha1a and GFRalpha1b genes has been shown in primary motor neurons, the kidney, and the enteric nervous system. To examine the activity of GFRalpha in the adult brain of a lower vertebrate, we have investigated the localization of GFRalpha1a and GFRalpha1b mRNA and the GFRalpha1 protein in zebrafish. GFRalpha1a and GFRalpha1b transcripts were observed in brain extracts by reverse transcription-polymerase chain reaction. Whole-mount in-situ hybridization experiments revealed a wide distribution of GFRalpha1a and GFRalpha1b mRNAs in various regions of the adult zebrafish brain. These included the olfactory bulbs, dorsal and ventral telencephalic area (telencephalon), preoptic area, dorsal and ventral thalamus, posterior tuberculum and hypothalamus (diencephalon), optic tectum (mesencephalon), cerebellum, and medulla oblongata (rhombencephalon). Finally, expression patterns of the GFRalpha1 protein, detected immunohistochemically, correlated well with the mRNA expression and provided further insights into translational activity at the neuroanatomical level. In conclusion, the current study demonstrated that the presence of GFRalpha1 persists beyond the embryonic development of the zebrafish brain and, together with the GDNF ligand, is probably implicated in the brain physiology of an adult teleost fish.

  1. Graphene Oxide Modulates B Cell Surface Phenotype and Impairs Immunoglobulin Secretion in Plasma Cell.

    Science.gov (United States)

    Xu, Shaohai; Xu, Shengmin; Chen, Shaopeng; Fan, Huadong; Luo, Xun; Yang, Xiaoyao; Wang, Jun; Yuan, Hang; Xu, An; Wu, Lijun

    2016-04-01

    Since discovery, graphene oxide (GO) has been used in all aspects of human life and revealed promising applications in biomedicine. Nevertheless, the potential risks of GO were always being revealed. Although GO was found to induce immune cell death and innate immune response, little is known regarding its toxicity to the specific adaptive immune system that is crucial for protecting against exotic invasion. The B-cell mediated adaptive immune system, which composed of highly specialized cells (B and plasma cell) and specific immune response (antibody response) is the focus in our present study. Using diverse standard immunological techniques, we found that GO modulated B cell surface phenotype, both costimulatory molecules (CD80, CD86 and especially CD40) and antigen presenting molecules (both classical and nonclassical) under the condition without causing cell death. Meanwhile, the terminal differentiated immunoglobulin (Ig) secreting plasma cell was affected by GO, which displayed a less secretion of Ig and more severe ER stress caused by the retention of the secreted form of Ig in cell compartment. The combined data reveal that GO has a particular adverse effect to B cell and the humoral immunity, directly demonstrating the potential risk of GO to the specific adaptive immunity.

  2. Endothelial cells downregulate apolipoprotein D expression in mural cells through paracrine secretion and Notch signaling.

    Science.gov (United States)

    Pajaniappan, Mohanasundari; Glober, Nancy K; Kennard, Simone; Liu, Hua; Zhao, Ning; Lilly, Brenda

    2011-09-01

    Endothelial and mural cell interactions are vitally important for proper formation and function of blood vessels. These two cell types communicate to regulate multiple aspects of vessel function. In studying genes regulated by this interaction, we identified apolipoprotein D (APOD) as one gene that is downregulated in mural cells by coculture with endothelial cells. APOD is a secreted glycoprotein that has been implicated in governing stress response, lipid metabolism, and aging. Moreover, APOD is known to regulate smooth muscle cells and is found in abundance within atherosclerotic lesions. Our data show that the regulation of APOD in mural cells is bimodal. Paracrine secretion by endothelial cells causes partial downregulation of APOD expression. Additionally, cell contact-dependent Notch signaling plays a role. NOTCH3 on mural cells promotes the downregulation of APOD, possibly through interaction with the JAGGED-1 ligand on endothelial cells. Our results show that NOTCH3 contributes to the downregulation of APOD and by itself is sufficient to attenuate APOD transcript expression. In examining the consequence of decreased APOD expression in mural cells, we show that APOD negatively regulates cell adhesion. APOD attenuates adhesion by reducing focal contacts; however, it has no effect on stress fiber formation. These data reveal a novel mechanism in which endothelial cells control neighboring mural cells through the downregulation of APOD, which, in turn, influences mural cell function by modulating adhesion.

  3. Ciliary Neurotrophic Factor Cell-Based Delivery Prevents Synaptic Impairment and Improves Memory in Mouse Models of Alzheimer's Disease

    NARCIS (Netherlands)

    Garcia, Pierre; Youssef, Ihsen; Utvik, Jo K.; Florent-Bechard, Sabrina; Barthelemy, Vanassa; Malaplate-Armand, Catherine; Kriem, Badreddine; Stenger, Christophe; Koziel, Violette; Olivier, Jean-Luc; Escanye, Marie-Christine; Hanse, Marine; Allouche, Ahmad; Desbene, Cedric; Yen, Frances T.; Bjerkvig, Rolf; Oster, Thierry; Niclou, Simone P.; Pillot, Thierry

    2010-01-01

    The development of novel therapeutic strategies for Alzheimer's disease (AD) represents one of the biggest unmet medical needs today. Application of neurotrophic factors able to modulate neuronal survival and synaptic connectivity is a promising therapeutic approach for AD. We aimed to determine whe

  4. Glial cell line-derived neurotrophic factor (GDNF) enhances sympathetic neurite growth in rat hearts at early developmental stages

    NARCIS (Netherlands)

    K. Miwa; J.K. Lee; Y. Takagishi; T. Opthof; X. Fu; I. Kodama

    2010-01-01

    Molecular signaling of sympathetic innervation of myocardium is an unresolved issue. The purpose of this study was to investigate the effect of neurotrophic factors on sympathetic neurite growth towards cardiomyocytes. Cardiomyocytes (CMs) and sympathetic neurons (SNs) were isolated from neonatal ra

  5. Heparanase regulates secretion, composition, and function of tumor cell-derived exosomes.

    Science.gov (United States)

    Thompson, Camilla A; Purushothaman, Anurag; Ramani, Vishnu C; Vlodavsky, Israel; Sanderson, Ralph D

    2013-04-05

    Emerging evidence indicates that exosomes play a key role in tumor-host cross-talk and that exosome secretion, composition, and functional capacity are altered as tumors progress to an aggressive phenotype. However, little is known regarding the mechanisms that regulate these changes. Heparanase is an enzyme whose expression is up-regulated as tumors become more aggressive and is associated with enhanced tumor growth, angiogenesis, and metastasis. We have discovered that in human cancer cells (myeloma, lymphoblastoid, and breast cancer), when expression of heparanase is enhanced or when tumor cells are exposed to exogenous heparanase, exosome secretion is dramatically increased. Heparanase enzyme activity is required for robust enhancement of exosome secretion because enzymatically inactive forms of heparanase, even when present in high amounts, do not dramatically increase exosome secretion. Heparanase also impacts exosome protein cargo as reflected by higher levels of syndecan-1, VEGF, and hepatocyte growth factor in exosomes secreted by heparanase-high expressing cells as compared with heparanase-low expressing cells. In functional assays, exosomes from heparanase-high cells stimulated spreading of tumor cells on fibronectin and invasion of endothelial cells through extracellular matrix better than did exosomes secreted by heparanase-low cells. These studies reveal that heparanase helps drive exosome secretion, alters exosome composition, and facilitates production of exosomes that impact both tumor and host cell behavior, thereby promoting tumor progression.

  6. Contraction induced secretion of VEGF from skeletal muscle cells is mediated by adenosine

    DEFF Research Database (Denmark)

    Høier, Birgitte; Olsen, Karina; Nyberg, Michael Permin

    2010-01-01

    The role of adenosine and contraction for secretion of VEGF in skeletal muscle was investigated in human subjects and rat primary skeletal muscle cells. Microdialysis probes were inserted into the thigh muscle of seven male subjects and dialysate was collected at rest, during infusion of adenosine...... and contraction caused secretion of VEGF (pcontraction induced secretion of VEGF protein was abolished by the A(2B) antagonist enprofyllin and markedly reduced by inhibition of PKA or MAPK. The results demonstrate that adenosine causes secretion of VEGF from human skeletal muscle cells...... and that the contraction induced secretion of VEGF is partially mediated via adenosine acting on A(2B) adenosine receptors. Moreover, the contraction induced secretion of VEGF protein from muscle is dependent on both PKA and MAPK activation, but only the MAPK pathway appears to be adenosine dependent....

  7. Differentiation of mouse embryonic stem cells into insulin-secreting cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Sui Jing; Jiang Fangxu; Shi Bingyin

    2011-01-01

    Regenerative medicine,including cell-replacement strategies,may have an important role in the treatment of type 1 diabetes which is associated with decreased islet cell mass. To date,significant progress has been made in generating insulin-secreting β cells from pluripotent mouse embryonic stem cells (ESCs).The aim of this study is to explore the potential of regulating the differentiation of ESCs into pancreatic endocrine cells capable of synthesizing the pancreatic hormones including insulin, glucagon, somatostatin and pancreatic polypeptide under proper conditions.Undifferentiated ES cell line was stably transfected with mouse RIP-YFP plasmid construction in serum-free medium using LipofectamineTM 2000 Reagents. We tested pancreatic specific gene expression and characterized these ESC-derived pancreatic endocrine cells. Most of these insulin-secreting cells co-expressed many of the phenotypic markers characteristic of β cells such as insulinl,insulin2,Islet1,MafA,insulinoma-associated antigen 1 (IA1) and so on,indicating a similar gene expression pattern to adult islet β cells in vivo. Characterization of this population revealed that it consisted predominantly of pancreatic endocrine cells that were able to undergo pancreatic specification under the appropriate conditions. We also demonstrated that zinc supplementation mediated up-regulation of insulin-secreting cells as an effective inducer promoted the development of ESC-derived diabetes therapy. In conclusion,this work not only established an efficient pancreatic differentiation strategy from ESCs to pancreatic endocrine lineage in vitro,but also leaded to the development of new strategies to derive transplantable islet-replacement β cells from embryonic stem cells for the future applications of a stem cell based therapy of diabetes.

  8. Neurotrophic Effect of Citrus Auraptene: Neuritogenic Activity in PC12 Cells

    Directory of Open Access Journals (Sweden)

    Mitsunari Nakajima

    2012-04-01

    Full Text Available The activation of extracellular signal-regulated kinases (ERK leads to a number of cellular changes associated with the development of long-term memory. Using cultured cortical neurons, we previously showed that the n-hexane extract prepared from the peels of Citrus grandis (Kawachi bankan induces the activation of ERK1/2 and that one of the compounds with this ability in the extract is 3,5,6,7,8,3′,4′-heptamethoxyflavone (HMF, a Citrus polymethoxyflavone. In fact, we found that HMF has the ability to rescue mice from drug-induced learning impairment. This hexane extract contains auraptene (AUR, a coumarin derivative with a monoterpene unit, together with HMF. The present study was designed to investigate the effect of AUR in vitro. Our results show that 1 AUR had the ability to induce the activation of ERK1/2 in not only cortical neurons but also the rat pheochromocytoma cell line (PC12 cells, which is a model system for studies on neuronal proliferation and differentiation; and 2 AUR had the ability to promote neurite outgrowth from PC12 cells.

  9. Chloroquine-Inducible Par-4 Secretion Is Essential for Tumor Cell Apoptosis and Inhibition of Metastasis

    Directory of Open Access Journals (Sweden)

    Ravshan Burikhanov

    2017-01-01

    Full Text Available The induction of tumor suppressor proteins capable of cancer cell apoptosis represents an attractive option for the re-purposing of existing drugs. We report that the anti-malarial drug, chloroquine (CQ, is a robust inducer of Par-4 secretion from normal cells in mice and cancer patients in a clinical trial. CQ-inducible Par-4 secretion triggers paracrine apoptosis of cancer cells and also inhibits metastatic tumor growth. CQ induces Par-4 secretion via the classical secretory pathway that requires the activation of p53. Mechanistically, p53 directly induces Rab8b, a GTPase essential for vesicle transport of Par-4 to the plasma membrane prior to secretion. Our findings indicate that CQ induces p53- and Rab8b-dependent Par-4 secretion from normal cells for Par-4-dependent inhibition of metastatic tumor growth.

  10. Oral treatment with laquinimod augments regulatory T-cells and brain-derived neurotrophic factor expression and reduces injury in the CNS of mice with experimental autoimmune encephalomyelitis.

    Science.gov (United States)

    Aharoni, Rina; Saada, Ravit; Eilam, Raya; Hayardeny, Liat; Sela, Michael; Arnon, Ruth

    2012-10-15

    Laquinimod is an orally active molecule that showed efficacy in clinical trials in multiple sclerosis. We studied its effects in the CNS, when administered by therapeutic regimen to mice inflicted with experimental autoimmune encephalomyelitis (EAE). Laquinimod reduced clinical and inflammatory manifestations and elevated the prevalence of T-regulatory cells in the brain. In untreated mice, in the chronic disease stage, brain derived neurotrophic factor (BDNF) expression was impaired. Laquinimod treatment restored BDNF expression to its level in healthy controls. Furthermore, CNS injury, manifested by astrogliosis, demyelination and axonal damages, was significantly reduced following laquinimod treatment, indicating its immunomodulatory and neuroprotective activity.

  11. Rab27A regulates exosome secretion from lung adenocarcinoma cells A549: involvement of EPI64.

    Science.gov (United States)

    Li, Wenhai; Hu, Yunsheng; Jiang, Tao; Han, Yong; Han, Guoliang; Chen, Jiakuan; Li, Xiaofei

    2014-11-01

    Exosomes are small membrane vesicles secreted into the extracellular compartment by exocytosis. The unique composition of exosomes can be transported to other cells which allow cells to exert biological functions at distant sites. However, in lung cancer, the regulation of exosome secretion was poorly understood. In this study, we employed human lung adenocarcinoma A549 cells to determine the exosome secretion and involved regulation mechanism. We found that Rab27A was expressed in A549 cells and the reduction of Rab27A by Rab27A-specific shRNA could significantly decrease the secretion of exosome by A549 cells. EPI64, a candidate GAP that is specific for Rab27, was also detected in A549 cells. By pull-down assay, we found that EPI64 participated in the exosome secretion of A549 cells by acting as a specific GAP for Rab27A, not Rab27B. Overexpression of EPI64 enhanced exosome secretion. Taken together, in A549 cells, EPI64 could regulate the exosome secretion by functioning as a GAP specific for Rab27A.

  12. Parallel secretion of pancreastatin and somatostatin from human pancreastatin producing cell line (QGP-1N).

    Science.gov (United States)

    Funakoshi, A; Tateishi, K; Kitayama, N; Jimi, A; Matsuoka, Y; Kono, A

    1993-05-01

    In this investigation we studied pancreastatin (PST) secretion from a human PST producing cell line (QGP-1N) in response to various secretagogues. Immunocytochemical study revealed the immunoreactivity of PST and somatostatin (SMT) in the same cells of a monolayer culture. Ki-ras DNA point mutation on codon 12 was found. Carbachol stimulated secretion of PST and SMT and intracellular Ca2+ mobilization in the range of 10(-6)-10(-4) M. The secretion and Ca2+ mobilization were inhibited by atropine, a muscarinic receptor antagonist. Phorbol ester and calcium ionophore (A23187) stimulated secretion of PST and SMT. The removal of extracellular calcium suppressed both secretions throughout stimulation with 10(-5) M carbachol. Fluoride, a well-known activator of guanine nucleotide binding (G) protein, stimulated intracellular Ca2+ mobilization and secretion of PST and SMT in a dose-dependent manner in the range of 5-40 mM. Also, 10(-5) M carbachol and 20 mM fluoride stimulated inositol 1,4,5-triphosphate production. However, cholecystokinin and gastrin-releasing peptide did not stimulate Ca2+ mobilization or secretion of the two peptides. These results suggest that secretion of PST and SMT from QGP-1N cells is regulated mainly by acetylcholine in a parallel fashion through muscarinic receptors coupled to the activation of polyphosphoinositide breakdown by a G-protein and that increases in intracellular Ca2+ and protein kinase C play an important role in stimulus-secretion coupling.

  13. Glucose decouples intracellular Ca2+ activity from glucagon secretion in mouse pancreatic islet alpha-cells.

    Directory of Open Access Journals (Sweden)

    Sylvain J Le Marchand

    Full Text Available The mechanisms of glucagon secretion and its suppression by glucose are presently unknown. This study investigates the relationship between intracellular calcium levels ([Ca(2+](i and hormone secretion under low and high glucose conditions. We examined the effects of modulating ion channel activities on [Ca(2+](i and hormone secretion from ex vivo mouse pancreatic islets. Glucagon-secreting α-cells were unambiguously identified by cell specific expression of fluorescent proteins. We found that activation of L-type voltage-gated calcium channels is critical for α-cell calcium oscillations and glucagon secretion at low glucose levels. Calcium channel activation depends on K(ATP channel activity but not on tetrodotoxin-sensitive Na(+ channels. The use of glucagon secretagogues reveals a positive correlation between α-cell [Ca(2+](i and secretion at low glucose levels. Glucose elevation suppresses glucagon secretion even after treatment with secretagogues. Importantly, this inhibition is not mediated by K(ATP channel activity or reduction in α-cell [Ca(2+](i. Our results demonstrate that glucose uncouples the positive relationship between [Ca(2+](i and secretory activity. We conclude that glucose suppression of glucagon secretion is not mediated by inactivation of calcium channels, but instead, it requires a calcium-independent inhibitory pathway.

  14. Effects of brain-derived neurotrophic factor on cell survival, differentiation and patterning of neuronal connections and Müller glia cells in the developing retina.

    Science.gov (United States)

    Pinzón-Duarte, Germán; Arango-González, Blanca; Guenther, Elke; Kohler, Konrad

    2004-03-01

    The aim of the present study was to determine the influence of brain-derived neurotrophic factor (BDNF) on survival, phenotype differentiation and network formation of retinal neurons and glia cells. To achieve a defined concentration and constant level of BDNF over several days, experiments were performed in an organotypic culture of the developing rat retina. After 6 days in vitro, apoptosis in the different cell layers was determined by TUNEL staining and cell-type-specific antibodies were used to identify distinct neuronal cell types and Müller cells. Cultured retinas treated with BDNF (100 ng BDNF/mL medium) were compared with untreated as well as with age-matched in vivo retinas. Quantitative morphometry was carried out using confocal microscopy. BDNF promoted the in vitro development and differentiation of the retina in general, i.e. the number of cells in the nuclear layers and the thickness of the plexiform layers were increased. For all neurons, the number of cells and the complexity of arborizations in the synaptic layers were clearly up-regulated by BDNF. In control cultures, the synaptic stratification of cone bipolar cells within the On- and Off-layer of the inner plexiform layer was disturbed and a strong reactivity of Müller cell glia was observed. These effects were not present in BDNF-treated cultures. Our data show that BDNF promotes the survival of retinal interneurons and plays an important role in establishing the phenotypes and the synaptic connections of a large number of neuronal types in the developing retina. Moreover, we show an effect of BDNF on Müller glia cells.

  15. Activated microglia induce bone marrow mesenchymal stem cells to produce glial cell-derived neurotrophic factor and protect neurons against oxygen-glucose deprivation injury

    Directory of Open Access Journals (Sweden)

    Bingke Lv

    2016-12-01

    Full Text Available In this study, we investigated interactions among microglia (MG, bone marrow mesenchymal stem cells (BMSCs and neurons in cerebral ischemia and the potential mechanisms using an in vitro oxygen-glucose deprivation (OGD model. Rat BMSCs were incubated with conditioned medium (CM from in vitro cultures of OGD-activated rat MG and murine BV2 MG cells. Effects of glial cell-derived neurotrophic factor (GDNF on rat neuron viability, apoptosis, lactate dehydrogenase (LDH leakage and mitochondrial membrane potential (MMP were analyzed in this model. OGD-activated MG promoted GDNF production by BMSCs (P < 0.01. TNFα, but not IL6 or IL1β, promoted GDNF production by BMSCs (P < 0.001. GDNF or CM pre-treated BMSCs elevated neuronal viability and suppressed apoptosis (P < 0.05 or P < 0.01; these effects were inhibited by the RET antibody. GDNF activated MEK/ERK and PI3K/AKT signaling but not JNK/c-JUN. Furthermore, GDNF upregulated B cell lymphoma 2 (BCL2 and heat shock 60 kDa protein 1 (HSP60 levels, suppressed LDH leakage, and promoted MMP. Thus, activated MG produce TNFα to stimulate GDNF production by BMSCs, which prevents and repairs OGD-induced neuronal injury, possibly via regulating MEK/ERK and PI3K/AKT signaling. These findings will facilitate the prevention and treatment of neuronal injury by cerebral ischemia.

  16. Secretion of goblet cell serine proteinase, ingobsin, is stimulated by vasoactive intestinal polypeptide and acetylcholine

    DEFF Research Database (Denmark)

    Poulsen, Steen Seier; Nexø, Ebba

    1987-01-01

    Ingobsin is localized to the intestinal goblet cells in the rat and in man. In the present study, we investigated the effect of vasoactive intestinal polypeptide (VIP) and acetylcholine on the secretion of ingobsin from the proximal duodenum. Intravenous infusion of VIP or acetylcholine increased...... the concentration of ingobsin in duodenal secretion, while the concentration in the duodenum was unchanged. Simultaneous infusion of VIP and acetylcholine increased the concentration of ingobsin in duodenal secretion and decreased the concentration of ingobsin in the duodenum. This study demonstrates that secretion...... of ingobsin from the proximal duodenum is exocrine and can be stimulated by VIP and acetylcholine....

  17. Estimation of adenosine triphosphate utilization of rat mast cells during and after anaphylactic histamine secretion

    DEFF Research Database (Denmark)

    Johansen, Torben

    1990-01-01

    Determination of the cellular content of adenosine triphosphate (ATP) and the rate of ATP-synthesis were used to estimate the cellular utilization of ATP in relation to anaphylactic histamine secretion. There was an increased rate of oxidative ATP-synthesis and a decreased cellular ATP content...... during the time period of histamine secretion and immediately after its completion. During secretion the additional ATP-utilization above the basal level of ATP-synthesis was 0.51 pmol/10(3) cells. 2.5 min after cell activation, the rate of additional ATP-utilization was 0.30 pmol/10(3) cells...

  18. Glial cell line-derived neurotrophic factor (GDNF) induces neuritogenesis in the cochlear spiral ganglion via neural cell adhesion molecule (NCAM).

    Science.gov (United States)

    Euteneuer, Sara; Yang, Kuo H; Chavez, Eduardo; Leichtle, Anke; Loers, Gabriele; Olshansky, Adel; Pak, Kwang; Schachner, Melitta; Ryan, Allen F

    2013-05-01

    Glial cell line-derived neurotrophic factor (GDNF) increases survival and neurite extension of spiral ganglion neurons (SGNs), the primary neurons of the auditory system, via yet unknown signaling mechanisms. In other cell types, signaling is achieved by the GPI-linked GDNF family receptor α1 (GFRα1) via recruitment of transmembrane receptors: Ret (re-arranged during transformation) and/or NCAM (neural cell adhesion molecule). Here we show that GDNF enhances neuritogenesis in organotypic cultures of spiral ganglia from 5-day-old rats and mice. Addition of GFRα1-Fc increases this effect. GDNF/GFRα1-Fc stimulation activates intracellular PI3K/Akt and MEK/Erk signaling cascades as detected by Western blot analysis of cultures prepared from rats at postnatal days 5 (P5, before the onset of hearing) and 20 (P20, after the onset of hearing). Both cascades mediate GDNF stimulation of neuritogenesis, since application of the Akt inhibitor Wortmannin or the Erk inhibitor U0126 abolished GDNF/GFRα1-Fc stimulated neuritogenesis in P5 rats. Since cultures of P5 NCAM-deficient mice failed to respond by neuritogenesis to GDNF/GFRα1-Fc, we conclude that NCAM serves as a receptor for GDNF signaling responsible for neuritogenesis in early postnatal spiral ganglion.

  19. Secretion of interferon gamma from human immune cells is altered by exposure to tributyltin and dibutyltin.

    Science.gov (United States)

    Lawrence, Shanieek; Reid, Jacqueline; Whalen, Margaret

    2015-05-01

    Tributyltin (TBT) and dibutyltin (DBT) are widespread environmental contaminants found in food, beverages, and human blood samples. Both of these butyltins (BTs) interfere with the ability of human natural killer (NK) cells to lyse target cells and alter secretion of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) from human immune cells in vitro. The capacity of BTs to interfere with secretion of other pro-inflammatory cytokines has not been examined. Interferon gamma (IFNγ) is a modulator of adaptive and innate immune responses, playing an important role in overall immune competence. This study shows that both TBT and DBT alter secretion of IFNγ from human immune cells. Peripheral blood cell preparations that were increasingly reconstituted were used to determine if exposures to either TBT or DBT affected IFNγ secretion and how the makeup of the cell preparation influenced that effect. IFNγ secretion was examined after 24 h, 48 h, and 6 day exposures to TBT (200 - 2.5 nM) and DBT (5 - 0.05 µM) in highly enriched human NK cells, a monocyte-depleted preparation of PBMCs, and monocyte-containing PBMCs. Both BTs altered IFNγ secretion from immune cells at most of the conditions tested (either increasing or decreasing secretion). However, there was significant variability among donors as to the concentrations and time points that showed changes as well as the baseline secretion of IFNγ. The majority of donors showed an increase in IFNγ secretion in response to at least one concentration of TBT or DBT at a minimum of one length of exposure.

  20. TRPC3 regulates release of brain-derived neurotrophic factor from human airway smooth muscle.

    Science.gov (United States)

    Vohra, Pawan K; Thompson, Michael A; Sathish, Venkatachalem; Kiel, Alexander; Jerde, Calvin; Pabelick, Christina M; Singh, Brij B; Prakash, Y S

    2013-12-01

    Exogenous brain-derived neurotrophic factor (BDNF) enhances Ca(2+) signaling and cell proliferation in human airway smooth muscle (ASM), especially with inflammation. Human ASM also expresses BDNF, raising the potential for autocrine/paracrine effects. The mechanisms by which ASM BDNF secretion occurs are not known. Transient receptor potential channels (TRPCs) regulate a variety of intracellular processes including store-operated Ca(2+) entry (SOCE; including in ASM) and secretion of factors such as cytokines. In human ASM, we tested the hypothesis that TRPC3 regulates BDNF secretion. At baseline, intracellular BDNF was present, and BDNF secretion was detectable by enzyme linked immunosorbent assay (ELISA) of cell supernatants or by real-time fluorescence imaging of cells transfected with GFP-BDNF vector. Exposure to the pro-inflammatory cytokine tumor necrosis factor-alpha (TNFα) (20ng/ml, 48h) or a mixture of allergens (ovalbumin, house dust mite, Alternaria, and Aspergillus extracts) significantly enhanced BDNF secretion and increased TRPC3 expression. TRPC3 knockdown (siRNA or inhibitor Pyr3; 10μM) blunted BDNF secretion, and prevented inflammation effects. Chelation of extracellular Ca(2+) (EGTA; 1mM) or intracellular Ca(2+) (BAPTA; 5μM) significantly reduced secreted BDNF, as did the knockdown of SOCE proteins STIM1 and Orai1 or plasma membrane caveolin-1. Functionally, secreted BDNF had autocrine effects suggested by phosphorylation of high-affinity tropomyosin-related kinase TrkB receptor, prevented by chelating extracellular BDNF with chimeric TrkB-Fc. These data emphasize the role of TRPC3 and Ca(2+) influx in the regulation of BDNF secretion by human ASM and the enhancing effects of inflammation. Given the BDNF effects on Ca(2+) and cell proliferation, BDNF secretion may contribute to altered airway structure and function in diseases such as asthma.

  1. The involvement of glucose-6-phosphatase in mucilage secretion by root cap cells of Zea mays

    Science.gov (United States)

    Moore, R.; McClelen, C. E.

    1985-01-01

    In order to determine the involvement of glucose-6-phosphatase in mucilage secretion by root cap cells, we have cytochemically localized the enzyme in columella and peripheral cells of root caps of Zea mays. Glucose-6-phosphatase is associated with the plasmalemma and cell wall of columella cells. As columella cells differentiate into peripheral cells and begin to produce and secrete mucilage, glucose-6-phosphatase staining intensifies and becomes associated with the mucilage and, to a lesser extent, the cell wall. Cells being sloughed from the cap are characterized by glucose-6-phosphatase staining being associated with the vacuole and plasmalemma. These changes in enzyme localization during cellular differentiation in root caps suggest that glucose-6-phosphatase is involved in the production and/or secretion of mucilage by peripheral cells of Z. mays.

  2. Insulin and norepinephrine regulate ghrelin secretion from a rat primary stomach cell culture.

    Science.gov (United States)

    Gagnon, Jeffrey; Anini, Younes

    2012-08-01

    Ghrelin is a peptide hormone primarily produced in the previously unidentified X/A endocrine cells of the stomach. Extensive studies have focused on the effects of ghrelin on growth hormone release and appetite regulation. However, the mechanisms regulating ghrelin secretion are less understood. In the present study, we developed a primary culture of newborn rat stomach cells to investigate the mechanisms regulating ghrelin synthesis and secretion. We demonstrated that this cell preparation secretes ghrelin in a regulated manner through the increase of cAMP, intracellular calcium, and activation of protein kinase C. Norepinephrine (NE) (0.1-10 μm) stimulated ghrelin secretion through the β1-adrenergic receptor via increased cAMP and protein kinase A activity, whereas acetylcholine had no effect. Because circulating ghrelin levels were previously shown to be inversely correlated with insulin levels, we investigated the effect of insulin on ghrelin secretion. We first demonstrated that ghrelin cells express the insulin receptor α- and β-subunits. Next, we determined that insulin (1-10 nm) inhibited both basal and NE-stimulated ghrelin secretion, caused an increase in phosphorylated serine-threonine kinase (AKT) and a reduction in intracellular cAMP, but did not alter proghrelin mRNA levels. The inhibitory effect of insulin was blocked by inhibiting phospho-inositol-3 kinase and AKT but not MAPK. Higher dose insulin (100 nm) did not suppress ghrelin secretion, which prompted the investigation of cellular insulin resistance by pretreating the cells with 100 nm insulin for 24 h. This caused a reduction in insulin receptor expression and prevented the insulin-mediated AKT activation and the suppression of ghrelin secretion with no impact on NE-stimulated ghrelin secretion. Our findings highlight the role of the sympathetic nervous system, insulin, and insulin resistance in the regulation of ghrelin secretion.

  3. Glial cell line-derived neurotrophic factor (GDNF) induced migration of spermatogonial cells in vitro via MEK and NF-kB pathways.

    Science.gov (United States)

    Huleihel, M; Fadlon, E; Abuelhija, A; Piltcher Haber, E; Lunenfeld, E

    2013-01-01

    Glial cell line-derived neurotrophic factor (GDNF) regulates spermatogonial stem cell (SSC) maintenance. In the present study, we examined the levels and the cellular origin of GDNF in mouse testes during age-development, and the capacity of GDNF to induce migration of enriched GFR-α1 positive cells in vitro. The involvement of MAP kinase (MEK) and NF-kB signal pathways were examined. Our results show high levels of GDNF in testicular tissue of one-week-old mice which significantly decreased with age when examined by ELISA, real time PCR (qPCR) and immunofluorescence staining (IF) analysis. GDNF receptor (GFR-α1) expression was similar to GDNF when examined by qPCR analysis. Only Sertoli cell cultures (SCs) from one-week-old mice produced GDNF compared to SCs from older mice. However, peritubular cells from all the examined ages did not produce GDNF. The addition of recombinant GDNF (rGDNF) or supernatant from SCs from one-week-old mice to GFR-α1 positive cells induced their migration in vitro. This effect was significantly reduced by the addition of inhibitors to MEK (PD98059, U0126), NF-kB (PDTC) and IkB protease inhibitor (TPCK). Our results show for the first time the capacity of rGDNF and supernatant from SCs to induce migration of enriched GFR-α1 positive cells, and the possible involvement of MEK, NF-kB and IkB in this process. This study may suggest a novel role for GDNF in the regulation SSC niches and spermatogenesis.

  4. Alpha cells secrete acetylcholine as a non-neuronal paracrine signal priming human beta cell function

    Science.gov (United States)

    Rodriguez-Diaz, Rayner; Dando, Robin; Jacques-Silva, M. Caroline; Fachado, Alberto; Molina, Judith; Abdulreda, Midhat; Ricordi, Camillo; Roper, Stephen D.; Berggren, Per-Olof; Caicedo, Alejandro

    2011-01-01

    Acetylcholine is a neurotransmitter that plays a major role in the function of the insulin secreting pancreatic beta cell1,2. Parasympathetic innervation of the endocrine pancreas, the islets of Langerhans, has been shown to provide cholinergic input to the beta cell in several species1,3,4, but the role of autonomic innervation in human beta cell function is at present unclear. Here we show that, in contrast to mouse islets, cholinergic innervation of human islets is sparse. Instead, we find that the alpha cells of the human islet provide paracrine cholinergic input to surrounding endocrine cells. Human alpha cells express the vesicular acetylcholine transporter and release acetylcholine when stimulated with kainate or a lowering in glucose concentration. Acetylcholine secretion by alpha cells in turn sensitizes the beta cell response to increases in glucose concentration. Our results demonstrate that in human islets acetylcholine is a paracrine signal that primes the beta cell to respond optimally to subsequent increases in glucose concentration. We anticipate these results to revise models about neural input and cholinergic signaling in the endocrine pancreas. Cholinergic signaling within the islet represents a potential therapeutic target in diabetes5, highlighting the relevance of this advance to future drug development. PMID:21685896

  5. Activation of Salivary Secretion: Coupling of Cell Volume and [Ca2+]i in Single Cells

    Science.gov (United States)

    Foskett, J. Kevin; Melvin, James E.

    1989-06-01

    High-resolution differential interference contrast microscopy and digital imaging of the fluorescent calcium indicator dye fura-2 were performed simultaneously in single rat salivary gland acinar cells to examine the effects of muscarinic stimulation on cell volume and cytoplasmic calcium concentration ([Ca2+]i). Agonist stimulation of fluid secretion is initially associated with a rapid tenfold increase in [Ca2+]i as well as a substantial cell shrinkage. Subsequent changes of cell volume in the continued presence of agonist are tightly coupled to dynamic levels of [Ca2+]i, even during [Ca2+]i oscillations. Experiments with Ca2+ chelators and ionophores showed that physiological elevations of [Ca2+]i are necessary and sufficient to cause changes in cell volume. The relation between [Ca2+]i and cell volume suggests that the latter reflects the secretory state of the acinar cell. Agonist-induced changes in [Ca2+]i, by modulating specific ion permeabilities, result in solute movement into or out of the cell. The resultant cell volume changes may be important in modulating salivary secretion.

  6. Effect of Rat Schwann Cell Secretion on Proliferation and Differentiation of Human Neural Stem Cells

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Objective To investigate the effect of rat Schwann cell secretion on the proliferation and differentiation of human embryonic neural stem cells (NSCs). Methods The samples were divided into three groups. In Group One, NSCs were cultured in DMED/F12 in which Schwann cells had grown for one day. In Group Two, NSCs and Schwann cells were co-cultured. In Group Three, NSCs were cultured in DMEM/F12. The morphology of NSCs was checked and b-tubulin, GalC, hoechst 33342 and GFAP labellings were detected. Results In Group One, all neural spheres were attached to the bottom and differentiated. The majority of them were b-tubulin positive while a few of cells were GFAP or GalC positive. In Group Two, neural spheres remained undifferentiatied and their proliferation was inhibited in places where Schwann cells were robust. In places where there were few Schwann cells, NSCs performed in a similar manner as in Group One. In Group Three, the cell growth state deteriorated day after day. On the 7th day, most NSCs died. Conclusion The secretion of rat Schwann cells has a growth supportive and differentiation-inducing effect on human NSCs.

  7. Glial Cell Line-Derived Neurotrophic Factor Family Members Reduce Microglial Activation via Inhibiting p38MAPKs-Mediated Inflammatory Responses

    Directory of Open Access Journals (Sweden)

    Uta Rickert

    2014-01-01

    Full Text Available Previous studies have shown that glial cell line-derived neurotrophic factor (GDNF family ligands (GFL are potent survival factors for dopaminergic neurons and motoneurons with therapeutic potential for Parkinson’s disease. However, little is known about direct influences of the GFL on microglia function, which are known to express part of the GDNF receptor system. Using RT-PCR and immunohistochemistrym we investigated the expression of the GDNF family receptor alpha 1 (GFR alpha and the coreceptor transmembrane receptor tyrosine kinase (RET in rat microglia in vitro as well as the effect of GFL on the expression of proinflammatory molecules in LPS activated microglia. We could show that GFL are able to regulate microglia functions and suggest that part of the well known neuroprotective action may be related to the suppression of microglial activation. We further elucidated the functional significance and pathophysiological implications of these findings and demonstrate that microglia are target cells of members of the GFL (GDNF and the structurally related neurotrophic factors neurturin (NRTN, artemin (ARTN, and persephin (PSPN.

  8. Ghrelin inhibits insulin secretion through the AMPK-UCP2 pathway in beta cells.

    Science.gov (United States)

    Wang, Ying; Nishi, Masahiro; Doi, Asako; Shono, Takeshi; Furukawa, Yasushi; Shimada, Takeshi; Furuta, Hiroto; Sasaki, Hideyuki; Nanjo, Kishio

    2010-04-16

    Ghrelin inhibits insulin secretion partly via induction of IA-2beta. However, the orexigenic effect of ghrelin is mediated by the AMP-activated protein kinase (AMPK)-uncoupling protein 2 (UCP2) pathway. Here, we demonstrate that ghrelin's inhibitory effect on insulin secretion also occurs through the AMPK-UCP2 pathway. Ghrelin increased AMPK phosphorylation and UCP2 mRNA expression in MIN6 insulinoma cells. Overexpression or downregulation of UCP2 attenuated or enhanced insulin secretion, respectively. Furthermore, AMPK activator had a similar effect to ghrelin on UCP2 and insulin secretion in MIN6 cells. In conclusion, ghrelin's inhibitory effect on insulin secretion is partly mediated by the AMPK-UCP2 pathway, which is independent of the IA-2beta pathway.

  9. Pavlovian Conditioning of Rat Mucosal Mast Cells to Secrete Rat Mast Cell Protease II

    Science.gov (United States)

    MacQueen, Glenda; Marshall, Jean; Perdue, Mary; Siegel, Shepard; Bienenstock, John

    1989-01-01

    Antigen (egg albumin) injections, which stimulate mucosal mast cells to secrete mediators, were paired with an audiovisual cue. After reexposure to the audiovisual cue, a mediator (rat mast cell protease II) was measured with a sensitive and specific assay. Animals reexposed to only the audiovisual cue released a quantity of protease not significantly different from animals reexposed to both the cue and the antigen; these groups released significantly more protease than animals that had received the cue and antigen in a noncontingent manner. The results support a role for the central nervous system as a functional effector of mast cell function in the allergic state.

  10. VEGF secretion during hypoxia depends on free radicals-induced Fyn kinase activity in mast cells

    Energy Technology Data Exchange (ETDEWEB)

    Garcia-Roman, Jonathan; Ibarra-Sanchez, Alfredo; Lamas, Monica [Departamento de Farmacobiologia, Centro de Investigacion y de Estudios Avanzados del IPN (Cinvestav, IPN) (Mexico); Gonzalez Espinosa, Claudia, E-mail: cgonzal@cinvestav.mx [Departamento de Farmacobiologia, Centro de Investigacion y de Estudios Avanzados del IPN (Cinvestav, IPN) (Mexico)

    2010-10-15

    Research highlights: {yields} Bone marrow-derived mast cells (BMMCs) secrete functional VEGF but do not degranulate after Cobalt chloride-induced hypoxia. {yields} CoCl{sub 2}-induced VEGF secretion in mast cells occurs by a Ca{sup 2+}-insensitive but brefeldin A and Tetanus toxin-sensitive mechanism. {yields} Trolox and N-acetylcysteine inhibit hypoxia-induced VEGF secretion but only Trolox inhibits Fc{epsilon}RI-dependent anaphylactic degranulation in mast cells. {yields} Src family kinase Fyn activation after free radical production is necessary for hypoxia-induced VEGF secretion in mast cells. -- Abstract: Mast cells (MC) have an important role in pathologic conditions such as asthma and chronic obstructive pulmonary disease (COPD), where hypoxia conduce to deleterious inflammatory response. MC contribute to hypoxia-induced angiogenesis producing factors such as vascular endothelial growth factor (VEGF), but the mechanisms behind the control of hypoxia-induced VEGF secretion in this cell type is poorly understood. We used the hypoxia-mimicking agent cobalt chloride (CoCl{sub 2}) to analyze VEGF secretion in murine bone marrow-derived mast cells (BMMCs). We found that CoCl{sub 2} promotes a sustained production of functional VEGF, able to induce proliferation of endothelial cells in vitro. CoCl{sub 2}-induced VEGF secretion was independent of calcium rise but dependent on tetanus toxin-sensitive vesicle-associated membrane proteins (VAMPs). VEGF exocytosis required free radicals formation and the activation of Src family kinases. Interestingly, an important deficiency on CoCl{sub 2}-induced VEGF secretion was observed in Fyn kinase-deficient BMMCs. Moreover, Fyn kinase was activated by CoCl{sub 2} in WT cells and this activation was prevented by treatment with antioxidants such as Trolox and N-acetylcysteine. Our results show that BMMCs are able to release VEGF under hypoxic conditions through a tetanus toxin-sensitive mechanism, promoted by free radicals

  11. Brain derived neurotrophic factor

    DEFF Research Database (Denmark)

    Mitchelmore, Cathy; Gede, Lene

    2014-01-01

    Brain Derived Neurotrophic Factor (BDNF) is a neurotrophin with important functions in neuronal development and neuroplasticity. Accumulating evidence suggests that alterations in BDNF expression levels underlie a variety of psychiatric and neurological disorders. Indeed, BDNF therapies are curre......Brain Derived Neurotrophic Factor (BDNF) is a neurotrophin with important functions in neuronal development and neuroplasticity. Accumulating evidence suggests that alterations in BDNF expression levels underlie a variety of psychiatric and neurological disorders. Indeed, BDNF therapies...

  12. TRPM5-mediated calcium uptake regulates mucin secretion from human colon goblet cells.

    Science.gov (United States)

    Mitrovic, Sandra; Nogueira, Cristina; Cantero-Recasens, Gerard; Kiefer, Kerstin; Fernández-Fernández, José M; Popoff, Jean-François; Casano, Laetitia; Bard, Frederic A; Gomez, Raul; Valverde, Miguel A; Malhotra, Vivek

    2013-05-28

    Mucin 5AC (MUC5AC) is secreted by goblet cells of the respiratory tract and, surprisingly, also expressed de novo in mucus secreting cancer lines. siRNA-mediated knockdown of 7343 human gene products in a human colonic cancer goblet cell line (HT29-18N2) revealed new proteins, including a Ca(2+)-activated channel TRPM5, for MUC5AC secretion. TRPM5 was required for PMA and ATP-induced secretion of MUC5AC from the post-Golgi secretory granules. Stable knockdown of TRPM5 reduced a TRPM5-like current and ATP-mediated Ca(2+) signal. ATP-induced MUC5AC secretion depended strongly on Ca(2+) influx, which was markedly reduced in TRPM5 knockdown cells. The difference in ATP-induced Ca(2+) entry between control and TRPM5 knockdown cells was abrogated in the absence of extracellular Ca(2+) and by inhibition of the Na(+)/Ca(2+) exchanger (NCX). Accordingly, MUC5AC secretion was reduced by inhibition of NCX. Thus TRPM5 activation by ATP couples TRPM5-mediated Na(+) entry to promote Ca(2+) uptake via an NCX to trigger MUC5AC secretion. DOI:http://dx.doi.org/10.7554/eLife.00658.001.

  13. 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.

  14. CELL-WALL GROWTH AND PROTEIN SECRETION IN FUNGI

    NARCIS (Netherlands)

    SIETSMA, JH; WOSTEN, HAB; WESSELS, JGH

    1995-01-01

    Secretion of proteins is a vital process in fungi. Because hyphal walls form a diffusion barrier for proteins, a mechanism different from diffusion probably exist to transport proteins across the wall. In Schizophyllum commune, evidence has been obtained for synthesis at the hyphal apex of wall comp

  15. Ionizing radiation induces tumor cell lysyl oxidase secretion

    DEFF Research Database (Denmark)

    Shen, Colette J; Sharma, Ashish; Vuong, Dinh-Van;

    2014-01-01

    BACKGROUND: Ionizing radiation (IR) is a mainstay of cancer therapy, but irradiation can at times also lead to stress responses, which counteract IR-induced cytotoxicity. IR also triggers cellular secretion of vascular endothelial growth factor, transforming growth factor beta and matrix...

  16. Optimizing neurotrophic factor combinations for neurite outgrowth

    Science.gov (United States)

    Deister, C.; Schmidt, C. E.

    2006-06-01

    Most neurotrophic factors are members of one of three families: the neurotrophins, the glial cell-line derived neurotrophic factor family ligands (GFLs) and the neuropoietic cytokines. Each family activates distinct but overlapping cellular pathways. Several studies have shown additive or synergistic interactions between neurotrophic factors from different families, though generally only a single combination has been studied. Because of possible interactions between the neurotrophic factors, the optimum concentration of a factor in a mixture may differ from the optimum when applied individually. Additionally, the effect of combinations of neurotrophic factors from each of the three families on neurite extension is unclear. This study examines the effects of several combinations of the neurotrophin nerve growth factor (NGF), the GFL glial cell-line derived neurotrophic factor (GDNF) and the neuropoietic cytokine ciliary neurotrophic factor (CNTF) on neurite outgrowth from young rat dorsal root ganglion (DRG) explants. The combination of 50 ng ml-1 NGF and 10 ng ml-1 of each GDNF and CNTF induced the highest level of neurite outgrowth at a 752 ± 53% increase over untreated DRGs and increased the longest neurite length to 2031 ± 97 µm compared to 916 ± 64 µm for untreated DRGs. The optimum concentrations of the three factors applied in combination corresponded to the optimum concentration of each factor when applied individually. These results indicate that the efficacy of future therapies for nerve repair would be enhanced by the controlled release of a combination of neurotrophins, GFLs and neuropoietic cytokines at higher concentrations than used in previous conduit designs.

  17. Energy metabolism in rat mast cells in relation to histamine secretion

    DEFF Research Database (Denmark)

    Johansen, T

    1987-01-01

    1. The relation between the energy metabolism and the secretory activity of rat peritoneal mast cells has been studied by determination of the cellular content of ATP and the rate of lactate production reflecting the rate of ATP synthesis under various experimental conditions. Secretion...... and the cellular ATP content at the time of cell activation was demonstrated. This may indicate a direct link between ATP and the secretory mechanism. 3. The possibility of an increased utilization of ATP during histamine secretion was explored in mast cells exposed to metabolic inhibitors. Incubation of mast...... cells with 2-deoxyglucose (2-DG) decreased the ATP content of the cells, and a long-lasting and stable level of mast cell ATP was observed. This is explained by a small decrease in the rate of ATP-synthesis by 2-DG. In 2-DG-treated cells secretion of histamine in response to compound 48...

  18. IGF-1 drives chromogranin A secretion via activation of Arf1 in human neuroendocrine tumour cells.

    Science.gov (United States)

    Münzberg, Christin; Höhn, Katharina; Krndija, Denis; Maaß, Ulrike; Bartsch, Detlef K; Slater, Emily P; Oswald, Franz; Walther, Paul; Seufferlein, Thomas; von Wichert, Götz

    2015-05-01

    Hypersecretion is the major symptom of functional neuroendocrine tumours. The mechanisms that contribute to this excessive secretion of hormones are still elusive. A key event in secretion is the exit of secretory products from the Golgi apparatus. ADP-ribosylation factor (Arf) GTPases are known to control vesicle budding and trafficking, and have a leading function in the regulation of formation of secretory granula at the Golgi. Here, we show that Arf1 is the predominant Arf protein family member expressed in the neuroendocrine pancreatic tumour cell lines BON and QGP-1. In BON cells Arf1 colocalizes with Golgi markers as well as chromogranin A, and shows significant basal activity. The inhibition of Arf1 activity or expression significantly impaired secretion of chromogranin A. Furthermore, we show that the insulin-like growth factor 1 (IGF-1), a major regulator of growth and secretion in BON cells, induces Arf1 activity. We found that activation of Arf1 upon IGF-1 receptor stimulation is mediated by MEK/ERK signalling pathway in BON and QGP-1 cells. Moreover, the activity of Arf1 in BON cells is mediated by autocrinely secreted IGF-1, and concomitantly, autocrine IGF1 secretion is maintained by Arf1 activity. In summary, our data indicate an important regulatory role for Arf1 at the Golgi in hypersecretion in neuroendocrine cancer cells.

  19. Combination of chondroitinase ABC, glial cell line-derived neurotrophic factor and Nogo A antibody delayed-release microspheres promotes the functional recovery of spinal cord injury.

    Science.gov (United States)

    Zhang, Yu; Gu, Zuchao; Qiu, Guixing; Song, Yueming

    2013-11-01

    Spinal cord injury (SCI) is one of the most devastating injuries for patients. Glial cell line-derived neurotrophic factor (GDNF) is an important neurotrophic factor for the regeneration of the spinal neuraxial bundle, but GDNF would degrade rapidly if the protein was injected into the site of injury; thus, it cannot exert its fullest effects. Therefore, we introduced a delivery system of GDNF, poly(lactide-co-glycolic acid) (PLGA) delayed-release microspheres, in the current study and observed the effect of PLGA-GDNF and the combination of PLGA-GDNF and another 2 agents PLGA-chondroitinase ABC (ChABC) and PLGA-Nogo A antibody in the treatment of SCI rats. Our results showed that PLGA-GDNF and the combination of chABC, GDNF, and Nogo A antibody microspheres could elevate the locomotor scores of SCI rats. The effect of PLGA-GDNF was much better than that of GDNF. The cortical somatosensory evoked potential was also improved by PLGA-GDNF and the combination of chABC, GDNF, and Nogo A antibody microspheres. Our results suggest that PLGA delayed-release microsphere may be a useful and effective tool in delivering protein agents into the injury sites of patients with SCI. This novel combination therapy may provide a new idea in promoting the functional recovery of the damaged spinal cord.

  20. Inhibition of mitochondrial gene transcription suppresses neurotensin secretion in the human carcinoid cell line BON.

    Science.gov (United States)

    Li, Nan; Wang, Qingding; Li, Jing; Wang, Xiaofu; Hellmich, Mark R; Rajaraman, Srinivasan; Greeley, George H; Townsend, Courtney M; Evers, B Mark

    2005-02-01

    Mitochondria, organelles essential for ATP production, play a central role in a number of cellular functions, including the regulation of insulin secretion. Neurotensin (NT), an important regulatory intestinal hormone, has been implicated in fatty acid translocation, gut motility and secretion, and intestinal cell growth; however, mechanisms regulating NT secretion have not been entirely defined. The purpose of this study was to determine the effect of inhibition of mitochondrial gene transcription on NT secretion. BON cells, a novel human carcinoid cell line that produces and secretes NT peptide and expresses the gene encoding NT (designated NT/N), were treated with ethidium bromide (EB; 0.05, 0.1, and 0.4 microg/ml), an inhibitor of DNA and RNA synthesis, or vehicle over a time course (1-4 days). Cells were then stimulated with either ACh (100 microM) or phorbol 12 myristate,13-acetate (PMA, 10 nM) for 30 min. Media and cells were extracted, and NT peptide measured by RIA. Treatment with EB had no effect on BON cell viability or cell cycle distribution over the 4-day course. In contrast, EB treatment produced a dose-dependent reduction of mitochondrial gene expression; however, NT/N gene expression was not altered. Mitochondrial inhibition by EB treatment suppressed NT secretion induced by ACh and PMA, both in a dose-dependent manner. EB-mediated inhibition of NT secretion and mitochondrial gene expression was reversed with removal of EB. Our results demonstrate that inhibition of mitochondrial gene transcription suppresses both ACh- and PMA-stimulated NT release. These findings are the first to demonstrate that mitochondrial function is important for agonist-mediated NT secretion.

  1. Effects of N-acetylcysteine on matrix metalloproteinase-9 secretion and cell migration of human corneal epithelial cells

    OpenAIRE

    Ramaesh, T; Ramaesh, K; Riley, S C; West, J.D.; Dhillon, B

    2012-01-01

    Matrix metalloproteinase-9 (MMP-9) secreted by corneal epithelial cells has a role in the remodelling of extracellular matrix and migration of epithelial cells. Elevated levels of MMP-9 activity in the ocular surface may be involved in the pathogenesis of corneal diseases. N-acetylcysteine (NAC) has been used to treat corneal diseases, including recurrent epithelial erosions. In this study, its effects on the MMP-9 secretion and human corneal epithelial (HCE) cell migration were evaluated in ...

  2. Caveolin-1-Mediated Expression and Secretion of Kallikrein 6 in Colon Cancer Cells

    Directory of Open Access Journals (Sweden)

    Rebecca S. Henkhaus

    2008-02-01

    Full Text Available Kallikreins are secreted proteases that may play a functional role and/or serve as a serum biomarker for the presence or progression of certain types of cancers. Kallikrein 6 (KLK6 has been shown to be upregulated in several types of cancers, including colon. The aims of this study were to elucidate pathways that influence KLK6 gene expression and KLK6 protein secretion in the HCT116 human colon cancer cells. Our data indicate a central role for caveolin-1 (CAV-1, the main structural protein of caveolae, in both KLK6 gene expression and protein secretion. Sucrose gradient subcellular fractionation reveals that CAV-1 and KLK6 colocalize to lipid raft domains in the plasma membrane of HCT116 cells. Furthermore, we show that CAV-1, although it does not directly interact with the KLK6 molecule, enhances KLK6 secretion from the cells. Deactivation of CAV-1, through SRC-mediated phosphorylation, decreased KLK6 secretion. We also demonstrate that, in colon cancer cells, CAV-1 increased the amount of phosphorylated AKT in cells by inhibiting the activity of the AKT-negative regulators PP1 and PP2A. This study demonstrates that proteins such as CAV-1 and AKT, which are known to be altered in colon cancer, affect KLK6 expression and KLK6 secretion.

  3. Human amniotic membrane-derived stromal cells (hAMSC) interact depending on breast cancer cell type through secreted molecules.

    Science.gov (United States)

    Kim, Sun-Hee; Bang, So Hee; Kang, So Yeong; Park, Ki Dae; Eom, Jun Ho; Oh, Il Ung; Yoo, Si Hyung; Kim, Chan-Wha; Baek, Sun Young

    2015-02-01

    Human amniotic membrane-derived stromal cells (hAMSC) are candidates for cell-based therapies. We examined the characteristics of hAMSC including the interaction between hAMSC and breast cancer cells, MCF-7, and MDA-MB-231. Human amniotic membrane-derived stromal cells showed typical MSC properties, including fibroblast-like morphology, surface antigen expression, and mesodermal differentiation. To investigate cell-cell interaction via secreted molecules, we cultured breast cancer cells in hAMSC-conditioned medium (hAMSC-CM) and analyzed their proliferation, migration, and secretome profiles. MCF-7 and MDA-MB-231 cells exposed to hAMSC-CM showed increased proliferation and migration. However, in hAMSC-CM, MCF-7 cells proliferated significantly faster than MDA-MB-231 cells. When cultured in hAMSC-CM, MCF-7 cells migrated faster than MDA-MB-231 cells. Two cell types showed different profiles of secreted factors. MCF-7 cells expressed much amounts of IL-8, GRO, and MCP-1 in hAMSC-CM. Human amniotic membrane-derived stromal cells interact with breast cancer cells through secreted molecules. Factors secreted by hAMSCs promote the proliferation and migration of MCF-7 breast cancer cells. For much safe cell-based therapies using hAMSC, it is necessary to study carefully about interaction between hAMSC and cancer cells.

  4. Trafficking of osteonectin by retinal pigment epithelial cells: evidence for basolateral secretion.

    Science.gov (United States)

    Ratnayaka, Arjuna; Paraoan, Luminita; Nelson, Glyn; Spiller, Dave G; White, Michael R H; Hiscott, Paul

    2007-01-01

    Osteonectin is a glycoprotein that modulates several aspects of cellular behaviour including proliferation and adhesion. The retinal pigment epithelium forms a continuous monolayer of polarised cells immediately bellow the neuroretina, and is integral to the homeostasis of photoreceptor cells. While osteonectin is expressed by normal retinal pigment epithelium in situ, its expression is significantly increased in retinal pigment epithelial cells associated with several common retinal diseases. This pattern of expression implies an important role for osteonectin in the biology of retinal pigment epithelial cells. However, the trafficking, processing, and eventual fate of osteonectin in these cells is not clear at present. Although the theoretical report of a leader sequence within the osteonectin open reading frame and its extracellular presence in some tissues indirectly support secretion of the protein, there is no direct experimental demonstration of the secretion route to date. As a first step towards understanding the role of osteonectin in retinal pigment epithelium, we studied the intracellular distribution and trafficking of the protein in living cells. Here, we present experimental evidence that a precursor osteonectin fusion protein is targeted to the endoplasmic reticulum/Golgi pathway, with a likely basal secretion in retinal pigment epithelial cells. In addition, we show that the precursor osteonectin protein having the leader sequence masked fails to undergo secretion leading to cell death, a phenotype which may be of relevance not only for retinal pathology, but also for other diseases such as the bone disorder known as pseudoachondroplasia that is associated with a lack of osteonectin secretion.

  5. Pleiotropic effects of cancer cells' secreted factors on human stromal (mesenchymal) stem cells

    DEFF Research Database (Denmark)

    Al-toub, Mashael; Almusa, Abdulaziz; Almajed, Mohammed

    2013-01-01

    INTRODUCTION: Studying cancer tumors' microenvironment may reveal a novel role in driving cancer progression and metastasis. The biological interaction between stromal (mesenchymal) stem cells (MSCs) and cancer cells remains incompletely understood. Herein, we investigated the effects of tumor...... cells' secreted factors as represented by a panel of human cancer cell lines (breast (MCF7 and MDA-MB-231); prostate (PC-3); lung (NCI-H522); colon (HT-29) and head & neck (FaDu)) on the biological characteristics of MSCs. METHODS: Morphological changes were assessed using fluorescence microscopy....... Changes in gene expression were assessed using Agilent microarray and qRT-PCR. GeneSpring 12.1 and DAVID tools were used for bioinformatic and signaling pathway analyses. Cell migration was assessed using a transwell migration system. SB-431542, PF-573228 and PD98059 were used to inhibit transforming...

  6. Basement membrane components secreted by mouse yolk sac carcinoma cell lines

    DEFF Research Database (Denmark)

    Damjanov, A; Wewer, U M; Tuma, B

    1990-01-01

    carcinoma respectively. Cell lines NE and ME were composed of a monomorphous cell population; however, the morphology of ME was growth-medium-dependent. LRD was composed of a heterogeneous cell population and formed embryoid bodies. NE secreted soluble laminin, osteonectin, entactin and fibronectin but did...

  7. Microvesicles secreted from equine amniotic cells and their potential role in in vitro cell tendon repair

    Directory of Open Access Journals (Sweden)

    Claudia Perrini

    2015-07-01

    Full Text Available The regenerative mechanisms ascribed to mesenchymal stem cells (MSCs are classified into 3 categories: differentiating into damaged cell types, supplying nutrients, and improving survival/functions of the endogenous cells via paracrine actions. However, because of the inhospitable microenvironment of the injured tissues, a proportion of the implanted MSCs may quickly die, suggesting that other mechanisms might be present. This notion is supported by the overlapping beneficial effect (in terms of time of healing resulted  after the injection of AMCs or of amniotic mesenchymal cells - conditioned medium (AMC-CM  in equine spontaneous injured tendons and ligaments. Microvesicles (MVs released by cells are an integral component of the cell-to-cell communication network involved in tissue regeneration.In the present study, MVs secreted by AMCs were investigated with Nanosigth instrument and TEM. Then, the in vitro incorporation of MVs into equine tendon cells was studied by a dose-response curve. Lastly, the ability of MVs to counteract an in vitro inflammatory process induced by lipolysaccaride on tendon cells was studied evaluating the expression of pro-inflammatory genes like metallopeptidase (MPP 1 and 13, and prostaglandin-endoperoxide synthase 2 (COX2. Results demonstrated that AMCs secreted MVs ranging in size from 100 to 1000 nm with a prevalence of 100-200 nm large MVs. Tendon cells were able to uptake them with an inverse relationship between concentration and time. The greatest incorporation was detectable at 40x106 MVs/ml after 72h. MVs induced down-regulation of MMP1 and MMP13, suggesting that they may have contributed, along with soluble factors, to in vivo tendon regeneration.

  8. Modulation of neurotrophic signaling pathways by polyphenols.

    Science.gov (United States)

    Moosavi, Fatemeh; Hosseini, Razieh; Saso, Luciano; Firuzi, Omidreza

    2016-01-01

    Polyphenols are an important class of phytochemicals, and several lines of evidence have demonstrated their beneficial effects in the context of a number of pathologies including neurodegenerative disorders such as Alzheimer's and Parkinson's disease. In this report, we review the studies on the effects of polyphenols on neuronal survival, growth, proliferation and differentiation, and the signaling pathways involved in these neurotrophic actions. Several polyphenols including flavonoids such as baicalein, daidzein, luteolin, and nobiletin as well as nonflavonoid polyphenols such as auraptene, carnosic acid, curcuminoids, and hydroxycinnamic acid derivatives including caffeic acid phentyl ester enhance neuronal survival and promote neurite outgrowth in vitro, a hallmark of neuronal differentiation. Assessment of underlying mechanisms, especially in PC12 neuronal-like cells, reveals that direct agonistic effect on tropomyosin receptor kinase (Trk) receptors, the main receptors of neurotrophic factors including nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) explains the action of few polyphenols such as 7,8-dihydroxyflavone. However, several other polyphenolic compounds activate extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/Akt pathways. Increased expression of neurotrophic factors in vitro and in vivo is the mechanism of neurotrophic action of flavonoids such as scutellarin, daidzein, genistein, and fisetin, while compounds like apigenin and ferulic acid increase cyclic adenosine monophosphate response element-binding protein (CREB) phosphorylation. Finally, the antioxidant activity of polyphenols reflected in the activation of Nrf2 pathway and the consequent upregulation of detoxification enzymes such as heme oxygenase-1 as well as the contribution of these effects to the neurotrophic activity have also been discussed. In conclusion, a better understanding of the neurotrophic effects of polyphenols and the

  9. Visualization of glucagon secretion from pancreatic α cells by bioluminescence video microscopy: Identification of secretion sites in the intercellular contact regions.

    Science.gov (United States)

    Yokawa, Satoru; Suzuki, Takahiro; Inouye, Satoshi; Inoh, Yoshikazu; Suzuki, Ryo; Kanamori, Takao; Furuno, Tadahide; Hirashima, Naohide

    2017-04-15

    We have firstly visualized glucagon secretion using a method of video-rate bioluminescence imaging. The fusion protein of proglucagon and Gaussia luciferase (PGCG-GLase) was used as a reporter to detect glucagon secretion and was efficiently expressed in mouse pancreatic α cells (αTC1.6) using a preferred human codon-optimized gene. In the culture medium of the cells expressing PGCG-GLase, luminescence activity determined with a luminometer was increased with low glucose stimulation and KCl-induced depolarization, as observed for glucagon secretion. From immunochemical analyses, PGCG-GLase stably expressed in clonal αTC1.6 cells was correctly processed and released by secretory granules. Luminescence signals of the secreted PGCG-GLase from the stable cells were visualized by video-rate bioluminescence microscopy. The video images showed an increase in glucagon secretion from clustered cells in response to stimulation by KCl. The secretory events were observed frequently at the intercellular contact regions. Thus, the localization and frequency of glucagon secretion might be regulated by cell-cell adhesion.

  10. Transplantation of neural stem cells overexpressing glial cell line-derived neurotrophic factor enhances Akt and Erk1/2 signaling and neurogenesis in rats after stroke

    Institute of Scientific and Technical Information of China (English)

    YUAN Miao; WEN Sheng-jun; YANG Chao-xian; PANG Yuan-guang; GAO Xiao-qing; LIU Xiao-qing; HUANG Liang

    2013-01-01

    Background Our previous studies have indicated that the beneficial effects of grafting neural stem cells (NSCs) overexpressing glial cell line-derived neurotrophic factor (GDNF) in rats after stroke.However,the underlying mechanisms are highly debatable.In this study,we investigated whether neurogenesis,Akt,and extracellular signalregulated kinase 1/2 (Erk1/2) signaling were involved in this process.Methods Transient ischemic stroke were induced by occluding middle cerebral artery for 2 hours and reperfusion.At 3 days after reperfusion,GDNF/NSCs,NSCs,and vehicle were administered.Immunohistochemical staining was used to evaluate neurogenesis by nestin antibody; phosphorylation of Akt and Erk1/2 was investigated by Western blotting analysis.Results Transplantation of GDNF/NSCs and NSCs significantly increased nestin-positive cells compared to control group (vehicle) from 1 to 7 weeks after reperfusion,and GDNF/NSCs showed stronger effect than NSCs at 2 and 3 weeks after reperfusion.Meanwhile,enhanced phosphorylation level of Erk1/2 was observed in the GDNF/NSCs and NSCs groups compared with control group,and phosphorylation level of Erk1/2 in GDNF/NSCs group was remarkably higher than that of NSCs group at any given time.In contrast,expression of mitogen-activated protein kinase phosphatase-1 (MKP-1),known as inhibitor of Erk1/2 signaling,was significantly decreased in the GDNF/NSCs and NSCs groups compared with the control group.Moreover,much enhanced and prolonged phosphorylation level of Akt of GDNF/NSCs group was detected compared with control and NSCs group.Conclusion Grafting GDNF/NSCs enhances neurogenesis and activates Akt and Erk1/2 signaling,that may provide the potential for GDNF/NSCs in stroke treatment.

  11. Vacuolar ATPase regulates surfactant secretion in rat alveolar type II cells by modulating lamellar body calcium.

    Directory of Open Access Journals (Sweden)

    Narendranath Reddy Chintagari

    Full Text Available Lung surfactant reduces surface tension and maintains the stability of alveoli. How surfactant is released from alveolar epithelial type II cells is not fully understood. Vacuolar ATPase (V-ATPase is the enzyme responsible for pumping H(+ into lamellar bodies and is required for the processing of surfactant proteins and the packaging of surfactant lipids. However, its role in lung surfactant secretion is unknown. Proteomic analysis revealed that vacuolar ATPase (V-ATPase dominated the alveolar type II cell lipid raft proteome. Western blotting confirmed the association of V-ATPase a1 and B1/2 subunits with lipid rafts and their enrichment in lamellar bodies. The dissipation of lamellar body pH gradient by Bafilomycin A1 (Baf A1, an inhibitor of V-ATPase, increased surfactant secretion. Baf A1-stimulated secretion was blocked by the intracellular Ca(2+ chelator, BAPTA-AM, the protein kinase C (PKC inhibitor, staurosporine, and the Ca(2+/calmodulin-dependent protein kinase II (CaMKII, KN-62. Baf A1 induced Ca(2+ release from isolated lamellar bodies. Thapsigargin reduced the Baf A1-induced secretion, indicating cross-talk between lamellar body and endoplasmic reticulum Ca(2+ pools. Stimulation of type II cells with surfactant secretagogues dissipated the pH gradient across lamellar bodies and disassembled the V-ATPase complex, indicating the physiological relevance of the V-ATPase-mediated surfactant secretion. Finally, silencing of V-ATPase a1 and B2 subunits decreased stimulated surfactant secretion, indicating that these subunits were crucial for surfactant secretion. We conclude that V-ATPase regulates surfactant secretion via an increased Ca(2+ mobilization from lamellar bodies and endoplasmic reticulum, and the activation of PKC and CaMKII. Our finding revealed a previously unrealized role of V-ATPase in surfactant secretion.

  12. Deconstructing brain-derived neurotrophic factor actions in adult brain circuits to bridge an existing informational gap in neuro-cell biology

    Institute of Scientific and Technical Information of China (English)

    Heather Bowling; Aditi Bhattacharya; Eric Klann; Moses V Chao

    2016-01-01

    Brain-derived neurotrophic factor (BDNF) plays an important role in neurodevelopment, synaptic plas-ticity, learning and memory, and in preventing neurodegeneration. Despite decades of investigations into downstream signaling cascades and changes in cellular processes, the mechanisms of how BDNF reshapes circuitsin vivo remain unclear. This informational gap partly arises from the fact that the bulk of studies into the molecular actions of BDNF have been performed in dissociated neuronal cultures, while the ma-jority of studies on synaptic plasticity, learning and memory were performed in acute brain slices orin vivo. A recent study by Bowling-Bhattacharya et al., measured the proteomic changes in acute adult hippocampal slices following treatment and reported changes in proteins of neuronal and non-neuronal origin that may in concert modulate synaptic release and secretion in the slice. In this paper, we place these ifndings into the context of existing literature and discuss how they impact our understanding of how BDNF can reshape the brain.

  13. HBpF-proBDNF: A New Tool for the Analysis of Pro-Brain Derived Neurotrophic Factor Receptor Signaling and Cell Biology

    Science.gov (United States)

    Gaub, Perrine; de Léon, Andrès; Gibon, Julien; Soubannier, Vincent; Dorval, Geneviève; Séguéla, Philippe; Barker, Philip A.

    2016-01-01

    Neurotrophins activate intracellular signaling pathways necessary for neuronal survival, growth and apoptosis. The most abundant neurotrophin in the adult brain, brain-derived neurotrophic factor (BDNF), is first synthesized as a proBDNF precursor and recent studies have demonstrated that proBDNF can be secreted and that it functions as a ligand for a receptor complex containing p75NTR and sortilin. Activation of proBDNF receptors mediates growth cone collapse, reduces synaptic activity, and facilitates developmental apoptosis of motoneurons but the precise signaling cascades have been difficult to discern. To address this, we have engineered, expressed and purified HBpF-proBDNF, an expression construct containing a 6X-HIS tag, a biotin acceptor peptide (BAP) sequence, a PreScission™ Protease cleavage site and a FLAG-tag attached to the N-terminal part of murine proBDNF. Intact HBpF-proBDNF has activities indistinguishable from its wild-type counterpart and can be used to purify proBDNF signaling complexes or to monitor proBDNF endocytosis and retrograde transport. HBpF-proBDNF will be useful for characterizing proBDNF signaling complexes and for deciphering the role of proBDNF in neuronal development, synapse function and neurodegenerative disease. PMID:26950209

  14. Deconstructing brain-derived neurotrophic factor actions in adult brain circuits to bridge an existing informational gap in neuro-cell biology

    Directory of Open Access Journals (Sweden)

    Heather Bowling

    2016-01-01

    Full Text Available Brain-derived neurotrophic factor (BDNF plays an important role in neurodevelopment, synaptic plasticity, learning and memory, and in preventing neurodegeneration. Despite decades of investigations into downstream signaling cascades and changes in cellular processes, the mechanisms of how BDNF reshapes circuits in vivo remain unclear. This informational gap partly arises from the fact that the bulk of studies into the molecular actions of BDNF have been performed in dissociated neuronal cultures, while the majority of studies on synaptic plasticity, learning and memory were performed in acute brain slices or in vivo. A recent study by Bowling-Bhattacharya et al., measured the proteomic changes in acute adult hippocampal slices following treatment and reported changes in proteins of neuronal and non-neuronal origin that may in concert modulate synaptic release and secretion in the slice. In this paper, we place these findings into the context of existing literature and discuss how they impact our understanding of how BDNF can reshape the brain.

  15. High LIN28A Expressing Ovarian Cancer Cells Secrete Exosomes That Induce Invasion and Migration in HEK293 Cells.

    Science.gov (United States)

    Enriquez, Vanessa A; Cleys, Ellane R; Da Silveira, Juliano C; Spillman, Monique A; Winger, Quinton A; Bouma, Gerrit J

    2015-01-01

    Epithelial ovarian cancer is the most aggressive and deadly form of ovarian cancer and is the most lethal gynecological malignancy worldwide; therefore, efforts to elucidate the molecular factors that lead to epithelial ovarian cancer are essential to better understand this disease. Recent studies reveal that tumor cells release cell-secreted vesicles called exosomes and these exosomes can transfer RNAs and miRNAs to distant sites, leading to cell transformation and tumor development. The RNA-binding protein LIN28 is a known marker of stem cells and when expressed in cancer, it is associated with poor tumor outcome. We hypothesized that high LIN28 expressing ovarian cancer cells secrete exosomes that can be taken up by nontumor cells and cause changes in gene expression and cell behavior associated with tumor development. IGROV1 cells were found to contain high LIN28A and secrete exosomes that were taken up by HEK293 cells. Moreover, exposure to these IGROV1 secreted exosomes led to significant increases in genes involved in Epithelial-to-Mesenchymal Transition (EMT), induced HEK293 cell invasion and migration. These changes were not observed with exosomes secreted by OV420 cells, which contain no detectable amounts of LIN28A or LIN28B. No evidence was found of LIN28A transfer from IGROV1 exosomes to HEK293 cells.

  16. Interactions between Trypanosoma cruzi secreted proteins and host cell signaling pathways

    Directory of Open Access Journals (Sweden)

    Renata Watanabe Costa

    2016-03-01

    Full Text Available Chagas disease is one of the prevalent neglected tropical diseases, affecting at least 6-7 million individuals in Latin America. It is caused by the protozoan parasite Trypanosoma cruzi (T. cruzi, which is transmitted to vertebrate hosts by blood-sucking insects. After infection, the parasite invades and multiplies in the myocardium, leading to acute myocarditis that kills around 5% of untreated individuals. T. cruzi secretes proteins that manipulate multiple host cell signaling pathways to promote host cell invasion. The primary secreted lysosomal peptidase in T. cruzi is cruzipain, which has been shown to modulate the host immune response. Cruzipain hinders macrophage activation during the early stages of infection by interrupting the NF-kB P65 mediated signaling pathway. This allows the parasite to survive and replicate, and may contribute to the spread of infection in acute Chagas disease. Another secreted protein P21, which is expressed in all of the developmental stages of T. cruzi, has been shown to modulate host phagocytosis signaling pathways. The parasite also secretes soluble factors that exert effects on host extracellular matrix, such as proteolytic degradation of collagens. Finally, secreted phospholipase A from T. cruzi contributes to lipid modifications on host cells and concomitantly activates the PKC signaling pathway. Here we present a brief review of the interaction between secreted proteins from T. cruzi and the host cells, emphasizing the manipulation of host signaling pathways during invasion.

  17. Interactions between Trypanosoma cruzi Secreted Proteins and Host Cell Signaling Pathways

    Science.gov (United States)

    Watanabe Costa, Renata; da Silveira, Jose F.; Bahia, Diana

    2016-01-01

    Chagas disease is one of the prevalent neglected tropical diseases, affecting at least 6–7 million individuals in Latin America. It is caused by the protozoan parasite Trypanosoma cruzi, which is transmitted to vertebrate hosts by blood-sucking insects. After infection, the parasite invades and multiplies in the myocardium, leading to acute myocarditis that kills around 5% of untreated individuals. T. cruzi secretes proteins that manipulate multiple host cell signaling pathways to promote host cell invasion. The primary secreted lysosomal peptidase in T. cruzi is cruzipain, which has been shown to modulate the host immune response. Cruzipain hinders macrophage activation during the early stages of infection by interrupting the NF-kB P65 mediated signaling pathway. This allows the parasite to survive and replicate, and may contribute to the spread of infection in acute Chagas disease. Another secreted protein P21, which is expressed in all of the developmental stages of T. cruzi, has been shown to modulate host phagocytosis signaling pathways. The parasite also secretes soluble factors that exert effects on host extracellular matrix, such as proteolytic degradation of collagens. Finally, secreted phospholipase A from T. cruzi contributes to lipid modifications on host cells and concomitantly activates the PKC signaling pathway. Here, we present a brief review of the interaction between secreted proteins from T. cruzi and the host cells, emphasizing the manipulation of host signaling pathways during invasion. PMID:27065960

  18. Ultrastructural Evidence of Exosome Secretion by Progenitor Cells in Adult Mouse Myocardium and Adult Human Cardiospheres

    Directory of Open Access Journals (Sweden)

    Lucio Barile

    2012-01-01

    Full Text Available The demonstration of beneficial effects of cell therapy despite the persistence of only few transplanted cells in vivo suggests secreted factors may be the active component of this treatment. This so-called paracrine hypothesis is supported by observations that culture media conditioned by progenitor cells contain growth factors that mediate proangiogenic and cytoprotective effects. Cardiac progenitor cells in semi-suspension culture form spherical clusters (cardiospheres that deliver paracrine signals to neighboring cells. A key component of paracrine secretion is exosomes, membrane vesicles that are stored intracellularly in endosomal compartments and are secreted when these structures fuse with the cell plasma membrane. Exosomes have been identified as the active component of proangiogenic effects of bone marrow CD34+ stem cells in mice and the regenerative effects of embryonic mesenchymal stem cells in infarcted hearts in pigs and mice. Here, we provide electron microscopic evidence of exosome secretion by progenitor cells in mouse myocardium and human cardiospheres. Exosomes are emerging as an attractive vector of paracrine signals delivered by progenitor cells. They can be stored as an “off-the-shelf” product. As such, exosomes have the potential for circumventing many of the limitations of viable cells for therapeutic applications in regenerative medicine.

  19. Localization of brain-derived neurotrophic factor to distinct terminals of mossy fiber axons implies regulation of both excitation and feedforward inhibition of CA3 pyramidal cells.

    Science.gov (United States)

    Danzer, Steve C; McNamara, James O

    2004-12-15

    Hippocampal dentate granule cells directly excite and indirectly inhibit CA3 pyramidal cells via distinct presynaptic terminal specializations of their mossy fiber axons. This mossy fiber pathway contains the highest concentration of brain-derived neurotrophic factor (BDNF) in the CNS, yet whether BDNF is positioned to regulate the excitatory and/or inhibitory pathways is unknown. To localize BDNF, confocal microscopy of green fluorescent protein transgenic mice was combined with BDNF immunohistochemistry. Approximately half of presynaptic granule cell-CA3 pyramidal cell contacts were found to contain BDNF. Moreover, enhanced neuronal activity virtually doubled the percentage of BDNF-immunoreactive terminals contacting CA3 pyramidal cells. To our surprise, BDNF was also found in mossy fiber terminals contacting inhibitory neurons. These studies demonstrate that mossy fiber BDNF is poised to regulate both direct excitatory and indirect feedforward inhibitory inputs to CA3 pyramdal cells and reveal that seizure activity increases the pool of BDNF-expressing granule cell presynaptic terminals contacting CA3 pyramidal cells.

  20. Lipid Rafts Are Physiologic Membrane Microdomains Necessary for the Morphogenic and Developmental Functions of Glial Cell Line-Derived Neurotrophic Factor In Vivo.

    Science.gov (United States)

    Tsui, Cynthia C; Gabreski, Nicole A; Hein, Sarah J; Pierchala, Brian A

    2015-09-23

    Glial cell line-derived neurotrophic factor (GDNF) promotes PNS development and kidney morphogenesis via a receptor complex consisting of the glycerophosphatidylinositol (GPI)-anchored, ligand binding receptor GDNF family receptor α1 (GFRα1) and the receptor tyrosine kinase Ret. Although Ret signal transduction in vitro is augmented by translocation into lipid rafts via GFRα1, the existence and importance of lipid rafts in GDNF-Ret signaling under physiologic conditions is unresolved. A knock-in mouse was produced that replaced GFRα1 with GFRα1-TM, which contains a transmembrane (TM) domain instead of the GPI anchor. GFRα1-TM still binds GDNF and promotes Ret activation but does not translocate into rafts. In Gfrα1(TM/TM) mice, GFRα1-TM is expressed, trafficked, and processed at levels identical to GFRα1. Although Gfrα1(+/TM) mice are viable, Gfrα1(TM/TM) mice display bilateral renal agenesis, lack enteric neurons in the intestines, and have motor axon guidance deficits, similar to Gfrα1(-/-) mice. Therefore, the recruitment of Ret into lipid rafts by GFRα1 is required for the physiologic functions of GDNF in vertebrates. Significance statement: Membrane microdomains known as lipid rafts have been proposed to be unique subdomains in the plasma membrane that are critical for the signaling functions of multiple receptor complexes. Their existence and physiologic relevance has been debated. Based on in vitro studies, lipid rafts have been reported to be necessary for the function of the Glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors. The receptor for GDNF comprises the lipid raft-resident, glycerophosphatidylinositol-anchored receptor GDNF family receptor α1 (GFRα1) and the receptor tyrosine kinase Ret. Here we demonstrate, using a knock-in mouse model in which GFRα1 is no longer located in lipid rafts, that the developmental functions of GDNF in the periphery require the translocation of the GDNF receptor complex

  1. Syntaxin-4 is essential for IgE secretion by plasma cells

    Energy Technology Data Exchange (ETDEWEB)

    Rahman, Arman; DeCourcey, Joseph; Larbi, Nadia Ben [Immunomodulation Group, School of Biotechnology, Dublin City University (Ireland); Loughran, Sinéad T.; Walls, Dermot [School of Biotechnology and National Centre for Sensor Research, Dublin City University (Ireland); Loscher, Christine E., E-mail: christine.loscher@dcu.ie [Immunomodulation Group, School of Biotechnology, Dublin City University (Ireland)

    2013-10-11

    Highlights: •Knock-down of syntaxin-4 in U266 plasma cells resulted in reduction of IgE secretion. •Knock-down of syntaxin-4 also leads to the accumulation of IgE in the cell. •Immuno-fluorescence staining shows co-localisation of IgE and syntaxin-4 in U266 cells. •Findings suggest a critical requirement for syntaxin-4 in IgE secretion from plasma cells. -- Abstract: The humoral immune system provides a crucial first defense against the invasion of microbial pathogens via the secretion of antigen specific immunoglobulins (Ig). The secretion of Ig is carried out by terminally differentiated B-lymphocytes called plasma cells. Despite the key role of plasma cells in the immune response, the mechanisms by which they constitutively traffic large volumes of Ig out of the cell is poorly understood. The involvement of Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in the regulation of protein trafficking from cells has been well documented. Syntaxin-4, a member of the Qa SNARE syntaxin family has been implicated in fusion events at the plasma membrane in a number of cells in the immune system. In this work we show that knock-down of syntaxin-4 in the multiple myeloma U266 human plasma cell line results in a loss of IgE secretion and accumulation of IgE within the cells. Furthermore, we show that IgE co-localises with syntaxin-4 in U266 plasma cells suggesting direct involvement in secretion at the plasma membrane. This study demonstrates that syntaxin-4 plays a critical role in the secretion of IgE from plasma cells and sheds some light on the mechanisms by which these cells constitutively traffic vesicles to the surface for secretion. An understanding of this machinery may be beneficial in identifying potential therapeutic targets in multiple myeloma and autoimmune disease where over-production of Ig leads to severe pathology in patients.

  2. Bone morphogenetic protein-4 enhances vascular endothelial growth factor secretion by human retinal pigment epithelial cells.

    Science.gov (United States)

    Vogt, Rhonda R; Unda, Richard; Yeh, Lee-Chuan C; Vidro, Eileen K; Lee, John C; Tsin, Andrew T

    2006-08-01

    Retinal pigment epithelial (RPE) cells secrete vascular endothelial growth factor (VEGF), a cytokine known to promote angiogenesis. Results from RNase protection assays (RPAs) show that RPE from non-diabetic human donors and from adult retinal pigment epithelium-19 (ARPE-19) cells expressed significant bone morphogenetic protein-4 (BMP-4) message. In addition, ARPE-19 cells cultured in high glucose (25 mM), compared to those in physiological glucose (5.5 mM) released significantly more BMP-4 into the conditioned media (CM). However, the effect of BMP-4 on the release of VEGF by ARPE-19 cells has not been studied. Accordingly, ARPE-19 cells were treated with BMP-4 to determine VEGF secretion. BMP-4 and VEGF levels in the CM and cell lysates were measured by enzyme-linked immunosorbent assay (ELISA). Cells treated with exogenous BMP-4 had higher VEGF in the CM and this treatment effect was dose- and time-dependent, while cell lysates had low levels of VEGF. Addition of cycloheximide (CHX) or actinomycin-D (ACT) significantly reduced VEGF secretion from cells treated with BMP-4, suggesting that the BMP-4-induced secretion of VEGF requires new RNA and protein synthesis. Our results suggest that BMP-4 may play a role in the regulation of ocular angiogenesis associated with diabetic retinopathy (DR) by stimulating VEGF release from RPE cells.

  3. Knockdown of flotillin-2 inhibits lung surfactant secretion by alveolar type Ⅱ cells

    Institute of Scientific and Technical Information of China (English)

    Narendranath Reddy Chintagari; Deming Gou; Lin Liu

    2008-01-01

    @@ Dear Editor, Lung surfactant is stored in lamellar bodies and exocytosed following fusion of the lamellar bodies with the plasma membrane of alveolar type Ⅱ (AT2) cells [1].A number of proteins have been shown to be involved in surfactant secretion including SNAREs,NSF,α-SNAP and annexin A2 [2,3].Lipid rafts enriched in SNAREs are crucial for surfactant secretion [4].

  4. Analysis of in vitro secretion profiles from adipose-derived cell populations

    Directory of Open Access Journals (Sweden)

    Blaber Sinead P

    2012-08-01

    Full Text Available Abstract Background Adipose tissue is an attractive source of cells for therapeutic purposes because of the ease of harvest and the high frequency of mesenchymal stem cells (MSCs. Whilst it is clear that MSCs have significant therapeutic potential via their ability to secrete immuno-modulatory and trophic cytokines, the therapeutic use of mixed cell populations from the adipose stromal vascular fraction (SVF is becoming increasingly common. Methods In this study we have measured a panel of 27 cytokines and growth factors secreted by various combinations of human adipose-derived cell populations. These were 1. co-culture of freshly isolated SVF with adipocytes, 2. freshly isolated SVF cultured alone, 3. freshly isolated adipocytes alone and 4. adherent adipose-derived mesenchymal stem cells (ADSCs at passage 2. In addition, we produced an ‘in silico’ dataset by combining the individual secretion profiles obtained from culturing the SVF with that of the adipocytes. This was compared to the secretion profile of co-cultured SVF and adipocytes. Two-tailed t-tests were performed on the secretion profiles obtained from the SVF, adipocytes, ADSCs and the ‘in silico’ dataset and compared to the secretion profiles obtained from the co-culture of the SVF with adipocytes. A p-value of  Results A co-culture of SVF and adipocytes results in a distinct secretion profile when compared to all other adipose-derived cell populations studied. This illustrates that cellular crosstalk during co-culture of the SVF with adipocytes modulates the production of cytokines by one or more cell types. No biologically relevant differences were detected in the proteomes of SVF cultured alone or co-cultured with adipocytes. Conclusions The use of mixed adipose cell populations does not appear to induce cellular stress and results in enhanced secretion profiles. Given the importance of secreted cytokines in cell therapy, the use of a mixed cell population such as the

  5. A combination of chondroitinase ABC, glial cell line-derived neurotrophic factor, and Nogo A antibody delayed-release microspheres for the treatment of spinal cord injury

    Institute of Scientific and Technical Information of China (English)

    Yu Zhang; Yueming Song

    2011-01-01

    The purpose of this study was to evaluate the effect of poly(lactide-co-glycolic acid) delayed-release microspheres, which were prepared using glial cell line-derived neurotrophic factor (GDNF), on the delayed-release, controllability, and protection of GDNF activity. The present study is the first to combine chondroitinase ABC, GDNF, and Nogo A antibody delayed-release microspheres for the treatment of spinal cord injury. Results show that the combined therapy of chondroitinase ABC,GDNF, and Nogo A antibody microspheres can increase the immunoreaction of neurofilament 200in the injured spinal cord, and this therapeutic effect was better than chondroitinase ABC, GDNF, or Nogo A antibody microspheres administered singularly.

  6. Glial cell line-derived neurotrophic factor up-regulates GTP-cyclohydrolase I activity and tetrahydrobiopterin levels in primary dopaminergic neurones

    DEFF Research Database (Denmark)

    Bauer, M; Suppmann, S; Meyer, M;

    2002-01-01

    Glial cell line-derived neurotrophic factor (GDNF) protects dopaminergic neurones against toxic and physical damage. In addition, GDNF promotes differentiation and structural integrity of dopaminergic neurones. Here we show that GDNF can support the function of primary dopaminergic neurones...... by triggering activation of GTP-cyclohydrolase I (GTPCH I), a key enzyme in catecholamine biosynthesis. GDNF stimulation of primary dopaminergic neurones expressing both tyrosine 3-monooxygenase and GTPCH I resulted in a dose-dependent doubling of GTPCH I activity, and a concomitant increase...... in tetrahydrobiopterin levels whereas tyrosine 3-monooxygenase activity was not altered. Actinomycin D, asan inhibitor of de novo biosynthesis, abolished any GDNF-mediated up-regulation of GTPCH I activity. However, GTPCH I mRNA levels in primary dopaminergic neurones were not altered by GDNF treatment, suggesting...

  7. Brain-derived neurotrophic factor is produced by skeletal muscle cells in response to contraction and enhances fat oxidation via activation of AMP-activated protein kinase

    DEFF Research Database (Denmark)

    Matthews, V B; Åström, Maj-Brit; Chan, M H S

    2009-01-01

    C12 skeletal muscle cells were electrically stimulated to mimic contraction. L6 myotubes and isolated rat extensor digitorum longus muscles were treated with BDNF and phosphorylation of the proteins AMP-activated protein kinase (AMPK) (Thr(172)) and acetyl coenzyme A carboxylase beta (ACCbeta) (Ser...... kinase (p44/42 Thr(202)/Tyr(204)) phosphorylation in these muscles. In addition, phosphorylation of ACCbeta was markedly elevated in the Bdnf electroporated muscles. CONCLUSIONS/INTERPRETATION: These data identify BDNF as a contraction-inducible protein in skeletal muscle that is capable of enhancing......AIMS/HYPOTHESIS: Brain-derived neurotrophic factor (BDNF) is produced in skeletal muscle, but its functional significance is unknown. We aimed to determine the signalling processes and metabolic actions of BDNF. METHODS: We first examined whether exercise induced BDNF expression in humans. Next, C2...

  8. Brain-derived neurotrophic factor inhibits osmotic swelling of rat retinal glial (Müller) and bipolar cells by activation of basic fibroblast growth factor signaling.

    Science.gov (United States)

    Berk, B-A; Vogler, S; Pannicke, T; Kuhrt, H; Garcia, T B; Wiedemann, P; Reichenbach, A; Seeger, J; Bringmann, A

    2015-06-04

    Water accumulation in retinal glial (Müller) and neuronal cells resulting in cellular swelling contributes to the development of retinal edema and neurodegeneration. Intravitreal administration of neurotrophins such as brain-derived neurotrophic factor (BDNF) is known to promote survival of retinal neurons. Here, we show that exogenous BDNF inhibits the osmotic swelling of Müller cell somata induced by superfusion of rat retinal slices or freshly isolated cells with a hypoosmotic solution containing barium ions. BDNF also inhibited the osmotic swelling of bipolar cell somata in retinal slices, but failed to inhibit the osmotic soma swelling of freshly isolated bipolar cells. The inhibitory effect of BDNF on Müller cell swelling was mediated by activation of tropomyosin-related kinase B (TrkB) and transactivation of fibroblast growth factor receptors. Exogenous basic fibroblast growth factor (bFGF) fully inhibited the osmotic swelling of Müller cell somata while it partially inhibited the osmotic swelling of bipolar cell somata. Isolated Müller cells displayed immunoreactivity of truncated TrkB, but not full-length TrkB. Isolated rod bipolar cells displayed immunoreactivities of both TrkB isoforms. Data suggest that the neuroprotective effect of exogenous BDNF in the retina is in part mediated by prevention of the cytotoxic swelling of retinal glial and bipolar cells. While BDNF directly acts on Müller cells by activation of TrkB, BDNF indirectly acts on bipolar cells by inducing glial release of factors like bFGF that inhibit bipolar cell swelling.

  9. The Vibrio parahaemolyticus Type III Secretion Systems manipulate host cell MAPK for critical steps in pathogenesis.

    LENUS (Irish Health Repository)

    Matlawska-Wasowska, Ksenia

    2010-12-01

    Vibrio parahaemolyticus is a food-borne pathogen causing inflammation of the gastrointestinal epithelium. Pathogenic strains of this bacterium possess two Type III Secretion Systems (TTSS) that deliver effector proteins into host cells. In order to better understand human host cell responses to V. parahaemolyticus, the modulation of Mitogen Activated Protein Kinase (MAPK) activation in epithelial cells by an O3:K6 clinical isolate, RIMD2210633, was investigated. The importance of MAPK activation for the ability of the bacterium to be cytotoxic and to induce secretion of Interleukin-8 (IL-8) was determined.

  10. Influence of elevated body temperature on circulating immunoglobulin-secreting cells

    DEFF Research Database (Denmark)

    Kappel, M; Barington, T; Gyhrs, A;

    1995-01-01

    . On another occasion they served as their own controls, being immersed into thermoneutral water (water temperature 34.5 degrees C) for 2 h. Blood samples were drawn before immersion, at body temperatures of 38, 39 and 39.5 degrees C, as well as 2 h after WI when their body temperatures were normalized....... In the control experiments, blood samples were drawn at identical time points. A significant increase in the number of IgM-secreting cells per fixed number of blood mononuclear cells (BMNC) occurred 2 h after WI, whereas the number of IgA-secreting cells per fixed number of BMNC did not change. When the possible...

  11. Evaluation of insulin expression and secretion in genetically engineered gut K and L-cells

    Directory of Open Access Journals (Sweden)

    Ahmad Zalinah

    2012-09-01

    Full Text Available Abstract Background Gene therapy could provide an effective treatment of diabetes. Previous studies have investigated the potential for several cell and tissue types to produce mature and active insulin. Gut K and L-cells could be potential candidate hosts for gene therapy because of their special features. Results In this study, we isolated gut K and L-cells to compare the potential of both cell types to produce insulin when exposed to similar conditions. The isolated pure K and L-cells were transfected with recombinant plasmids encoding insulin and with specific promoters for K or L-cells. Insulin expression was studied in response to glucose or meat hydrolysate. We found that glucose and meat hydrolysate efficiently induced insulin secretion from K and L-cells. However, the effects of meat hydrolysate on insulin secretion were more potent in both cells compared with glucose. Results of enzyme-linked immunosorbent assays showed that L-cells secreted more insulin compared with K-cells regardless of the stimulator, although this difference was not statistically significant. Conclusion The responses of K and L-cells to stimulation with glucose or meat hydrolysate were generally comparable. Therefore, both K and L-cells show similar potential to be used as surrogate cells for insulin gene expression in vitro. The potential use of these cells for diabetic gene therapy warrants further investigation.

  12. The F309S mutation increases factor VIII secretion in human cell line

    Directory of Open Access Journals (Sweden)

    Daianne Maciely Carvalho Fantacini

    2016-06-01

    Full Text Available ABSTRACT OBJECTIVES: The capacity of a human cell line to secrete recombinant factor VIII with a F309S point mutation was investigated, as was the effect of the addition of chemical chaperones (betaine and sodium-4-phenylbutyrate on the secretion of factor VIII. METHODS: This work used a vector with a F309S mutation in the A1 domain to investigate FVIII production in the HEK 293 human cell line. Factor VIII activity was measured by chromogenic assay. Furthermore, the effects of chemical drugs on the culture were evaluated. RESULTS: The addition of the F309S mutation to a previously described FVIII variant increased FVIII secretion by 4.5 fold. Moreover, the addition of betaine or sodium-4-phenylbutyrate increased the secretion rate of FVIIIΔB proteins in HEK 293 cells, but the same effect was not seen for FVIIIΔB-F309S indicating that all the recombinant protein produced had been efficiently secreted. CONCLUSION: Bioengineering factor VIII expressed in human cells may lead to an efficient production of recombinant factor VIII and contribute toward low-cost coagulation factor replacement therapy for hemophilia A. FVIII-F309S produced in human cells can be effective in vivo.

  13. Ca²⁺ signaling and regulation of fluid secretion in salivary gland acinar cells.

    Science.gov (United States)

    Ambudkar, Indu S

    2014-06-01

    Neurotransmitter stimulation of plasma membrane receptors stimulates salivary gland fluid secretion via a complex process that is determined by coordinated temporal and spatial regulation of several Ca(2+) signaling processes as well as ion flux systems. Studies over the past four decades have demonstrated that Ca(2+) is a critical factor in the control of salivary gland function. Importantly, critical components of this process have now been identified, including plasma membrane receptors, calcium channels, and regulatory proteins. The key event in activation of fluid secretion is an increase in intracellular [Ca(2+)] ([Ca(2+)]i) triggered by IP3-induced release of Ca(2+) from ER via the IP3R. This increase regulates the ion fluxes required to drive vectorial fluid secretion. IP3Rs determine the site of initiation and the pattern of [Ca(2+)]i signal in the cell. However, Ca(2+) entry into the cell is required to sustain the elevation of [Ca(2+)]i and fluid secretion. This Ca(2+) influx pathway, store-operated calcium influx pathway (SOCE), has been studied in great detail and the regulatory mechanisms as well as key molecular components have now been identified. Orai1, TRPC1, and STIM1 are critical components of SOCE and among these, Ca(2+) entry via TRPC1 is a major determinant of fluid secretion. The receptor-evoked Ca(2+) signal in salivary gland acinar cells is unique in that it starts at the apical pole and then rapidly increases across the cell. The basis for the polarized Ca(2+) signal can be ascribed to the polarized arrangement of the Ca(2+) channels, transporters, and signaling proteins. Distinct localization of these proteins in the cell suggests compartmentalization of Ca(2+) signals during regulation of fluid secretion. This chapter will discuss new concepts and findings regarding the polarization and control of Ca(2+) signals in the regulation of fluid secretion.

  14. Signaling of glial cell line-derived neurotrophic factor and its receptor GFRα1 induce Nurr1 and Pitx3 to promote survival of grafted midbrain-derived neural stem cells in a rat model of Parkinson disease.

    Science.gov (United States)

    Lei, Zhinian; Jiang, Yu; Li, Tao; Zhu, Jianbao; Zeng, Shuilin

    2011-09-01

    Glial cell line-derived neurotrophic factor (GDNF) and its receptor GFRα1 have been implicated in the survival of ventral midbrain dopaminergic (DA) neurons, but the molecular mechanisms bywhich GDNF generates DA neurons in grafted midbrain-derived neural stem cells (mNSCs) are not understood. Midbrain-derived neural stem cells isolated from rat embryonic mesencephalon (embryonic day 12) were treated with GDNF or in combination with GFRα1 small interfering RNA. Reverse transcription-polymerase chain reaction, Western blot, and immunocytochemistry were used totest the expression of the orphan nuclear receptor Nurr1 and thetranscription factor Pitx3 and newborn tyrosine hydroxylase (TH)-positive cells. Treatment of mNSCs with GDNF increased mNSCs' sphere diameter, reduced expression of caspase 3, and increased expression of Bcl-2. Glial cell line-derived neurotrophic factor-treated mNSCs enhanced Nurr1 and Pitx3 expression and the fraction of TH-, TH/Pitx3-, and TH/Nurr1-positive cells in culture. Grafted GDNF-treated mNSCs significantly decreased apomorphine-induced rotation behavior in 6-hydroxydopamine-lesioned rats. Glialcell line-derived neurotrophic factor-treated mNSCs showed increased numbers of TH/Pitx3- and TH/Nurr1-postivie cells. The effect elicited by GDNF was inhibited by small interfering RNA-mediated knockdown of GFRα1. Our data demonstrate the contribution of GDNF to DA neuron development and may also elucidate pathogenetic mechanisms in Parkinson disease and contribute to the development of novel therapies for the disorder.

  15. Molecular mechanisms of lipoapoptosis and metformin protection in GLP-1 secreting cells

    DEFF Research Database (Denmark)

    Kappe, Camilla; Holst, Jens Juul; Zhang, Qimin

    2012-01-01

    Evidence is emerging that elevated serum free fatty acids (hyperlipidemia) contribute to the pathogenesis of type-2-diabetes, and lipotoxicity is observed in many cell types. We recently published data indicating lipotoxic effects of simulated hyperlipidemia also in GLP-1-secreting cells, where t...

  16. p16(Ink4a)-induced senescence of pancreatic beta cells enhances insulin secretion.

    Science.gov (United States)

    Helman, Aharon; Klochendler, Agnes; Azazmeh, Narmen; Gabai, Yael; Horwitz, Elad; Anzi, Shira; Swisa, Avital; Condiotti, Reba; Granit, Roy Z; Nevo, Yuval; Fixler, Yaakov; Shreibman, Dorin; Zamir, Amit; Tornovsky-Babeay, Sharona; Dai, Chunhua; Glaser, Benjamin; Powers, Alvin C; Shapiro, A M James; Magnuson, Mark A; Dor, Yuval; Ben-Porath, Ittai

    2016-04-01

    Cellular senescence is thought to contribute to age-associated deterioration of tissue physiology. The senescence effector p16(Ink4a) is expressed in pancreatic beta cells during aging and limits their proliferative potential; however, its effects on beta cell function are poorly characterized. We found that beta cell-specific activation of p16(Ink4a) in transgenic mice enhances glucose-stimulated insulin secretion (GSIS). In mice with diabetes, this leads to improved glucose homeostasis, providing an unexpected functional benefit. Expression of p16(Ink4a) in beta cells induces hallmarks of senescence--including cell enlargement, and greater glucose uptake and mitochondrial activity--which promote increased insulin secretion. GSIS increases during the normal aging of mice and is driven by elevated p16(Ink4a) activity. We found that islets from human adults contain p16(Ink4a)-expressing senescent beta cells and that senescence induced by p16(Ink4a) in a human beta cell line increases insulin secretion in a manner dependent, in part, on the activity of the mechanistic target of rapamycin (mTOR) and the peroxisome proliferator-activated receptor (PPAR)-γ proteins. Our findings reveal a novel role for p16(Ink4a) and cellular senescence in promoting insulin secretion by beta cells and in regulating normal functional tissue maturation with age.

  17. Intestinal epithelial cell secretion of RELM-beta protects against gastrointestinal worm infection

    Science.gov (United States)

    IL-4 and IL-13 protect against parasitic helminths, but little is known about the mechanism of host protection. We show that IL-4/IL-13 confer immunity against worms by inducing intestinal epithelial cells (IEC) to differentiate into goblet cells that secrete resistin-like molecule beta (RELMB). R...

  18. PAX4 promotes PDX1-induced differentiation of mesenchymal stem cells into insulin-secreting cells

    Science.gov (United States)

    Xu, Lifa; Xu, Congjing; Zhou, Shuping; Liu, Xueke; Wang, Jian; Liu, Xinkuang; Qian, Suping; Xin, Yingru; Gao, Yi; Zhu, Yongqiang; Tang, Xiaolong

    2017-01-01

    A shortage of postmortem pancreatic tissue for islet isolation impedes the application of cell replacement therapy in patients with diabetes. As an alternative for islet cell transplantation, transcription factors, including PDX1, PAX4, and neurogenin-3, that aid in the formation of insulin-producing β cells during development have been investigated. The present study evaluated the effects of PAX4 and PDX1 on the differentiation of mesenchymal stem cells (MSCs) into insulin-producing β-like cells in vitro using recombinant adenoviruses carrying PDX1 or PDX1 plus PAX4. RT-PCR, Western blot, and immunofluorescence assays were used to detect the expression levels of relevant genes and proteins, and enzyme-linked immunosorbent assays were used to determine the amount of insulin and C-peptide secreted by the virus-infected cells following stimulation with high glucose. The results showed that PAX4 markedly enhanced the propensity of PDX1-positive MSCs to form mature islet-like clusters and functional insulin-producing β-like cells. Our findings provide a novel foundation for generating β-like cells from MSCs with PAX4 and PDX1 for future clinical application.

  19. CaSR function in the intestine: Hormone secretion, electrolyte absorption and secretion, paracrine non-canonical Wnt signaling and colonic crypt cell proliferation.

    Science.gov (United States)

    Macleod, R John

    2013-06-01

    Expression and function of the CaSR have been shown in some mammalian taste buds and basal cells of the esophagus. Signaling cascades responsible for CaSR-mediated stimulation of H(+)-K(+)-ATPase on human parietal cells have been defined. Transgenic mice and reductionistic cell culture models have shown that the CaSR promotes gastrin secretion from G cells, cholecystokinin (CCK) secretion from duodenal I cells and BMP-2 secretion from sub-epithelial myofibroblasts. In addition, the CaSR mediates a novel paracrine relationship between myofibroblasts and overlying epithelial cells in the colon. Thus, CaSR activators stimulate secretion of Wnt5a from myofibroblasts and expression of the Wnt5a receptor Ror2 in epithelial cells. CaSR-mediated Wnt5a/Ror2 engagement stimulates epithelial differentiation and reduces expression of the receptor for tumor necrosis factor (TNFR1). CaSR activators also modulate intestinal motility, inhibit Cl(-) secretion and stimulate Na(+) absorption in both the small intestine and colon. Colonic epithelia from conditional and global CaSR knockout mice exhibit increased proliferation with increased Wnt/β-catenin signaling, demonstrating that the CaSR negatively modulates colonic epithelial growth.

  20. Neurotrophic factors in sponal pain transmission

    NARCIS (Netherlands)

    J.L.M. Jongen (Joost)

    2006-01-01

    textabstractHet doel van dit proefschrift was de betrokkenheid van Glial Cell Line-Derived Neurotrophic Factor (GDNF) bij spinale pijntransmissie te bestuderen. In Hoofdstuk 1 worden enkele recente ontwikkelen betreffende de organisatie van het nociceptieve systeem besproken, gevolgd door een besc

  1. Secreted autotransporter toxin (Sat) triggers autophagy in epithelial cells that relies on cell detachment.

    Science.gov (United States)

    Liévin-Le Moal, Vanessa; Comenge, Yannick; Ruby, Vincent; Amsellem, Raymonde; Nicolas, Valérie; Servin, Alain L

    2011-07-01

    The secreted autotransporter toxin, Sat, which belongs to the subfamily of serine protease autotransporters of Enterobacteriaceae, acts as a virulence factor in extraintestinal and intestinal pathogenic strains of Escherichia coli. We observed that HeLa cells exposed to the cell-free culture supernatant of recombinant strain AAEC185p(Sat-IH11128) producing the Sat toxin (CFCS(Sat) ), displayed dramatic disorganization of the F-actin cytoskeleton before loosening cell-to-cell junctions and detachment. Examination of the effect of Sat on GFP-microtubule-associated protein light chain 3 (LC3) HeLa cells revealed that CFCS(Sat) -induced autophagy follows CFCS(Sat) -induced F-actin cytoskeleton rearrangement. The induced autophagy shows an acceleration of the autophagy flux soon after Sat treatment, followed later by a blockade of the flux leading to the accumulation of large GFP-LC3-positive vacuoles in the cell cytoplasm. CFCS(Sat) did not induce cell detachment in autophagy-deficient mouse embryonic fibroblasts in contrast with wild-type mouse embryonic fibroblasts. The CFCS(Sat) -induced large GFP-LC3 dots do not display the characteristics of autophagolysosomes including expression of cathepsin D and Lamp-1 and 2 proteins, and Lysotracker Red- and DQ-BSA-positive labelling. We provide evidences that CFCS(Sat) -induced autophagy is not a cell response intended to get rid of the intracellular toxin. By a pharmacological blockers approach, we found that the blockade of Erk1/2 and p38 MAPKs, but not JNK, inhibited the CFCS(Sat) -induced autophagy and cell detachment whereas phosphatidylinositol-3 kinase blockers inhibiting canonical autophagy were inactive. When attached CFCS(Sat) -treated cells start to detach they showed caspase-independent cell death and rearrangements of the focal adhesion-associated vinculin and paxillin. Collectively, our results support that Sat triggers autophagy in epithelial cells that relies on its cell-detachment effect.

  2. Morphine Attenuates Apically-Directed Cytokine Secretion from Intestinal Epithelial Cells in Response to Enteric Pathogens

    Directory of Open Access Journals (Sweden)

    Amanda J. Brosnahan

    2014-04-01

    Full Text Available Epithelial cells represent the first line of host immune defense at mucosal surfaces. Although opioids appear to increase host susceptibility to infection, no studies have examined opioid effects on epithelial immune functions. We tested the hypothesis that morphine alters vectorial cytokine secretion from intestinal epithelial cell (IPEC-J2 monolayers in response to enteropathogens. Both entero-adherent Escherichia coli O157:H7 and entero-invasive Salmonella enterica serovar Typhimurium increased apically-directed IL-6 secretion and bi-directional IL-8 secretion from epithelial monolayers, but only IL-6 secretion evoked by E. coli was reduced by morphine acting through a naloxone-sensitive mechanism. Moreover, the respective type 4 and 5 Toll-like receptor agonists, lipopolysaccharide and flagellin, increased IL-8 secretion from monolayers, which was also attenuated by morphine pretreatment. These results suggest that morphine decreases cytokine secretion and potentially phagocyte migration and activation directed towards the mucosal surface; actions that could increase host susceptibility to some enteric infections.

  3. A somatostatin-secreting cell line established from a human pancreatic islet cell carcinoma (somatostatinoma): release experiment and immunohistochemical study.

    Science.gov (United States)

    Iguchi, H; Hayashi, I; Kono, A

    1990-06-15

    Production and secretion of somatostatin (SRIF) were studied using a carcinoembryonic antigen (CEA)-producing cell line (QGP-1) established from a human pancreatic islet cell carcinoma. High concentrations of SRIF (274 +/- 51 ng/mg of protein, mean +/- SD, n = 5) and CEA (3083 +/- 347 ng/mg of protein, mean +/- SD, n = 5) were present in QGP-1 cells, and the basal secretion rates of SRIF and CEA by the cells (n = 5) were 46.4 +/- 4.8 and 1690 +/- 78 pg/10(5) cells/h, respectively. Immunohistochemical studies revealed the presence of SRIF in xenografts of QGP-1 cells and colocalization of SRIF and CEA. Secretion of SRIF by QGP-1 cells was stimulated in the presence of high K+ (50 mmol) and theophylline (10 mmol), but arginine (10 mmol) and glucose (300 mg/dl) had no effect on the SRIF secretion. The QGP-1 cell line may be useful for studying the regulation mechanism of SRIF secretion.

  4. Oxytocin and dopamine stimulate ghrelin secretion by the ghrelin-producing cell line, MGN3-1 in vitro.

    OpenAIRE

    Iwakura, Hiroshi; Ariyasu, Hiroyuki; Hosoda, Hiroshi; Yamada, Go; Hosoda, Kiminori; Nakao, Kazuwa; Kangawa, Kenji; Akamizu, Takashi

    2011-01-01

    To understand the physiological role of ghrelin, it is crucial to study both the actions of ghrelin and the regulation of ghrelin secretion. Although ghrelin actions have been extensively revealed, the direct factors regulating ghrelin secretion by ghrelin-producing cells (X/A-like cells), however, is not fully understood. In this study, we examined the effects of peptide hormones and neurotransmitters on in vitro ghrelin secretion by the recently developed ghrelin-producing cell line MGN3-1....

  5. Glucose stimulates calcium-activated chloride secretion in small intestinal cells.

    Science.gov (United States)

    Yin, Liangjie; Vijaygopal, Pooja; MacGregor, Gordon G; Menon, Rejeesh; Ranganathan, Perungavur; Prabhakaran, Sreekala; Zhang, Lurong; Zhang, Mei; Binder, Henry J; Okunieff, Paul; Vidyasagar, Sadasivan

    2014-04-01

    The sodium-coupled glucose transporter-1 (SGLT1)-based oral rehydration solution (ORS) used in the management of acute diarrhea does not substantially reduce stool output, despite the fact that glucose stimulates the absorption of sodium and water. To explain this phenomenon, we investigated the possibility that glucose might also stimulate anion secretion. Transepithelial electrical measurements and isotope flux measurements in Ussing chambers were used to study the effect of glucose on active chloride and fluid secretion in mouse small intestinal cells and human Caco-2 cells. Confocal fluorescence laser microscopy and immunohistochemistry measured intracellular changes in calcium, sodium-glucose linked transporter, and calcium-activated chloride channel (anoctamin 1) expression. In addition to enhancing active sodium absorption, glucose increased intracellular calcium and stimulated electrogenic chloride secretion. Calcium imaging studies showed increased intracellular calcium when intestinal cells were exposed to glucose. Niflumic acid, but not glibenclamide, inhibited glucose-stimulated chloride secretion in mouse small intestines and in Caco-2 cells. Glucose-stimulated chloride secretion was not seen in ileal tissues incubated with the intracellular calcium chelater BAPTA-AM and the sodium-potassium-2 chloride cotransporter 1 (NKCC1) blocker bumetanide. These observations establish that glucose not only stimulates active Na absorption, a well-established phenomenon, but also induces a Ca-activated chloride secretion. This may explain the failure of glucose-based ORS to markedly reduce stool output in acute diarrhea. These results have immediate potential to improve the treatment outcomes for acute and/or chronic diarrheal diseases by replacing glucose with compounds that do not stimulate chloride secretion.

  6. Intrathecal IgG synthesis and autoantibody-secreting cells in multiple sclerosis

    DEFF Research Database (Denmark)

    Sellebjerg, Finn; Jensen, Claus V; Christiansen, Michael

    2000-01-01

    We studied intrathecal IgG synthesis and autoantibody-secreting cells in 148 patients with possible onset symptoms of MS (POSMS) or clinically definite MS (CDMS). In POSMS intrathecal synthesis of IgG oligoclonal bands and abnormalities on T2-weighted magnetic resonance imaging were associated...... but the former were more prevalent. The cerebrospinal fluid (CSF) leukocyte count and the number of anti-protelipid protein antibody-secreting cells in cerebrospinal fluid (CSF) correlated with disease activity in POSMS. Intrathecal IgG synthesis levels and the number of anti-myelin basic protein antibody......-secreting cells in CSF correlated with disease activity in CDMS. Our results support recent reports of pathogenetic heterogeneity and a pathogenetic role of the antibody response in MS...

  7. Migration of antibody secreting cells towards CXCL12 depends on the isotype that forms the BCR

    Science.gov (United States)

    Achatz-Straussberger, Gertrude; Zaborsky, Nadja; Königsberger, Sebastian; Luger, Elke O.; Lamers, Marinus; Crameri, Reto; Achatz, Gernot

    2010-01-01

    Truncation of the cytoplasmic tail of membrane-bound IgE in vivo results in lower serum IgE levels, decreased numbers of IgE-secreting plasma cells and the abrogation of specific secondary immune responses. Here we present mouse strain KN1 that expresses a chimeric ε-γ1 BCR, consisting of the extracellular domains of the ε gene and the trans-membrane and cytoplasmic domains of the γ1 gene. Thus, differences in the IgE immune response of KN1 mice reflect the influence of the “γ1-mediated signalling” of mIgE bearing B cells. KN1 mice show an increased serum IgE level, resulting from an elevated number of IgE-secreting cells. Although the primary IgE immune response in KN1 mice is inconspicuous, the secondary response is far more robust. Most strikingly, IgE-antibody secreting cells with “γ1-signalling history” migrate more efficiently towards the chemokine CXCL12, which guides plasmablasts to plasma cell niches, than IgE-antibody secreting cells with WT “ε-signalling history”. We conclude that IgE plasmablasts have an intrinsic, lower chance to contribute to the long-lived plasma cell pool than IgG1 plasmablasts. PMID:18925577

  8. Human neural stem cells genetically modified to overexpress brain-derived neurotrophic factor promote functional recovery and neuroprotection in a mouse stroke model.

    Science.gov (United States)

    Lee, Hong J; Lim, In J; Lee, Min C; Kim, Seung U

    2010-11-15

    Intracerebral hemorrhage (ICH) is a lethal stroke type; mortality approaches 50%, and current medical therapy against ICH shows only limited effectiveness, so an alternative approach is required, such as stem cell-based cell therapy. Previously we have shown that intravenously transplanted human neural stem cells (NSCs) selectively migrate to the brain and promote functional recovery in rat ICH model, and others have shown that intracerebral infusion of brain-derived neurotrophic factor (BDNF) results in improved structural and functional outcome from cerebral ischemia. We postulated that human NSCs overexpressing BDNF transplanted into cerebral cortex overlying ICH lesion could provide improved survival of grafted NSCs and increased angiogenesis and behavioral recovery in mouse ICH model. ICH was induced in adult mice by injection of bacterial collagenase into striatum. The HB1.F3.BDNF (F3.BDNF) human NSC line produces sixfold higher amounts of BDNFF over the parental F3 cell line in vitro, induces behavioral improvement, and produces a threefold increase in cell survival at 2 weeks and 8 weeks posttransplantation. Brain transplantation of human NSCs overexpressing BDNF provided differentiation and survival of grafted human NSCs and renewed angiogenesis of host brain and functional recovery of ICH animals. These results indicate that the F3.BDNF human NSCs should be of great value as a cellular source for experimental studies involving cellular therapy for human neurological disorders, including ICH.

  9. Brain-derived neurotrophic factor induces migration of endothelial cells through a TrkB-ERK-integrin αVβ3-FAK cascade.

    Science.gov (United States)

    Matsuda, Shinji; Fujita, Tsuyoshi; Kajiya, Mikihito; Takeda, Katsuhiro; Shiba, Hideki; Kawaguchi, Hiroyuki; Kurihara, Hidemi

    2012-05-01

    Brain-derived neurotrophic factor (BDNF) promotes the regeneration of periodontal tissue. Since angiogenesis is important for tissue regeneration, investigating effect of BDNF on endothelial cell function may help to reveal its mechanism, whereby, BDNF promotes periodontal tissue regeneration. In this study, we examined the influence of BDNF on migration in human microvascular endothelial cells (HMVECs), focusing on the effects on extracellular signal-regulated kinase (ERK), integrin α(V)β(3), and focal adhesion kinase (FAK). The migration of endothelial cells was assessed with a modified Boyden chamber and a wound healing assay. The expression of integrin α(V)β(3) and the phosphorylation of ERK and FAK were analyzed by immunoblotting and immunofluorescence microscopy. BDNF (25 ng/ml) induced cell migration. PD98059, an ERK inhibitor, K252a, a specific inhibitor for TrkB, a high affinity receptor of BDNF, and an anti-integrin α(V)β(3) antibody suppressed the BDNF-induced migration. BDNF increased the levels of integrin α(V)β(3) and phosphorylated ERK1/2 and FAK. The ERK inhibitor and TrkB inhibitor also reduced levels of integrin α(V)β(3) and phosphorylated FAK. We propose that BDNF stimulates endothelial cell migration by a process involving TrkB/ERK/integrin α(V)β(3)/FAK, and this may help to enhance the regeneration of periodontal tissue.

  10. Brain-derived neurotrophic factor levels influence the balance of migration and differentiation of subventricular zone cells, but not guidance to the olfactory bulb.

    Science.gov (United States)

    Petridis, Athanasios K; El Maarouf, Abderrahman

    2011-02-01

    New progenitor cells in the subventricular zone (SVZ) migrate rostrally and differentiate into interneurons in the olfactory bulb (OB) throughout life. Brain-derived neurotrophic factor (BDNF) may influence the normal progression of this migration. In the present study, mouse SVZ explant cultures were used to investigate how BDNF modulates the behavior of these migrating progenitors. Concentrations of BDNF in the physiological range (e.g. 1ng/mL) stimulated migration, whereas doses of 10 ng/mL or higher induced SVZ cell differentiation and reduced migration. Pharmacological inhibition of the mitogen-activated protein kinase (MAPK) pathway blocked the BDNF-induced differentiation of SVZ progenitors, indicating that differentiation of SVZ progenitors in response to high-dose BDNF is initiated through MAPK. Physiological concentrations of BDNF, like the presence of polysialic acid in the tissue, stimulated migration of cells from the explant without affecting the speed at which this occurs. Interestingly, in vivo immunohistochemical and molecular analysis showed similar levels of BDNF in both the SVZ and OB; that is, there was no positive gradient attracting SVZ cells towards the OB. Our data show that SVZ cells respond differently to different concentrations of BDNF.

  11. Effect of glial cell line-derived neurotrophic factor on behavior and key members of the brain serotonin system in mouse strains genetically predisposed to behavioral disorders.

    Science.gov (United States)

    Naumenko, Vladimir S; Bazovkina, Daria V; Semenova, Alina A; Tsybko, Anton S; Il'chibaeva, Tatyana V; Kondaurova, Elena M; Popova, Nina K

    2013-12-01

    The effect of glial cell line-derived neurotrophic factor (GDNF) on behavior and on the serotonin (5-HT) system of a mouse strain predisposed to depressive-like behavior, ASC/Icg (Antidepressant Sensitive Cataleptics), in comparison with the parental "nondepressive" CBA/Lac mice was studied. Within 7 days after acute administration, GDNF (800 ng, i.c.v.) decreased cataleptic immobility but increased depressive-like behavioral traits in both investigated mouse strains and produced anxiolytic effects in ASC mice. The expression of the gene encoding the key enzyme for 5-HT biosynthesis in the brain, tryptophan hydroxylase-2 (Tph-2), and 5-HT1A receptor gene in the midbrain as well as 5-HT2A receptor gene in the frontal cortex were increased in GDNF-treated ASC mice. At the same time, GDNF decreased 5-HT1A and 5-HT2A receptor gene expression in the hippocampus of ASC mice. GDNF failed to change Tph2, 5-HT1A , or 5-HT2A receptor mRNA levels in CBA mice as well as 5-HT transporter gene expression and 5-HT1A and 5-HT2A receptor functional activity in both investigated mouse strains. The results show 1) a GDNF-induced increase in the expression of key genes of the brain 5-HT system, Tph2, 5-HT1A , and 5-HT2A receptors, and 2) significant genotype-dependent differences in the 5-HT system response to GDNF treatment. The data suggest that genetically defined cross-talk between neurotrophic factors and the brain 5-HT system underlies the variability in behavioral response to GDNF.

  12. The synergistic effect of nanotopography and sustained dual release of hydrophobic and hydrophilic neurotrophic factors on human mesenchymal stem cell neuronal lineage commitment.

    Science.gov (United States)

    Teo, Benjamin Kim Kiat; Tan, Guo-Dong Sean; Yim, Evelyn K F

    2014-08-01

    A combination of nanotopography and controlled release is a potential platform for neuronal tissue engineering applications. Previous studies showed that combining both physical and chemical guidance was more effective than individual cues in the directional promotion of neurite outgrowth. Nanotopography can direct human mesenchymal stem cells (hMSCs) into neuronal lineage, while controlled release of neurotrophic factors can deliver temporally controlled biochemical signals. Hypothesizing that the synergistic effect will enhance neuronal lineage commitment of hMSCs, a fabrication method for multiple neurotrophic factors delivery from a single nanopatterned (350 nm gratings), poly-ɛ-caprolactone (PCL) film was developed and evaluated. Our results showed a synergistic effect on hMSC differentiation cultured on substrates with both nanotopographical and biochemical cues. The protein/drug encapsulation into PCL nanopatterned films was first optimized using a hydrophilic model protein, bovine serum albumin. The hydrophobic retinoic acid (RA) molecule was directly incorporated into PCL films. To achieve sustained release, hydrophilic nerve growth factor (NGF) was first encapsulated within polyelectrolyte complexation fibers before they were embedded within the nanopatterned PCL film. Our results showed that nanotopography on the fabricated polymer films remained intact, while release of bioactive RA and NGF was sustained over a period of 3 weeks. Under the combinatorial effect of physical and biochemical cues, we observed an enhanced upregulation of neuronal genes such as microtubule-associated protein 2 (MAP2) and neurofilament light (NFL) as compared with sustained delivery of individual cues and bolus delivery. Quantitative polymerase chain reaction analysis showed that MAP2 and NFL gene upregulation in hMSCs was most pronounced on the nanogratings with sustained release of both RA and NGF. The fabricated platforms supported the sustained delivery of multiple

  13. Glioblastoma cell-secreted interleukin-8 induces brain endothelial cell permeability via CXCR2.

    Directory of Open Access Journals (Sweden)

    Julie Dwyer

    Full Text Available Glioblastoma constitutes the most aggressive and deadly of brain tumors. As yet, both conventional and molecular-based therapies have met with limited success in treatment of this cancer. Among other explanations, the heterogeneity of glioblastoma and the associated microenvironment contribute to its development, as well as resistance and recurrence in response to treatments. Increased vascularity suggests that tumor angiogenesis plays an important role in glioblastoma progression. However, the molecular crosstalk between endothelial and glioblastoma cells requires further investigation. To examine the effects of glioblastoma-derived signals on endothelial homeostasis, glioblastoma cell secretions were collected and used to treat brain endothelial cells. Here, we present evidence that the glioblastoma secretome provides pro-angiogenic signals sufficient to disrupt VE-cadherin-mediated cell-cell junctions and promote endothelial permeability in brain microvascular endothelial cells. An unbiased angiogenesis-specific antibody array screen identified the chemokine, interleukin-8, which was further demonstrated to function as a key factor involved in glioblastoma-induced permeability, mediated through its receptor CXCR2 on brain endothelia. This underappreciated interface between glioblastoma cells and associated endothelium may inspire the development of novel therapeutic strategies to induce tumor regression by preventing vascular permeability and inhibiting angiogenesis.

  14. Root border cells and secretions as critical elements in plant host defense.

    Science.gov (United States)

    Driouich, Azeddine; Follet-Gueye, Marie-Laure; Vicré-Gibouin, Maïté; Hawes, Martha

    2013-08-01

    Border cells and border-like cells are released from the root tip as individual cells and small aggregates, or as a group of attached cells. These are viable components of the root system that play a key role in controlling root interaction with living microbes of the rhizosphere. As their separation from root tip proceeds, the cells synthesize and secrete a hydrated mucilage that contains polysaccharides, secondary metabolites, antimicrobial proteins and extracellular DNA (exDNA). This exDNA-based matrix seems to function in root defense in a way similar to that of recently characterized neutrophil extracellular traps (NETs) in mammalian cells. This review discusses the role of the cells and secreted compounds in the protection of root tip against microbial infections.

  15. Secreted microvesicular miR-31 inhibits osteogenic differentiation of mesenchymal stem cells

    DEFF Research Database (Denmark)

    Weilner, Sylvia; Schraml, Elisabeth; Wieser, Matthias

    2016-01-01

    Damage to cells and tissues is one of the driving forces of aging and age-related diseases. Various repair systems are in place to counteract this functional decline. In particular, the property of adult stem cells to self-renew and differentiate is essential for tissue homeostasis and regeneration....... However, their functionality declines with age (Rando, 2006). One organ that is notably affected by the reduced differentiation capacity of stem cells with age is the skeleton. Here, we found that circulating microvesicles impact on the osteogenic differentiation capacity of mesenchymal stem cells....... As a potential source of its secretion, we identified senescent endothelial cells, which are known to increase during aging in vivo (Erusalimsky, 2009). Endothelial miR-31 is secreted within senescent cell-derived microvesicles and taken up by mesenchymal stem cells where it inhibits osteogenic differentiation...

  16. Decidual-secreted factors alter invasive trophoblast membrane and secreted proteins implying a role for decidual cell regulation of placentation.

    Directory of Open Access Journals (Sweden)

    Ellen Melaleuca Menkhorst

    Full Text Available Inadequate or inappropriate implantation and placentation during the establishment of human pregnancy is thought to lead to first trimester miscarriage, placental insufficiency and other obstetric complications. To create the placental blood supply, specialized cells, the 'extravillous trophoblast' (EVT invade through the differentiated uterine endometrium (the decidua to engraft and remodel uterine spiral arteries. We hypothesized that decidual factors would regulate EVT function by altering the production of EVT membrane and secreted factors. We used a proteomics approach to identify EVT membrane and secreted proteins regulated by decidual cell factors. Human endometrial stromal cells were decidualized in vitro by treatment with estradiol (10(-8 M, medroxyprogesterone acetate (10(-7 M and cAMP (0.5 mM for 14 days. Conditioned media (CM was collected on day 2 (non-decidualized CM and 14 (decidualized CM of treatment. Isolated primary EVT cultured on Matrigel™ were treated with media control, non-decidualized or decidualized CM for 16 h. EVT CM was fractionated for proteins <30 kDa using size-exclusion affinity nanoparticles (SEAN before trypsin digestion and HPLC-MS/MS. 43 proteins produced by EVT were identified; 14 not previously known to be expressed in the placenta and 12 which had previously been associated with diseases of pregnancy including preeclampsia. Profilin 1, lysosome associated membrane glycoprotein 1 (LAMP1, dipeptidyl peptidase 1 (DPP1/cathepsin C and annexin A2 expression by interstitial EVT in vivo was validated by immunhistochemistry. Decidual CM regulation in vitro was validated by western blotting: decidualized CM upregulated profilin 1 in EVT CM and non-decidualized CM upregulated annexin A2 in EVT CM and pro-DPP1 in EVT cell lysate. Here, non-decidualized factors induced protease expression by EVT suggesting that non-decidualized factors may induce a pro-inflammatory cascade. Preeclampsia is a pro

  17. The role of brain-derived neurotrophic factor in the regulation of cell growth and gene expression in melanotrope cells of Xenopus laevis.

    NARCIS (Netherlands)

    Jenks, B.G.; Kuribara, M.; Kidane, A.H.; Kramer, B.M.; Roubos, E.W.; Scheenen, W.J.

    2012-01-01

    Brain-derived neurotrophic factor (BDNF) is, despite its name, also found outside the central nervous system (CNS), but the functional significance of this observation is largely unknown. This review concerns the expression of BDNF in the pituitary gland. While the presence of the neurotrophin in th

  18. Self-inducible secretion of glucagon-like peptide-1 (GLP-1) that allows MIN6 cells to maintain insulin secretion and insure cell survival.

    Science.gov (United States)

    Nakashima, Koji; Shimoda, Masashi; Hamamoto, Sumiko; Tatsumi, Fuminori; Hirukawa, Hidenori; Tawaramoto, Kazuhito; Kanda, Yukiko; Kaku, Kohei

    2012-02-26

    Based on the hypothesis that MIN6 cells could produce glucagon-like peptide-1 (GLP-1) to maintain cell survival, we analyzed the effects of GLP-1 receptor agonist, exendin-4 (Ex4), and antagonist, exendin-(9-39) (Ex9) on cell function and cell differentiation. MIN6 cells expressed proglucagon mRNAs and produced GLP-1, which was accelerated by Ex4 and suppressed by Ex9. Moreover, Ex4 further enhanced glucose-stimulated GLP-1 secretion, suggesting autocrine loop-contributed amplification of the GLP-1 signal. Ex4 up-regulated cell differentiation- and cell function-related CREBBP, Pdx-1, Pax6, proglucagon, and PC1/3 gene expressions. The confocal laser scanning images revealed that GLP-1 positive cells were dominant in the early stage of cells, but positive for insulin were more prominent in the mature stage of cells. Ex4 accelerated cell viability, while Ex9 and anti-GLP-1 receptor antibody enhanced cell apoptosis. MIN6 cells possess a mechanism of GLP-1 signal amplification in an autocrine fashion, by which the cells maintained insulin production and cell survival.

  19. Stimulation of Hepatoma Cell Invasiveness and Metastatic Potential by Proteins Secreted From Irradiated Nonparenchymal Cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhou Leyuan [Department of Radiation Oncology, Zhongshan Hospital, Fudan University, Shanghai (China); Wang Zhiming [Department of Medical Oncology, Zhongshan Hospital, Fudan University, Shanghai (China); Gao Yabo [Department of Radiation Oncology, Zhongshan Hospital, Fudan University, Shanghai (China); Wang Lingyan [Experimental Research Center, Zhongshan Hospital, Fudan University, Shanghai (China); Zeng Zhaochong, E-mail: zeng.zhaochong@zs-hospital.sh.cn [Department of Radiation Oncology, Zhongshan Hospital, Fudan University, Shanghai (China)

    2012-11-01

    Purpose: To determine whether factors secreted by irradiated liver nonparenchymal cells (NPCs) may influence invasiveness and/or metastatic potential of hepatocellular carcinoma (HCC) cells and to elucidate a possible mechanism for such effect. Methods and Materials: Primary rat NPCs were cultured and divided into irradiated (10-Gy X-ray) and nonirradiated groups. Forty-eight hours after irradiation, conditioned medium from irradiated (SR) or nonirradiated (SnonR) cultures were collected and added to sublethally irradiated cultures of the hepatoma McA-RH7777 cell line. Then, hepatoma cells were continuously passaged for eight generations (RH10Gy-SR and RH10Gy-SnonR). The invasiveness and metastatic potential of McA-RH7777, RH10Gy-SnonR, and RH10Gy-SR cells were evaluated using an in vitro gelatinous protein (Matrigel) invasion and an in vivo metastasis assay. In addition, SR and SnonR were tested using rat cytokine antibody arrays and enzyme-linked immunosorbent assay (ELISA). Results: In vitro gelatinous protein invasion assay indicated that the numbers of invading cells was significantly higher in RH10Gy-SR (40 {+-} 4.74) than in RH10Gy-SnonR (30.6 {+-} 3.85) cells, and lowest in McA-RH7777 (11.4 {+-} 3.56) cells. The same pattern was observed in vivo in a lung metastasis assay, as evaluated by number of metastatic lung nodules seen with RH10Gy-SR (28.83 {+-} 5.38), RH10Gy-SnonR (22.17 {+-} 4.26), and McA-RH7777 (8.3 {+-} 3.8) cells. Rat cytokine antibody arrays and ELISA demonstrated that metastasis-promoting cytokines (tumor necrosis factor-{alpha} and interleukin-6), circulating growth factors (vascular endothelial growth factor and epidermal growth factor), and metalloproteinases (MMP-2 and MMP-9) were upregulated in SR compared with SnonR. Conclusions: Radiation can increase invasiveness and metastatic potential of sublethally irradiated hepatoma cells, and soluble mediators released from irradiated NPCs promote this potential. Increased secretion of

  20. EEN regulates the proliferation and survival of multiple myeloma cells by potentiating IGF-1 secretion

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Er-Wen [Guangzhou Institute of Forensic Science, Guangzhou (China); Department of Forensic Pathology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou (China); Xue, Sheng-Jiang [Department of Forensic Pathology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou (China); Li, Xiao-Yan [Department of Pharmacy, The Third Affiliated Hospital, Sun Yat-Sen University, Guangzhou (China); Xu, Suo-Wen [Department of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou (China); Cheng, Jian-Ding; Zheng, Jin-Xiang [Department of Forensic Pathology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou (China); Shi, He; Lv, Guo-Li; Li, Zhi-Gang; Li, Yue; Liu, Chang-Hui; Chen, Xiao-Hui; Liu, Hong [Guangzhou Institute of Forensic Science, Guangzhou (China); Li, Jie, E-mail: mdlijie@sina.com [Department of Anaesthesiology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou (China); Liu, Chao, E-mail: liuchaogaj@21cn.com [Guangzhou Institute of Forensic Science, Guangzhou (China)

    2014-05-02

    Highlights: • Levels of EEN expression paralleled with the rate of cell proliferation. • EEN was involved in the proliferation and survival of multiple myeloma (MM) cells. • EEN regulated the activity of IGF-1-Akt/mTOR pathway. • EEN regulated proliferation and survival of MM cells by enhancing IGF-1 secretion. - Abstract: The molecular mechanisms of multiple myeloma are not well defined. EEN is an endocytosis-regulating molecule. Here we report that EEN regulates the proliferation and survival of multiple myeloma cells, by regulating IGF-1 secretion. In the present study, we observed that EEN expression paralleled with cell proliferation, EEN accelerated cell proliferation, facilitated cell cycle transition from G1 to S phase by regulating cyclin-dependent kinases (CDKs) pathway, and delayed cell apoptosis via Bcl2/Bax-mitochondrial pathway. Mechanistically, we found that EEN was indispensable for insulin-like growth factor-1 (IGF-1) secretion and the activation of protein kinase B-mammalian target of rapamycin (Akt-mTOR) pathway. Exogenous IGF-1 overcame the phenotype of EEN depletion, while IGF-1 neutralization overcame that of EEN over-expression. Collectively, these data suggest that EEN may play a pivotal role in excessive cell proliferation and insufficient cell apoptosis of bone marrow plasma cells in multiple myeloma. Therefore, EEN may represent a potential diagnostic marker or therapeutic target for multiple myeloma.

  1. Effects of sodium cromoglycate and nedocromil sodium on histamine secretion from human lung mast cells.

    Science.gov (United States)

    Leung, K B; Flint, K C; Brostoff, J; Hudspith, B N; Johnson, N M; Lau, H Y; Liu, W L; Pearce, F L

    1988-01-01

    Sodium cromoglycate and nedocromil sodium produced a dose dependent inhibition of histamine secretion from human pulmonary mast cells obtained by bronchoalveolar lavage and by enzymatic dissociation of lung parenchyma. Both compounds were significantly more active against the lavage cells than against the dispersed lung cells, and nedocromil sodium was an order of magnitude more effective than sodium cromoglycate against both cell types. Tachyphylaxis was observed with the parenchymal cells but not with the lavage cells. Nedocromil sodium and sodium cromoglycate also inhibited histamine release from the lavage cells of patients with sarcoidosis and extrinsic asthma. PMID:2462755

  2. Polarized secretion of newly synthesized lipoproteins by the Caco-2 human intestinal cell line.

    Science.gov (United States)

    Traber, M G; Kayden, H J; Rindler, M J

    1987-11-01

    Lipoprotein secretion by Caco-2 cells, a human intestinal cell line, was studied in cells grown on inserts containing a Millipore filter (0.45 micron), separating secretory products from the apical and basolateral membranes into separate chambers. Under these conditions, as observed by electron microscopy, the cells formed a monolayer of columnar epithelial cells with microvilli on the apical surface and tight junctions between cells. The electrical resistances of the cell monolayers were 250-500 ohms/cm2. Both 14C-labeled lipids and 35S-labeled proteins were used to assess lipoprotein secretion. After a 24-hr incubation with [14C]oleic acid, 60-80% of the secreted triglyceride (TG) was in the basolateral chamber; 40% of the TG was present in the d less than 1.006 g/ml (chylomicron + VLDL) fraction and 50% in the 1.006 less than d less than 1.063 g/ml (LDL) fraction. After a 4-hr incubation with [35S]methionine, apolipoproteins were found to be major secretory products with 75-100% secreted to the basolateral chamber. Apolipoproteins B-100, B-48, E, A-I, A-IV, and C-III were identified by immunoprecipitation. The d less than 1.006 g/ml fraction was found to contain all of the major apolipoproteins, while the LDL fraction contained primarily apoB-100 and apoE; the HDL (1.063 less than d less than 1.21 g/ml) fraction principally contained apoA-I and apoA-IV. Mn-heparin precipitated all of the [35S]methionine-labeled apoB-100 and B-48 and a majority of the other apolipoproteins, and 80% of the [14C]oleic acid-labeled triglyceride, but only 15% of the phospholipid, demonstrating that Caco-2 cells secrete triglyceride-rich lipoproteins containing apoB. Secretion of lipoproteins was dependent on the lipid content of the medium; prior incubation with lipoprotein-depleted serum specifically reduced the secretion of lipoproteins, while addition of both LDL and oleic acid to the medium maintained the level of apoB-100, B-48, and A-IV secretion to that observed in the control

  3. Purification and Cultivation of Human Pituitary Growth Hormones Secreting Cells

    Science.gov (United States)

    Hymer, W. C.; Todd, P.; Grindeland, R.; Lanham, W.; Morrison, D.

    1985-01-01

    The rat and human pituitary gland contains a mixture of hormone producing cell types. The separation of cells which make growth hormone (GH) is attempted for the purpose of understanding how the hormone molecule is made within the pituitary cell; what form(s) it takes within the cell; and what form(s) GH assumes as it leaves the cell. Since GH has a number of biological targets (e.g., muscle, liver, bone), the assessment of the activities of the intracellular/extracellular GH by new and sensitive bioassays. GH cells contained in the mixture was separated by free flow electrophoresis. These experiments show that GH cells have different electrophoretic mobilities. This is relevant to NASA since a lack of GH could be a prime causative factor in muscle atrophy. Further, GH has recently been implicated in the etiology of motion sickness in space. Continous flow electrophoresis experiment on STS-8 showed that GH cells could be partially separated in microgravity. However, definitive cell culture studies could not be done due to insufficient cell recoveries.

  4. Acute overexpression of lactate dehydrogenase-A perturbs beta-cell mitochondrial metabolism and insulin secretion.

    Science.gov (United States)

    Ainscow, E K; Zhao, C; Rutter, G A

    2000-07-01

    Islet beta-cells express low levels of lactate dehydrogenase and have high glycerol phosphate dehydrogenase activity. To determine whether this configuration favors oxidative glucose metabolism via mitochondria in the beta-cell and is important for beta-cell metabolic signal transduction, we have determined the effects on glucose metabolism and insulin secretion of acute overexpression of the skeletal muscle isoform of lactate dehydrogenase (LDH)-A. Monitored in single MIN6 beta-cells, LDH hyperexpression (achieved by intranuclear cDNA microinjection or adenoviral infection) diminished the response to glucose of both phases of increases in mitochondrial NAD(P)H, as well as increases in mitochondrial membrane potential, cytosolic free ATP, and cystolic free Ca2+. These effects were observed at all glucose concentrations, but were most pronounced at submaximal glucose levels. Correspondingly, adenoviral vector-mediated LDH-A overexpression reduced insulin secretion stimulated by 11 mmol/l glucose and the subsequent response to stimulation with 30 mmol/l glucose, but it was without significant effect when the concentration of glucose was raised acutely from 3 to 30 mmol/l. Thus, overexpression of LDH activity interferes with normal glucose metabolism and insulin secretion in the islet beta-cell type, and it may therefore be directly responsible for insulin secretory defects in some forms of type 2 diabetes. The results also reinforce the view that glucose-derived pyruvate metabolism in the mitochondrion is critical for glucose-stimulated insulin secretion in the beta-cell.

  5. Ghrelin secretion stimulated by β1-adrenergic receptors in cultured ghrelinoma cells and in fasted mice

    Science.gov (United States)

    Zhao, Tong-Jin; Sakata, Ichiro; Liang, Guosheng; Richardson, James A.; Brown, Michael S.; Goldstein, Joseph L.; Zigman, Jeffrey M.

    2010-01-01

    Ghrelin, an octanoylated peptide hormone produced in the stomach, rises dramatically in mouse plasma during chronic severe calorie deprivation, an event that is essential to maintain life. The mechanism for this increase is not understood. Here, we study the control of ghrelin secretion in tissue culture cells derived from mice bearing ghrelinomas induced by a tissue-specific SV40 T-antigen transgene. We found that the ghrelin-secreting cells express high levels of mRNA encoding β1-adrenergic receptors. Addition of norepinephrine or epinephrine to the culture medium stimulated ghrelin secretion, and this effect was blocked by atenolol, a selective β1-adrenergic antagonist. When WT mice were treated with reserpine to deplete adrenergic neurotransmitters from sympathetic neurons, the fasting-induced increase in plasma ghrelin was blocked. Inhibition was also seen following atenolol administration. We conclude that ghrelin secretion during fasting is induced by adrenergic agents released by sympathetic neurons and acting directly on β1 receptors on the ghrelin-secreting cells of the stomach. PMID:20713709

  6. Metformin directly inhibits ghrelin secretion through AMP-activated protein kinase in rat primary gastric cells.

    Science.gov (United States)

    Gagnon, J; Sheppard, E; Anini, Y

    2013-03-01

    The antidiabetic drug Metformin causes weight loss in both diabetic and non-diabetic individuals. Metformin treatment is also associated with lower circulating levels of the orexigenic hormone ghrelin. To test whether Metformin directly affects ghrelin cells, rat primary stomach cells were treated with Metformin and the levels of ghrelin secretion, proghrelin gene expression and activation of adenosine monophosphate-activated protein kinase (AMPK) were examined. Metformin significantly reduced ghrelin secretion and proghrelin mRNA production and both these effects were blocked by co-incubation with the AMPK inhibitor compound C. Furthermore, the AMPK activator 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR) significantly inhibited ghrelin secretion. Additionally, ghrelin cells were shown to express AMPK. Finally, Metformin treatment caused a significant increase in the level of phosphorylated (active) AMPK. Our results show that Metformin directly inhibits stomach ghrelin production and secretion through AMPK. This reduction in ghrelin secretion may be one of the key components in Metformin's mechanism of weight loss.

  7. Ghrelin secretion stimulated by {beta}1-adrenergic receptors in cultured ghrelinoma cells and in fasted mice.

    Science.gov (United States)

    Zhao, Tong-Jin; Sakata, Ichiro; Li, Robert Lin; Liang, Guosheng; Richardson, James A; Brown, Michael S; Goldstein, Joseph L; Zigman, Jeffrey M

    2010-09-07

    Ghrelin, an octanoylated peptide hormone produced in the stomach, rises dramatically in mouse plasma during chronic severe calorie deprivation, an event that is essential to maintain life. The mechanism for this increase is not understood. Here, we study the control of ghrelin secretion in tissue culture cells derived from mice bearing ghrelinomas induced by a tissue-specific SV40 T-antigen transgene. We found that the ghrelin-secreting cells express high levels of mRNA encoding beta(1)-adrenergic receptors. Addition of norepinephrine or epinephrine to the culture medium stimulated ghrelin secretion, and this effect was blocked by atenolol, a selective beta(1)-adrenergic antagonist. When WT mice were treated with reserpine to deplete adrenergic neurotransmitters from sympathetic neurons, the fasting-induced increase in plasma ghrelin was blocked. Inhibition was also seen following atenolol administration. We conclude that ghrelin secretion during fasting is induced by adrenergic agents released by sympathetic neurons and acting directly on beta(1) receptors on the ghrelin-secreting cells of the stomach.

  8. Repair of spinal cord injury by neural stem cells transfected with brain-derived neurotrophic factor-green fluorescent protein in rats A double effect of stem cells and growth factors

    Institute of Scientific and Technical Information of China (English)

    Yansong Wang; Gang Lü

    2010-01-01

    Brain-derived neurotrophic factor(BDNF)can significantly promote nerve regeneration and repair.High expression of the BDNF-green fluorescent protein(GFP)gene persists for a long time after transfection into neural stem cells.Nevertheless,little is known about the biological characteristics of BDNF-GFP modified nerve stem cells in vivo and their ability to induce BDNF expression or repair spinal cord injury.In the present study,we transplanted BDNF-GFP transgenic neural stem cells into a hemisection model of rats.Rats with BDNF-GFP stem cells exhibited significantly increased BDNF expression and better locomotor function compared with stem cells alone.Cellular therapy with BDNF-GFP transgenic stem cells can improve outcomes better than stem cells alone and may have therapeutic potential for spinal cord injury.

  9. SALMON SOFT ROE DNA ON BLOOD CELLS SECRETION OF CYTOKINES IN HEALTHY DONORS

    Directory of Open Access Journals (Sweden)

    L. N. Fedjanina

    2005-01-01

    Full Text Available Abstract. Salmon soft roe DNA influence on healthy donors blood cells secretion of early hemopoietic factors (IL-3, GM-CSF, TNFα as well as biologically active substance influence on cytokine balance of Тh1 and Тh2 responses (IFNγ, IL-10 in vitro was studied. It is established, that DNA has modulatory effect on secretion of all investigated cytokines - IL-3, GM-CSF, TNFα, INFγ and IL-10 by blood cells of healthy donors, increases their initially low concentration, reduces initially high and does not have essential influence at an average level of their secretion. Under action of DNA IFNγ level (stimulation index=3,3 increases more significantly than IL-10 level (stimulation index =1,9. Thus, salmon soft roe DNA possesses immunomodulatory properties.

  10. Internal Ca2+ mobilization and secretion in bovine adrenal chromaffin cells

    DEFF Research Database (Denmark)

    Cheek, T R; Thastrup, Ole

    1989-01-01

    )-mobilizing muscarinic agonists to induce secretion reflects the fact that the 50 nM rise in [Ca2+]i they elicit is insufficient to trigger the exocytotic machinery. A recent report, however, has demonstrated that some of the nicotine-induced rise in [Ca2+]i could originate from the InsP3-releasable Ca2......+ store. The role of this Ca2+ store in secretion from bovine adrenal chromaffin cells is therefore unclear. In order to investigate in more detail the role of the InsP3-sensitive Ca2+ store in secretion from these cells, we have used a combination of an InsP3-mobilizing muscarinic agonist...

  11. Alendronate inhibits cell invasion and MMP-2 secretion in human chondrosarcoma cell line

    Institute of Scientific and Technical Information of China (English)

    Te-jen LAI; Yi-chin FONG; Sheng-feng HSU; Temao LI; Horng-chaung HSU; Jaung-geng LIN; Chin-jung HSU; Ming-chih CHOU; Meng-chih LEE; Shun-fa YANG

    2007-01-01

    Aim: Chondrosarcoma is a malignant primary bone tumor that responds poorly to both chemotherapy and radiation therapy. The aim of the present study was to investigate the effect of alendronate, a bisphosphonate, on the invasion and migration of human chondrosarcoma cells (JJ012). Methods: JJ012 cells were treated with alendronate of various concentrations up to 100 μmol/L for a speci-fied period, and then gelatin zymography and matrigel invasion assay was per-formed to study the effects of alendronate on matrix metalloproteinase (MMP)-2 activity and the invasion ability of JJ012 cells, respectively. Results: Our data showed that alendronate exerted a dose- and time-dependent inhibitory effect on the invasion and migration of JJ012 cells. Furthermore, gelatin zymography and RT-PCR showed that alendronate treatment decreased the activity and mRNA levels of MMP-2 in a concentration-dependent manner. Conclusion: Our find-ings suggest that alendronate may reduce MMP-2 secretion at the transcriptional and translational levels, and inhibit the invasion of chondrosarcoma cell. Therefore,alendronate may be a potential candidate for the systemic therapy of chondro-sarcomas, as well as other malignant diseases.

  12. Secretion of SerpinB2 from endothelial cells activated with inflammatory stimuli

    Energy Technology Data Exchange (ETDEWEB)

    Boncela, Joanna; Przygodzka, Patrycja [Institute of Medical Biology, Polish Academy of Sciences, Lodz (Poland); Wyroba, Elzbieta [Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw (Poland); Papiewska-Pajak, Izabela [Department of Molecular and Medical Biophysics, Medical University of Lodz (Poland); Cierniewski, Czeslaw S., E-mail: czeslaw.cierniewski@umed.lodz.pl [Institute of Medical Biology, Polish Academy of Sciences, Lodz (Poland); Department of Molecular and Medical Biophysics, Medical University of Lodz (Poland)

    2013-05-01

    Due to the lack of an N-terminal signal peptide, SerpinB2 (plasminogen activator inhibitor type 2) accumulates in cells and only a small percentage of it is secreted. The extracellular concentration of SerpinB2 significantly increases during inflammation. In the present study we investigated the mechanism with which SerpinB2 can be secreted from endothelial cells activated with LPS. We evaluated the intracellular distribution of SerpinB2 by double immunogold labeling followed by a high resolution electron microscopy analysis. We found that SerpinB2 gathers in the vesicular structures and in the endothelial cell periphery. These vesicles stained positive for the trans-Golgi network marker TGN46, which is consistent with their formation by the endoplasmatic reticulum (ER) and Golgi-dependent pathways. SerpinB2 was delivered to the plasma membrane, apparently together with TGN46 in the same vesicles, which after fusion with the membranes released cargo. Secretion of SerpinB2 was partially inhibited by brefeldin A. The secreted SerpinB2 was predominantly in its nonglycosylated 43 kDa form as evaluated by Western immunoblotting. Our data suggest that increased expression of SerpinB2 by an inflammatory stimulus is sufficient to generate structures that resemble secretory vesicles. These vesicles may represent the mechanism by which high local concentrations of SerpinB2 are released at inflammation sites from endothelial cells. - Highlights: ► LPS stimulates generation of secretory vesicles containing SerpinB2. ► SerpinB2 concentrates in TGN46 positive vesicles close to the plasma membrane. ► Brefeldin A inhibits secretion of SerpinB2. ► The secreted SerpinB2 was predominantly in its nonglycosylated 43 kDa.

  13. Unsaturated fatty acids induce mesenchymal stem cells to increase secretion of angiogenic mediators.

    Science.gov (United States)

    Smith, Andria N; Muffley, Lara A; Bell, Austin N; Numhom, Surawej; Hocking, Anne M

    2012-09-01

    Mesenchymal stem cells (MSC) represent emerging cell-based therapies for diabetes and associated complications. Ongoing clinical trials are using exogenous MSC to treat type 1 and 2 diabetes, cardiovascular disease and non-healing wounds due to diabetes. The majority of these trials are aimed at exploiting the ability of these multipotent mesenchymal stromal cells to release soluble mediators that reduce inflammation and promote both angiogenesis and cell survival at sites of tissue damage. Growing evidence suggests that MSC secretion of soluble factors is dependent on tissue microenvironment. Despite the contribution of fatty acids to the metabolic environment of type 2 diabetes, almost nothing is known about their effects on MSC secretion of growth factors and cytokines. In this study, human bone marrow-derived MSC were exposed to linoleic acid, an omega-6 polyunsaturated fatty acid, or oleic acid, a monounsaturated fatty acid, for seven days in the presence of 5.38 mM glucose. Outcomes measured included MSC proliferation, gene expression, protein secretion and chemotaxis. Linoleic and oleic acids inhibited MSC proliferation and altered MSC expression and secretion of known mediators of angiogenesis. Both unsaturated fatty acids induced MSC to increase secretion of interleukin-6, VEGF and nitric oxide. In addition, linoleic acid but not oleic acid induced MSC to increase production of interleukin-8. Collectively these data suggest that exposure to fatty acids may have functional consequences for MSC therapy. Fatty acids may affect MSC engraftment to injured tissue and MSC secretion of cytokines and growth factors that regulate local cellular responses to injury.

  14. [Therapeutic potential of human mesenchymal stromal cells secreted components: a problem with standartization].

    Science.gov (United States)

    Sagaradze, G D; Grigorieva, O A; Efimenko, A Yu; Chaplenko, A A; Suslina, S N; Sysoeva, V Yu; Kalinina, N I; Akopyan, Zh A; Tkachuk, V A

    2015-01-01

    Regenerative medicine approaches, such as replacement of damaged tissue by ex vivo manufactured constructions or stimulation of endogenous reparative and regenerative processes to treat different diseases, are actively developing. One of the major tools for regenerative medicine are stem and progenitor cells, including multipotent mesenchymal stem/stromal cells (MSC). Because the paracrine action of bioactive factors secreted by MSC is considered as a main mechanism underlying MSC regenerative effects, application of MSC extracellular secreted products could be a promising approach to stimulate tissue regeneration; it also has some advantages compared to the injection of the cells themselves. However, because of the complexity of composition and multiplicity of mechanisms of action distinguished the medicinal products based on bioactive factors secreted by human MSC from the most of pharmaceuticals, it is important to develop the approaches to their standardization and quality control. In the current study, based on the literature data and guidelines as well as on our own experimental results, we provided rationalization for nomenclature and methods of quality control for the complex of extracellular products secreted by human adipose-derived MSC on key indicators, such as "Identification", "Specific activity" and "Biological safety". Developed approaches were tested on the samples of conditioned media contained products secreted by MSC isolated from subcutaneous adipose tissue of 30 donors. This strategy for the standardization of innovative medicinal products and biomaterials based on the bioactive extracellular factors secreted by human MSC could be applicable for a wide range of bioactive complex products, produced using the different types of stem and progenitor cells.

  15. Beta-cell specific deletion of Dicer1 leads to defective insulin secretion and diabetes mellitus.

    Directory of Open Access Journals (Sweden)

    Martins Kalis

    Full Text Available Mature microRNAs (miRNAs, derived through cleavage of pre-miRNAs by the Dicer1 enzyme, regulate protein expression in many cell-types including cells in the pancreatic islets of Langerhans. To investigate the importance of miRNAs in mouse insulin secreting β-cells, we have generated mice with a β-cells specific disruption of the Dicer1 gene using the Cre-lox system controlled by the rat insulin promoter (RIP. In contrast to their normoglycaemic control littermates (RIP-Cre(+/- Dicer1(Δ/wt, RIP-Cre(+/-Dicer1(flox/flox mice (RIP-Cre Dicer1(Δ/Δ developed progressive hyperglycaemia and full-blown diabetes mellitus in adulthood that recapitulated the natural history of the spontaneous disease in mice. Reduced insulin gene expression and concomitant reduced insulin secretion preceded the hyperglycaemic state and diabetes development. Immunohistochemical, flow cytometric and ultrastructural analyses revealed altered islet morphology, marked decreased β-cell mass, reduced numbers of granules within the β-cells and reduced granule docking in adult RIP-Cre Dicer1(Δ/Δ mice. β-cell specific Dicer1 deletion did not appear to disrupt fetal and neonatal β-cell development as 2-week old RIP-Cre Dicer1(Δ/Δ mice showed ultrastructurally normal β-cells and intact insulin secretion. In conclusion, we have demonstrated that a β-cell specific disruption of the miRNAs network, although allowing for apparently normal β-cell development, leads to progressive impairment of insulin secretion, glucose homeostasis and diabetes development.

  16. Brain-Derived Neurotrophic Factor Loaded PS80 PBCA Nanocarrier for In Vitro Neural Differentiation of Mouse Induced Pluripotent Stem Cells

    Science.gov (United States)

    Chung, Chiu-Yen; Lin, Martin Hsiu-Chu; Lee, I-Neng; Lee, Tsong-Hai; Lee, Ming-Hsueh; Yang, Jen-Tsung

    2017-01-01

    Brain derived neurotrophic factor (BDNF) can induce neural differentiation in stem cells and has the potential for repair of the nervous system. In this study, a polysorbate 80-coated polybutylcyanoacrylate nanocarrier (PS80 PBCA NC) was constructed to deliver plasmid DNAs (pDNAs) containing BDNF gene attached to a hypoxia-responsive element (HRE-cmvBDNF). The hypoxia-sensing mechanism of BDNF expression and inductiveness of the nano-formulation on mouse induced pluripotent stem cells (iPSCs) to differentiate into neurons following hypoxia was tested in vitro with immunofluorescent staining and Western blotting. The HRE-cmvBDNF appeared to adsorb onto the surface of PS80 PBCA NC, with a resultant mean diameter of 92.6 ± 1.0 nm and zeta potential of −14.1 ± 1.1 mV. HIF-1α level in iPSCs was significantly higher in hypoxia, which resulted in a 51% greater BDNF expression when transfected with PS80 PBCA NC/HRE-cmvBDNF than those without hypoxia. TrkB and phospho-Akt were also elevated which correlated with neural differentiation. The findings suggest that PS80 PBCA NC too can be endocytosed to serve as an efficient vector for genes coupled to the HRE in hypoxia-sensitive cells, and activation of the PI3/Akt pathway in iPSCs by BDNF is capable of neural lineage specification. PMID:28335495

  17. Adenoviral-mediated glial cell line-derived neurotrophic factor gene transfer has a protective effect on sciatic nerve following constriction-induced spinal cord injury.

    Science.gov (United States)

    Chou, An-Kuo; Yang, Ming-Chang; Tsai, Hung-Pei; Chai, Chee-Yin; Tai, Ming-Hong; Kwan, Aij-Li; Hong, Yi-Ren

    2014-01-01

    Neuropathic pain due to peripheral nerve injury may be associated with abnormal central nerve activity. Glial cell-line-derived neurotrophic factor (GDNF) can help attenuate neuropathic pain in different animal models of nerve injury. However, whether GDNF can ameliorate neuropathic pain in the spinal cord dorsal horn (SCDH) in constriction-induced peripheral nerve injury remains unknown. We investigated the therapeutic effects of adenoviral-mediated GDNF on neuropathic pain behaviors, microglial activation, pro-inflammatory cytokine expression and programmed cell death in a chronic constriction injury (CCI) nerve injury animal model. In this study, neuropathic pain was produced by CCI on the ipsilateral SCDH. Mechanical allodynia was examined with von Frey filaments and thermal sensitivity was tested using a plantar test apparatus post-operatively. Target proteins GDNF-1, GDNFRa-1, MMP2, MMP9, p38, phospho-p38, ED1, IL6, IL1β, AIF, caspase-9, cleaved caspase-9, caspase-3, cleaved caspase-3, PARP, cleaved PARP, SPECTRIN, cleaved SPECTRIN, Beclin-1, PKCσ, PKCγ, iNOS, eNOS and nNOS were detected. Microglial activity was measured by observing changes in immunoreactivity with OX-42. NeuN and TUNEL staining were used to reveal whether apoptosis was attenuated by GDNF. Results showed that administrating GDNF began to attenuate both allodynia and thermal hyperalgesia at day 7. CCI-rats were found to have lower GDNF and GDNFRa-1 expression compared to controls, and GDNF re-activated their expression. Also, GDNF significantly down-regulated CCI-induced protein expression except for MMP2, eNOS and nNOS, indicating that the protective action of GDNF might be associated with anti-inflammation and prohibition of microglia activation. Immunocytochemistry staining showed that GDNF reduced CCI-induced neuronal apoptosis. In sum, GDNF enhanced the neurotrophic effect by inhibiting microglia activation and cytokine production via p38 and PKC signaling. GDNF could be a good

  18. Soluble adenylyl cyclase is an acid-base sensor in epithelial base-secreting cells.

    Science.gov (United States)

    Roa, Jinae N; Tresguerres, Martin

    2016-08-01

    Blood acid-base regulation by specialized epithelia, such as gills and kidney, requires the ability to sense blood acid-base status. Here, we developed primary cultures of ray (Urolophus halleri) gill cells to study mechanisms for acid-base sensing without the interference of whole animal hormonal regulation. Ray gills have abundant base-secreting cells, identified by their noticeable expression of vacuolar-type H(+)-ATPase (VHA), and also express the evolutionarily conserved acid-base sensor soluble adenylyl cyclase (sAC). Exposure of cultured cells to extracellular alkalosis (pH 8.0, 40 mM HCO3 (-)) triggered VHA translocation to the cell membrane, similar to previous reports in live animals experiencing blood alkalosis. VHA translocation was dependent on sAC, as it was blocked by the sAC-specific inhibitor KH7. Ray gill base-secreting cells also express transmembrane adenylyl cyclases (tmACs); however, tmAC inhibition by 2',5'-dideoxyadenosine did not prevent alkalosis-dependent VHA translocation, and tmAC activation by forskolin reduced the abundance of VHA at the cell membrane. This study demonstrates that sAC is a necessary and sufficient sensor of extracellular alkalosis in ray gill base-secreting cells. In addition, this study indicates that different sources of cAMP differentially modulate cell biology.

  19. IL13 activates autophagy to regulate secretion in airway epithelial cells.

    Science.gov (United States)

    Dickinson, John D; Alevy, Yael; Malvin, Nicole P; Patel, Khushbu K; Gunsten, Sean P; Holtzman, Michael J; Stappenbeck, Thaddeus S; Brody, Steven L

    2016-01-01

    Cytokine modulation of autophagy is increasingly recognized in disease pathogenesis, and current concepts suggest that type 1 cytokines activate autophagy, whereas type 2 cytokines are inhibitory. However, this paradigm derives primarily from studies of immune cells and is poorly characterized in tissue cells, including sentinel epithelial cells that regulate the immune response. In particular, the type 2 cytokine IL13 (interleukin 13) drives the formation of airway goblet cells that secrete excess mucus as a characteristic feature of airway disease, but whether this process is influenced by autophagy was undefined. Here we use a mouse model of airway disease in which IL33 (interleukin 33) stimulation leads to IL13-dependent formation of airway goblet cells as tracked by levels of mucin MUC5AC (mucin 5AC, oligomeric mucus/gel forming), and we show that these cells manifest a block in mucus secretion in autophagy gene Atg16l1-deficient mice compared to wild-type control mice. Similarly, primary-culture human tracheal epithelial cells treated with IL13 to stimulate mucus formation also exhibit a block in MUC5AC secretion in cells depleted of autophagy gene ATG5 (autophagy-related 5) or ATG14 (autophagy-related 14) compared to nondepleted control cells. Our findings indicate that autophagy is essential for airway mucus secretion in a type 2, IL13-dependent immune disease process and thereby provide a novel therapeutic strategy for attenuating airway obstruction in hypersecretory inflammatory diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis lung disease. Taken together, these observations suggest that the regulation of autophagy by Th2 cytokines is cell-context dependent.

  20. Activated human T cells secrete exosomes that participate in IL-2 mediated immune response signaling.

    Directory of Open Access Journals (Sweden)

    Jessica Wahlgren

    Full Text Available It has previously been shown that nano-meter sized vesicles (30-100 nm, exosomes, secreted by antigen presenting cells can induce T cell responses thus showing the potential of exosomes to be used as immunological tools. Additionally, activated CD3⁺ T cells can secrete exosomes that have the ability to modulate different immunological responses. Here, we investigated what effects exosomes originating from activated CD3⁺ T cells have on resting CD3⁺ T cells by studying T cell proliferation, cytokine production and by performing T cell and exosome phenotype characterization. Human exosomes were generated in vitro following CD3⁺ T cell stimulation with anti-CD28, anti-CD3 and IL-2. Our results show that exosomes purified from stimulated CD3⁺ T cells together with IL-2 were able to generate proliferation in autologous resting CD3⁺ T cells. The CD3⁺ T cells stimulated with exosomes together with IL-2 had a higher proportion of CD8⁺ T cells and had a different cytokine profile compared to controls. These results indicate that activated CD3⁺ T cells communicate with resting autologous T cells via exosomes.

  1. Neuron-like differentiation of adult rat bone marrow stromal cells induced by transforming growth factor-beta and brain-derived neurotrophic factor

    Institute of Scientific and Technical Information of China (English)

    Chang Liu; Xifan Mei; Gang Lü; Yansong Wang; Quanshuang Li; Zhanpeng Guo

    2009-01-01

    BACKGROUND: It has been demonstrated that transforming growth factor-β (TGF-β) and brain-derived neurotrophic factor (BDNF) can induce stem cell differentiation into neuron-like cells.OBJECTIVE: To investigate the efficacy of TGF-β and BDNF at inducing the differentiation of adult rat bone marrow stromal cells (BMSCs) into neuron-like cells, both in combination or alone.DESIGN, TIME AND SETTING: A comparative observation experiment was performed at the Department of Orthopedics, First Affiliated Hospital of Liaoning Medical University between October 2007 and January 2008.MATERIALS: TGF-βand BDNF were purchased from Sigma, USA; mouse anti-rat neuron specific enolase, neurofilament and glial fibrillary acidic protein were purchased from Beijing HMHL Biochem Ltd., China.METHODS: BMSCs were isolated from rats aged 4 weeks and incubated with TGF-β(1μg/L) and/or BDNF (50μg/mL).MAIN OUTCOME MEASURES: Expression of neuron-specific enolase, neurofilament and glial fibrillary acidic protein were determined by immunocytochemistry.RESULTS: BMSCs differentiated into neuron-like cells following induction of TGF-β and BDNF, and expressed both neuron-specific enolase and neurofilament. The percent of positive cells was significantly greater in the combination group than those induced with TGF-β or BDNF alone (P<0.01).CONCLUSION: Treatment of BMSCs with a combination of TGF-β and BDNF induced differentiation into neuron-like cells, with the induction being significantly greater than with TGF-β or BDNF alone.

  2. Bacillus subtilis: from soil bacterium to super-secreting cell factory

    Directory of Open Access Journals (Sweden)

    van Dijl Jan Maarten

    2013-01-01

    Full Text Available Abstract The biotechnology industry has become a key element in modern societies. Within this industry, the production of recombinant enzymes and biopharmaceutical proteins is of major importance. The global markets for such recombinant proteins are growing rapidly and, accordingly, there is a continuous need for new production platforms that can deliver protein products in greater yields, with higher quality and at lower costs. This calls for the development of next-generation super-secreting cell factories. One of the microbial cell factories that can meet these challenges is the Gram-positive bacterium Bacillus subtilis, an inhabitant of the upper layers of the soil that has the capacity to secrete proteins in the gram per litre range. The engineering of B. subtilis into a next-generation super-secreting cell factory requires combined Systems and Synthetic Biology approaches. In this way, the bacterial protein secretion machinery can be optimized from the single molecule to the network level while, at the same time, taking into account the balanced use of cellular resources. Although highly ambitious, this is an achievable objective due to recent advances in functional genomics and Systems- and Synthetic Biological analyses of B. subtilis cells.

  3. Palmitic acid and linoleic acid metabolism in Caco-2 cells: Different triglyceride synthesis and lipoprotein secretion

    NARCIS (Netherlands)

    van Greevenbroek, M.M.J.; Voorhout, W.F.; Erkelens, D.W.; van Meer, G.; de Bruin, T.W.A.

    1995-01-01

    Polarized monolayers of intestinal Caco-2 cells were used to study the effects of saturated palmitic acid (16:0) and polyunsaturated linoleic acid (18:2) on triglyceride synthesis and lipoprotein secretion. Monolayers were incubated for 24 h, at the apical or lumenal side, with palmitic acid (16:0)

  4. Secretion of SerpinB2 from endothelial cells activated with inflammatory stimuli.

    Science.gov (United States)

    Boncela, Joanna; Przygodzka, Patrycja; Wyroba, Elzbieta; Papiewska-Pajak, Izabela; Cierniewski, Czeslaw S

    2013-05-01

    Due to the lack of an N-terminal signal peptide, SerpinB2 (plasminogen activator inhibitor type 2) accumulates in cells and only a small percentage of it is secreted. The extracellular concentration of SerpinB2 significantly increases during inflammation. In the present study we investigated the mechanism with which SerpinB2 can be secreted from endothelial cells activated with LPS. We evaluated the intracellular distribution of SerpinB2 by double immunogold labeling followed by a high resolution electron microscopy analysis. We found that SerpinB2 gathers in the vesicular structures and in the endothelial cell periphery. These vesicles stained positive for the trans-Golgi network marker TGN46, which is consistent with their formation by the endoplasmatic reticulum (ER) and Golgi-dependent pathways. SerpinB2 was delivered to the plasma membrane, apparently together with TGN46 in the same vesicles, which after fusion with the membranes released cargo. Secretion of SerpinB2 was partially inhibited by brefeldin A. The secreted SerpinB2 was predominantly in its nonglycosylated 43kDa form as evaluated by Western immunoblotting. Our data suggest that increased expression of SerpinB2 by an inflammatory stimulus is sufficient to generate structures that resemble secretory vesicles. These vesicles may represent the mechanism by which high local concentrations of SerpinB2 are released at inflammation sites from endothelial cells.

  5. The Comparison of Adipose Stem Cell and Placental Stem Cell in Secretion Characteristics and in Facial Antiaging

    Directory of Open Access Journals (Sweden)

    Yan Xu

    2016-01-01

    Full Text Available Background. Mesenchymal stem cells are the most commonly used seed cells in biomedical research and tissue engineering. Their secretory proteins have also been proven to play an important role in tissue healing. Methods. We isolated adipose stem cells and placental stem cells and performed analysis examining characteristics. The secretory proteins were extracted from conditioned medium and analyzed by MALDI-TOF/TOF. The antiaging effect of conditioned mediums was evaluated by the results of facial skin application. Results. Adipose stem cells and placental stem cells were found to be very similar in their surface markers and multipotency. The specific proteins secreted from adipose stem cells were more adept at cell adhesion, migration, wound healing, and tissue remodeling, while the proteins secreted by placental stem cells were more adept at angiogenesis, cell proliferation, differentiation, cell survival, immunomodulation, and collagen degradation. While these two types of conditioned medium could improve the facial index, the improvement of Melanin index after injection of the adipose stem cell conditioned medium was much more significant. Conclusion. The results suggest that the secreted proteins are ideal cell-free substances for regeneration medicine, especially in the antiaging field.

  6. Neurotrophic Effects of Mu Bie Zi (Momordica cochinchinensis) Seed Elucidated by High-Throughput Screening of Natural Products for NGF Mimetic Effects in PC-12 Cells

    Science.gov (United States)

    Mazzio, E.; Georges, B.; McTier, O.; Soliman, Karam F. A.

    2015-01-01

    Post-mitotic central nervous system (CNS) neurons have limited capacity for regeneration, creating a challenge in the development of effective therapeutics for spinal cord injury or neurodegenerative diseases. Furthermore, therapeutic use of human neurotrophic agents such as nerve growth factor (NGF) are limited due to hampered transport across the blood brain barrier (BBB) and a large number of peripheral side effects (e.g. neuro-inflammatory pain/tissue degeneration etc.). Therefore, there is a continued need for discovery of small molecule NGF mimetics that can penetrate the BBB and initiate CNS neuronal outgrowth/regeneration. In the current study, we conduct an exploratory high-through-put (HTP) screening of 1144 predominantly natural/herb products (947 natural herbs/plants/spices, 29 polyphenolics and 168 synthetic drugs) for ability to induce neurite outgrowth in PC12 dopaminergic cells grown on rat tail collagen, over 7 days. The data indicate a remarkably rare event-low hit ratio with only 1/1144 tested substances (<111.25 µg/mL) being capable of inducing neurite outgrowth in a dose dependent manner, identified as; Mu Bie Zi, Momordica cochinchinensis seed extract (MCS). To quantify the neurotrophic effects of MCS, 36 images (n = 6) (average of 340 cells per image), were numerically assessed for neurite length, neurite count/cell and min/max neurite length in microns (µm) using Image J software. The data show neurite elongation from 0.07 ± 0.02 µm (controls) to 5.5 ± 0.62 µm (NGF 0.5 μg/mL) and 3.39 ± 0.45 µm (138 μg/mL) in MCS, where the average maximum length per group extended from 3.58 ± 0.42 µm (controls) to 41.93 ± 3.14 µm (NGF) and 40.20 ± 2.72 µm (MCS). Imaging analysis using immunocytochemistry (ICC) confirmed that NGF and MCS had similar influence on 3-D orientation/expression of 160/200 kD neurofilament, tubulin and F-actin. These latent changes were associated with early rise in phosphorylated extracellular signal

  7. Galectin-3 Binding Protein Secreted by Breast Cancer Cells Inhibits Monocyte-Derived Fibrocyte Differentiation.

    Science.gov (United States)

    White, Michael J V; Roife, David; Gomer, Richard H

    2015-08-15

    To metastasize, tumor cells often need to migrate through a layer of collagen-containing scar tissue which encapsulates the tumor. A key component of scar tissue and fibrosing diseases is the monocyte-derived fibrocyte, a collagen-secreting profibrotic cell. To test the hypothesis that invasive tumor cells may block the formation of the fibrous sheath, we determined whether tumor cells secrete factors that inhibit monocyte-derived fibrocyte differentiation. We found that the human metastatic breast cancer cell line MDA-MB-231 secretes activity that inhibits human monocyte-derived fibrocyte differentiation, whereas less aggressive breast cancer cell lines secrete less of this activity. Purification indicated that Galectin-3 binding protein (LGALS3BP) is the active factor. Recombinant LGALS3BP inhibits monocyte-derived fibrocyte differentiation, and immunodepletion of LGALS3BP from MDA-MB 231 conditioned media removes the monocyte-derived fibrocyte differentiation-inhibiting activity. LGALS3BP inhibits the differentiation of monocyte-derived fibrocytes from wild-type mouse spleen cells, but not from SIGN-R1(-/-) mouse spleen cells, suggesting that CD209/SIGN-R1 is required for the LGALS3BP effect. Galectin-3 and galectin-1, binding partners of LGALS3BP, potentiate monocyte-derived fibrocyte differentiation. In breast cancer biopsies, increased levels of tumor cell-associated LGALS3BP were observed in regions of the tumor that were invading the surrounding stroma. These findings suggest LGALS3BP and galectin-3 as new targets to treat metastatic cancer and fibrosing diseases.

  8. Megakaryocytes regulate hematopoietic stem cell quiescence via Cxcl4 secretion

    Science.gov (United States)

    Bruns, Ingmar; Lucas, Daniel; Pinho, Sandra; Ahmed, Jalal; Lambert, Michele P.; Kunisaki, Yuya; Scheiermann, Christoph; Schiff, Lauren; Poncz, Mortimer; Bergman, Aviv; Frenette, Paul S.

    2014-01-01

    In the bone marrow (BM), hematopoietic stem cells (HSCs) lodge in specialized microenvironments that tightly control their proliferative state to adapt to the varying needs for replenishment of blood cells while also preventing exhaustion1. All putative niche cells suggested thus far have a non-hematopoietic origin2-8. Thus, it remains unclear how feedback from mature cells is conveyed to HSCs to adjust proliferation. Here we show that megakaryocytes (Mk) can directly regulate HSC pool size. Three-dimensional whole-mount imaging revealed that endogenous HSCs are frequently located adjacent to Mk in a non-random fashion. Selective in vivo depletion of Mk resulted in specific loss of HSC quiescence and led to a marked expansion of functional HSCs. Gene expression analyses revealed that Mk were the source of chemokine C-X-C motif ligand 4 (Cxcl4, also named platelet factor 4, Pf4) in the BM and Cxcl4 injection reduced HSC numbers via increased quiescence. By contrast, Cxcl4−/− mice exhibited increased HSC numbers and proliferation. Combined use of whole-mount imaging and computational modelling was highly suggestive of a megakaryocytic niche capable of influencing independently HSC maintenance by regulating quiescence. Thus, these results indicate that a terminally differentiated HSC progeny contributes to niche activity by directly regulating HSC behavior. PMID:25326802

  9. Combination effects of epidermal growth factor and glial cell line-derived neurotrophic factor on the in vitro developmental potential of porcine oocytes

    DEFF Research Database (Denmark)

    Valleh, Mehdi Vafaye; Rasmussen, Mikkel Aabech; Hyttel, Poul

    2016-01-01

    of improving this issue, the single and combined effects of epidermal growth factor (EGF) and glial cell line-derived neurotrophic factor (GDNF) on oocyte developmental competence were investigated. Porcine cumulus–oocyte cell complexes (COCs) were matured in serum-free medium supplemented with EGF (0, 10...... or 50 ng/ml) and/or GDNF (0, 10 or 50 ng/ml) for 44 h, and subsequently subjected to fertilization and cultured for 7 days in vitro. The in vitro-formed blastocysts derived from selected growth factor groups (i.e. EGF = 50 ng/ml; GDNF = 50 ng/ml; EGF = 50 ng/ml + GDNF = 50 ng/ml) were also used for m......RNA expression analysis, or were subjected to Hoechst staining. The results showed that the addition of EGF and/or GDNF during oocyte maturation dose dependently enhanced oocyte developmental competence. Compared with the embryos obtained from control or single growth factor-treated oocytes, treatment...

  10. Glial cell line-derived neurotrophic factor protects against high-fat diet-induced hepatic steatosis by suppressing hepatic PPAR-γ expression.

    Science.gov (United States)

    Mwangi, Simon Musyoka; Peng, Sophia; Nezami, Behtash Ghazi; Thorn, Natalie; Farris, Alton B; Jain, Sanjay; Laroui, Hamed; Merlin, Didier; Anania, Frank; Srinivasan, Shanthi

    2016-01-15

    Glial cell line-derived neurotrophic factor (GDNF) protects against high-fat diet (HFD)-induced hepatic steatosis in mice, however, the mechanisms involved are not known. In this study we investigated the effects of GDNF overexpression and nanoparticle delivery of GDNF in mice on hepatic steatosis and fibrosis and the expression of genes involved in the regulation of hepatic lipid uptake and de novo lipogenesis. Transgenic overexpression of GDNF in liver and other metabolically active tissues was protective against HFD-induced hepatic steatosis. Mice overexpressing GDNF had significantly reduced P62/sequestosome 1 protein levels suggestive of accelerated autophagic clearance. They also had significantly reduced peroxisome proliferator-activated receptor-γ (PPAR-γ) and CD36 gene expression and protein levels, and lower expression of mRNA coding for enzymes involved in de novo lipogenesis. GDNF-loaded nanoparticles were protective against short-term HFD-induced hepatic steatosis and attenuated liver fibrosis in mice with long-standing HFD-induced hepatic steatosis. They also suppressed the liver expression of steatosis-associated genes. In vitro, GDNF suppressed triglyceride accumulation in Hep G2 cells through enhanced p38 mitogen-activated protein kinase-dependent signaling and inhibition of PPAR-γ gene promoter activity. These results show that GDNF acts directly in the liver to protect against HFD-induced cellular stress and that GDNF may have a role in the treatment of nonalcoholic fatty liver disease.

  11. The mechanisms of insulin secretion and calcium signaling in pancreatic β-cells exposed to fluoroquinolones.

    Science.gov (United States)

    Bito, Motoki; Tomita, Takashi; Komori, Mika; Taogoshi, Takanori; Kimura, Yasuhiro; Kihira, Kenji

    2013-01-01

    Fluoroquinolones reportedly induce hypoglycemia through stimulation of insulin secretion from pancreatic β-cells via inhibition of K(ATP) channels and activation of L-type voltage-dependent Ca(2+) channels. In physiological condition, the cytosolic Ca(2+) concentration ([Ca(2+)](c)) is also regulated by release of Ca(2+) from intracellular Ca(2+) stores. In this study, we investigated the mechanism of insulin secretion induced by fluoroquinolones, with respect to intracellular Ca(2+) stores. Even where the absence of supplemental extracellular Ca(2+), insulin secretion and [Ca(2+)](c) were increased by gatifloxacin, levofloxacin or tolbutamide. Insulin secretion and the rise of [Ca(2+)](c) induced by fluoroquinolones were reduced by depleting of Ca(2+) in endoplasmic reticumum (ER) by thapsigargin, and inhibiting ryanodine receptor of ER by dantrolene. Inhibition of inositol 1,4,5-triphosphate receptor of ER by xestospongin C suppressed insulin secretion induced by fluoroquinolones, whereas it did not affect [Ca(2+)](c). Destruction of acidic Ca(2+) stores such as lysosome and lysosome-related organelles by glycyl-L-phenylalanine-2-nephthylamide (GPN) did not affect insulin secretion and the rise of [Ca(2+)](c) induced by fluoroquinolones. The increase in insulin and [Ca(2+)](c) induced by tolbutamide were reduced by thapsigargin, dantrolene, and GPN but not by xestospongin C. In conclusion, fluoroquinolones induces Ca(2+) release from ER mediated by the ryanodine receptor, and the reaction might involve in insulin secretion. Sulfonylureas induce Ca(2+) release from GPN-sensitive acidic Ca(2+) stores, but fluoroquinolones did not.

  12. Regulation of retinoschisin secretion in Weri-Rb1 cells by the F-actin and microtubule cytoskeleton.

    Directory of Open Access Journals (Sweden)

    Eiko Kitamura

    Full Text Available Retinoschisin is encoded by the gene responsible for X-linked retinoschisis (XLRS, an early onset macular degeneration that results in a splitting of the inner layers of the retina and severe loss in vision. Retinoschisin is predominantly expressed and secreted from photoreceptor cells as a homo-oligomer protein; it then associates with the surface of retinal cells and maintains the retina cellular architecture. Many missense mutations in the XLRS1 gene are known to cause intracellular retention of retinoschisin, indicating that the secretion process of the protein is a critical step for its normal function in the retina. However, the molecular mechanisms underlying retinoschisin's secretion remain to be fully elucidated. In this study, we investigated the role of the F-actin cytoskeleton in the secretion of retinoschisin by treating Weri-Rb1 cells, which are known to secrete retinoschisin, with cytochalasin D, jasplakinolide, Y-27632, and dibutyryl cGMP. Our results show that cytochalasin D and jasplakinolide inhibit retinoschisin secretion, whereas Y-27632 and dibutyryl cGMP enhance secretion causing F-actin alterations. We also demonstrate that high concentrations of taxol, which hyperpolymerizes microtubules, inhibit retinoschisin secretion. Our data suggest that retinoschisin secretion is regulated by the F-actin cytoskeleton, that cGMP or inhibition of ROCK alters F-actin structure enhancing the secretion, and that the microtubule cytoskeleton is also involved in this process.

  13. Pro-neurotrophins secreted from retinal ganglion cell axons are necessary for ephrinA-p75NTR-mediated axon guidance

    Directory of Open Access Journals (Sweden)

    Broom Emma R

    2010-11-01

    Full Text Available Abstract Background Retinotectal map formation develops via topographically specific guidance and branching of retinal axons in their target area. This process is controlled, in part, by reverse signalling of ephrinAs expressed on retinal axons. As glycosylphosphatidylinositol-anchored molecules, ephrinAs require transmembrane co-receptors to exert this function, for which the two neurotrophin receptors, p75NTR and TrkB, were recently proposed. Results We show here that the ligands for these receptors, the brain-derived neurotrophic factor precursor (proBDNF and its processed form, BDNF, respectively, control the branching of retinal axons antagonistically, which they mediate by inducing the corresponding neurotrophin receptor-ephrinA complexes. Moreover, scavenging proneurotrophins, by adding antibodies specific for the pro-domain of proBNDF or a soluble extracellular domain of p75NTR, abolish repellent ephrinA reverse signalling in the stripe assay. Conclusions This indicates that retinal cells secrete proneurotrophins, inducing the ephrinA-p75NTR interaction and enabling repellent axon guidance. The antagonistic functions of proBDNF and BDNF raise the possibility that topographic branching is controlled by local control of processing of proneurotrophins.

  14. Rapamycin promotes Schwann cell migration and nerve growth factor secretion

    OpenAIRE

    Liu, Fang; Zhang, Haiwei; Zhang, Kaiming; Wang, Xinyu; Li, Shipu; Yin, Yixia

    2014-01-01

    Rapamycin, similar to FK506, can promote neural regeneration in vitro. We assumed that the mechanisms of action of rapamycin and FK506 in promoting peripheral nerve regeneration were similar. This study compared the effects of different concentrations of rapamycin and FK506 on Schwann cells and investigated effects and mechanisms of rapamycin on improving peripheral nerve regeneration. Results demonstrated that the lowest rapamycin concentration (1.53 nmol/L) more significantly promoted Schwa...

  15. Modification of N-glycosylation sites allows secretion of bacterial chondroitinase ABC from mammalian cells.

    Science.gov (United States)

    Muir, Elizabeth M; Fyfe, Ian; Gardiner, Sonya; Li, Li; Warren, Philippa; Fawcett, James W; Keynes, Roger J; Rogers, John H

    2010-01-15

    Although many eukaryotic proteins have been secreted by transfected bacterial cells, little is known about how a bacterial protein is treated as it passes through the secretory pathway when expressed in a eukaryotic cell. The eukaryotic N-glycosylation system could interfere with folding and secretion of prokaryotic proteins whose sequence has not been adapted for glycosylation in structurally appropriate locations. Here we show that such interference does indeed occur for chondroitinase ABC from the bacterium Proteus vulgaris, and can be overcome by eliminating potential N-glycosylation sites. Chondroitinase ABC was heavily glycosylated when expressed in mammalian cells or in a mammalian translation system, and this process prevented secretion of functional enzyme. Directed mutagenesis of selected N-glycosylation sites allowed efficient secretion of active chondroitinase. As these proteoglycans are known to inhibit regeneration of axons in the mammalian central nervous system, the modified chondroitinase gene is a potential tool for gene therapy to promote neural regeneration, ultimately in human spinal cord injury.

  16. Taurine reduces the secretion of apolipoprotein B100 and lipids in HepG2 cells

    Directory of Open Access Journals (Sweden)

    Nagao Koji

    2008-10-01

    Full Text Available Abstract Background Higher concentrations of serum lipids and apolipoprotein B100 (apoB are major individual risk factors of atherosclerosis and coronary heart disease. Therefore ameliorative effects of food components against the diseases are being paid attention in the affluent countries. The present study was undertaken to investigate the effect of taurine on apoB secretion and lipid metabolism in human liver model HepG2 cells. Results The results demonstrated that an addition of taurine to the culture media reduces triacylglycerol (TG-mass in the cells and the medium. Similarly, cellular cholesterol-mass was decreased. Taurine inhibited the incorporation of [14C] oleate into cellular and medium TG, suggesting the inhibition of TG synthesis. In addition, taurine reduced the synthesis of cellular cholesterol ester and its secretion, suggesting the inhibition of acyl-coenzyme A:cholesterol acyltransferase activity. Furthermore, taurine reduced the secretion of apoB, which is a major protein component of very low-density lipoprotein. Conclusion This is a first report to demonstrate that taurine inhibits the secretion of apoB from HepG2 cells.

  17. Ixodes scapularis saliva mitigates inflammatory cytokine secretion during Anaplasma phagocytophilum stimulation of immune cells

    Directory of Open Access Journals (Sweden)

    Chen Gang

    2012-10-01

    Full Text Available Abstract Background Ixodes scapularis saliva enables the transmission of infectious agents to the mammalian host due to its immunomodulatory, anesthetic and anti-coagulant properties. However, how I. scapularis saliva influences host cytokine secretion in the presence of the obligate intracellular rickettsial pathogen Anaplasma phagocytophilum remains elusive. Methods Bone marrow derived macrophages (BMDMs were stimulated with pathogen associated molecular patterns (PAMPs and A. phagocytophilum. Cytokine secretion was measured in the presence and absence of I. scapularis saliva. Human peripheral blood mononuclear cells (PBMCs were also stimulated with Tumor Necrosis Factor (TNF-α in the presence and absence of I. scapularis saliva and interleukin (IL-8 was measured. Results I. scapularis saliva inhibits inflammatory cytokine secretion by macrophages during stimulation of Toll-like (TLR and Nod-like receptor (NLR signaling pathways. The effect of I. scapularis saliva on immune cells is not restricted to murine macrophages because decreasing levels of interleukin (IL-8 were observed after TNF-α stimulation of human peripheral blood mononuclear cells. I. scapularis saliva also mitigates pro-inflammatory cytokine response by murine macrophages during challenge with A. phagocytophilum. Conclusions These findings suggest that I. scapularis may inhibit inflammatory cytokine secretion during rickettsial transmission at the vector-host interface.

  18. Aldosterone induced galectin-3 secretion in vitro and in vivo: from cells to humans.

    Directory of Open Access Journals (Sweden)

    Yen-Hung Lin

    Full Text Available Patients with primary aldosteronism are associated with increased myocardial fibrosis. Galectin-3 is one of the most important mediators between macrophage activation and myocardial fibrosis.To investigate whether aldosterone induces galectin-3 secretion in vitro and in vivo.We investigated the possible molecular mechanism of aldosterone-induced galectin-3 secretion in macrophage cell lines (THP-1 and RAW 264.7 cells. Aldosterone induced galectin-3 secretion through mineralocorticoid receptors via the PI3K/Akt and NF-κB transcription signaling pathways. In addition, aldosterone-induced galectin-3 expression enhanced fibrosis-related factor expression in fibroblasts. We observed that galectin-3 mRNA from peripheral blood mononuclear cells and serum galectin-3 levels were both significantly increased in mice implanted with aldosterone pellets on days 7 and 14. We then conducted a prospective preliminary clinical study to investigate the association between aldosterone and galectin-3. Patients with aldosterone-producing adenoma had a significantly higher plasma galectin-3 level than patients with essential hypertension. One year after adrenalectomy, the plasma galectin-3 level had decreased significantly in the patients with aldosterone-producing adenoma.This study demonstrated that aldosterone could induce galectin-3 secretion in vitro and in vivo.

  19. Regulation of Pancreatic Beta Cell Stimulus-Secretion Coupling by microRNAs

    Directory of Open Access Journals (Sweden)

    Jonathan L. S. Esguerra

    2014-11-01

    Full Text Available Increased blood glucose after a meal is countered by the subsequent increased release of the hypoglycemic hormone insulin from the pancreatic beta cells. The cascade of molecular events encompassing the initial sensing and transport of glucose into the beta cell, culminating with the exocytosis of the insulin large dense core granules (LDCVs is termed “stimulus-secretion coupling.” Impairment in any of the relevant processes leads to insufficient insulin release, which contributes to the development of type 2 diabetes (T2D. The fate of the beta cell, when exposed to environmental triggers of the disease, is determined by the possibility to adapt to the new situation by regulation of gene expression. As established factors of post-transcriptional regulation, microRNAs (miRNAs are well-recognized mediators of beta cell plasticity and adaptation. Here, we put focus on the importance of comprehending the transcriptional regulation of miRNAs, and how miRNAs are implicated in stimulus-secretion coupling, specifically those influencing the late stages of insulin secretion. We suggest that efficient beta cell adaptation requires an optimal balance between transcriptional regulation of miRNAs themselves, and miRNA-dependent gene regulation. The increased knowledge of the beta cell transcriptional network inclusive of non-coding RNAs such as miRNAs is essential in identifying novel targets for the treatment of T2D.

  20. Continuous plant cell perfusion culture: bioreactor characterization and secreted enzyme production.

    Science.gov (United States)

    Su, Wei Wen; Arias, Renee

    2003-01-01

    Culture perfusion is widely practiced in mammalian cell processes to enhance secreted antibody production. Here, we report the development of an efficient continuous perfusion process for the cultivation of plant cell suspensions. The key to this process is a perfusion bioreactor that incorporates an annular settling zone into a stirred-tank bioreactor to achieve continuous cell/medium separation via gravitational sedimentation. From washout experiments, we found that under typical operating conditions (e.g., 200 rpm and 0.3 vvm) the liquid phase in the entire perfusion bioreactor was homogeneous despite the presence of the cylindrical baffle. Using secreted acid phosphatase (APase) produced in Anchusa officinalis cell culture as a model we have studied the perfusion cultures under complete or partial cell retention. The perfusion culture was operated under phosphate limitation to stimulate APase production. Successful operation of the perfusion process over four weeks has been achieved in this work. When A. officinalis cells were grown in the perfusion reactor and perfused at up to 0.4 vvd with complete cell retention, a cell dry weight exceeding 20 g/l could be achieved while secreted APase productivity leveled off at approximately 300 units/l/d. The culture became extremely dense with the maximum packed cell volume (PCV) surpassing 70%. In comparison, the maximum cell dry weight and overall secreted APase productivity in a typical batch culture were 10-12 g/l and 100-150 units/l/d, respectively. Operation of the perfusion culture under extremely high PCV for a prolonged period, however, led to declined oxygen uptake and reduced viability. Subsequently, cell removal via a bleed stream at up to 0.11 vvd was tested and shown to stabilize the culture at a PCV below 60%. With culture bleeding, both specific oxygen uptake rate and viability were shown to increase. This also led to a higher cell dry weight exceeding 25 g/l, and further improvement of secreted APase

  1. Androgen - secreting steroid cell tumor of the ovary

    Directory of Open Access Journals (Sweden)

    Paras Ratilal Udhreja

    2014-01-01

    Full Text Available Steroid cell tumors (SCTs, not otherwise specified of the ovary are rare subgroup of sex cord tumors, which account for less than 0.1% of all ovarian tumors and also that will present at any age. The majority of these tumors produce steroids with testosterone being the most common. A case of a 28-year-old woman who presented with symptoms of virilization is reported. Although SCTs are generally benign, there is a risk for malignant transformation. Surgery is the most important and hallmark treatment.

  2. Leptin differentially regulates NPY secretion in hypothalamic cell lines through distinct intracellular signal transduction pathways.

    Science.gov (United States)

    Dhillon, Sandeep S; Belsham, Denise D

    2011-04-11

    Leptin acts as a key peripheral hormone in distinct neurons in the hypothalamus to modulate both reproductive function and energy homeostasis. The control of neuropeptide Y (NPY) secretion is an example of a process that can be differentially regulated by leptin. In order to further understand these distinct modulatory effects, we have used immortalized, neuronal hypothalamic cell lines expressing NPY, mHypoE-38 and mHypoE-46. We found that these cell lines express the endogenous leptin receptor, ObRb, and secrete detectable levels of NPY. We exposed the neurons to 100nM leptin for 1h and determined that the basal levels of NPY in the cell lines were differentially regulated: NPY secretion was inhibited in mHypoE-46 neurons, whereas NPY secretion was induced in the mHypoE-38 neurons. In order to determine the mechanisms involved in the divergent regulation of NPY release, we analyzed the activity of a number of signaling components using phospho-specific antibodies directed towards specific proteins in the MAP kinase, PI3K, and AMPK pathways, among others. We found that leptin activated a different combination of second messengers in each cell line. Importantly, we could link the regulation of NPY secretion to different signaling pathways, AMPK in the mHypoE-46 and both MAPK and PI3K in the mHypoE-38 neurons. This is the first demonstration that leptin can specifically regulate individual NPY neuron secretory responses through distinct signaling pathways.

  3. JNK mitogen-activated protein kinase limits calcium-dependent chloride secretion across colonic epithelial cells.

    LENUS (Irish Health Repository)

    Donnellan, Fergal

    2010-01-01

    Neuroimmune agonists induce epithelial Cl(-) secretion through elevations in intracellular Ca2+ or cAMP. Previously, we demonstrated that epidermal growth factor receptor (EGFR) transactivation and subsequent ERK MAPK activation limits secretory responses to Ca2+-dependent, but not cAMP-dependent, agonists. Although JNK MAPKs are also expressed in epithelial cells, their role in regulating transport function is unknown. Here, we investigated the potential role for JNK in regulating Cl(-) secretion in T(84) colonic epithelial cells. Western blot analysis revealed that a prototypical Ca2+-dependent secretagogue, carbachol (CCh; 100 microM), induced phosphorylation of both the 46-kDa and 54-kDa isoforms of JNK. This effect was mimicked by thapsigargin (TG), which specifically elevates intracellular Ca2+, but not by forskolin (FSK; 10 microM), which elevates cAMP. CCh-induced JNK phosphorylation was attenuated by the EGFR inhibitor, tyrphostin-AG1478 (1 microM). Pretreatment of voltage-clamped T(84) cells with SP600125 (2 microM), a specific JNK inhibitor, potentiated secretory responses to both CCh and TG but not to FSK. The effects of SP600125 on CCh-induced secretion were not additive with those of the ERK inhibitor, PD98059. Finally, in apically permeabilized T(84) cell monolayers, SP600125 potentiated CCh-induced K+ conductances but not Na+\\/K+ATPase activity. These data demonstrate a novel role for JNK MAPK in regulating Ca2+ but not cAMP-dependent epithelial Cl(-) secretion. JNK activation is mediated by EGFR transactivation and exerts its antisecretory effects through inhibition of basolateral K+ channels. These data further our understanding of mechanisms regulating epithelial secretion and underscore the potential for exploitation of MAPK-dependent signaling in treatment of intestinal transport disorders.

  4. Baseline Goblet Cell Mucin Secretion in the Airways Exceeds Stimulated Secretion over Extended Time Periods, and Is Sensitive to Shear Stress and Intracellular Mucin Stores.

    Directory of Open Access Journals (Sweden)

    Yunxiang Zhu

    Full Text Available Airway mucin secretion studies have focused on goblet cell responses to exogenous agonists almost to the exclusion of baseline mucin secretion (BLMS. In human bronchial epithelial cell cultures (HBECCs, maximal agonist-stimulated secretion exceeds baseline by ~3-fold as measured over hour-long periods, but mucin stores are discharged completely and require 24 h for full restoration. Hence, over 24 h, total baseline exceeds agonist-induced secretion by several-fold. Studies with HBECCs and mouse tracheas showed that BLMS is highly sensitive to mechanical stresses. Harvesting three consecutive 1 h baseline luminal incubations with HBECCs yielded equal rates of BLMS; however, lengthening the middle period to 72 h decreased the respective rate significantly, suggesting a stimulation of BLMS by the gentle washes of HBECC luminal surfaces. BLMS declined exponentially after washing HBECCs (t1/2 = 2.75 h, to rates approaching zero. HBECCs exposed to low perfusion rates exhibited spike-like increases in BLMS when flow was jumped 5-fold: BLMS increased >4 fold, then decreased within 5 min to a stable plateau at 1.5-2-fold over control. Higher flow jumps induced proportionally higher BLMS increases. Inducing mucous hyperplasia in HBECCs increased mucin production, BLMS and agonist-induced secretion. Mouse tracheal BLMS was ~6-fold higher during perfusion, than when flow was stopped. Munc13-2 null mouse tracheas, with their defect of accumulated cellular mucins, exhibited similar BLMS as WT, contrary to predictions of lower values. Graded mucous metaplasia induced in WT and Munc13-2 null tracheas with IL-13, caused proportional increases in BLMS, suggesting that naïve Munc13-2 mouse BLMS is elevated by increased mucin stores. We conclude that BLMS is, [i] a major component of mucin secretion in the lung, [ii] sustained by the mechanical activity of a dynamic lung, [iii] proportional to levels of mucin stores, and [iv] regulated differentially from agonist

  5. Insulin-secreting β cells require a post-genomic concept

    Institute of Scientific and Technical Information of China (English)

    Fang-Xu; Jiang; Grant; Morahan

    2016-01-01

    Pancreatic insulin-secretingβcells are essential in maintaining normal glucose homeostasis accomplished byhighly specialized transcription of insulin gene,of which occupies up to 40%their transcriptome.Deficiency of these cells causes diabetes mellitus,a global public health problem.Although tremendous endeavors have been made to generate insulin-secreting cells from human pluripotent stem cells(i.e.,primitive cells capable of giving rise to all cell types in the body),a regenerative therapy to diabetes has not yet been established.Furthermore,the nomenclature ofβcells has become inconsistent,confusing and controversial due to the lack of standardized positive controls of developmental stagematched in vivo cells.In order to minimize this negative impact and facilitate critical research in this field,a postgenomic concept of pancreaticβcells might be helpful.In this review article,we will briefly describe howβcells were discovered and islet lineage is developed that may help understand the cause of nomenclatural controversy,suggest a post-genomic definition and finally provide a conclusive remark on future research of this pivotal cell.

  6. Hepatic stellate cells secreted hepatocyte growth factor contributes to the chemoresistance of hepatocellular carcinoma.

    Directory of Open Access Journals (Sweden)

    Guofeng Yu

    Full Text Available As the main source of extracellular matrix proteins in tumor stroma, hepatic stellate cells (HSCs have a great impact on biological behaviors of hepatocellular carcinoma (HCC. In the present study, we have investigated a mechanism whereby HSCs modulate the chemoresistance of hepatoma cells. We used human HSC line lx-2 and chemotherapeutic agent cisplatin to investigate their effects on human HCC cell line Hep3B. The results showed that cisplatin resistance in Hep3B cells was enhanced with LX-2 CM (cultured medium exposure in vitro as well as co-injection with LX-2 cells in null mice. Meanwhile, in presence of LX-2 CM, Hep3B cells underwent epithelial to mesenchymal transition (EMT and upregulation of cancer stem cell (CSC -like properties. Besides, LX-2 cells synthesized and secreted hepatic growth factor (HGF into the CM. HGF receptor tyrosine kinase mesenchymal-epithelial transition factor (Met was activated in Hep3B cells after LX-2 CM exposure. The HGF level of LX-2 CM could be effectively reduced by using HGF neutralizing antibody. Furthermore, depletion of HGF in LX-2 CM abolished its effects on activation of Met as well as promotion of the EMT, CSC-like features and cisplatin resistance in Hep3B cells. Collectively, secreting HGF into tumor milieu, HSCs may decrease hepatoma cells sensitization to chemotherapeutic agents by promoting EMT and CSC-like features via HGF/Met signaling.

  7. Glial cell-derived neurotrophic factor mRNA expression in a rat model of spinal cord injury following bone marrow stromal cell transplantation

    Institute of Scientific and Technical Information of China (English)

    Lei Li; Gang Lü; Yanfeng Wang; Hong Gao; Xin Xu; Lunhao Bai; Huan Wang

    2008-01-01

    BACKGROUND: Several animal experiments utilizing bone marrow stromal cell (BMSC) transplantation for the treatment of spinal cord injury have proposed a hypothesis that BMSC transplantation effects are associated with increased glial cell-derived neurotrophic factor (GDNF) expression.OBJECTIVE: To confirm the effects of BMSC transplantation on GDNF mRNA expression in rats with spinal cord injury by reverse transcription-polymerase chain reaction (RT-PCR).DESIGN, TIME AND SETTING: The present molecular, cell biology experiment was performed at the Key Laboratory of Children's Congenital Malformation, Ministry of Health of China & Department of Developmental Biology, Basic Medical College, China Medical University between March 2006 and May 2007.MATERIALS: Sixty healthy Wistar rats aged 2--4-months and of either gender were included in this study. Spinal cord injury was induced in all rats by hemisection ofT9 on the left side. RT-PCR kits were purchased from TaKaRa Company, China. Type 9600 RCR amplifier was provided by PerkinElmer Company, USA. METHODS: Three rats were selected for BMSC culture and subsequent transplantation (after three passages). Of the remaining 57 rats, nine were selected for sham-operation (sham-operated group), where only the T9 spinal cord was exposed without hemisection. A total of 48 rats were randomly and evenly divided into BMSC transplantation and model groups. In the BMSC transplantation group, following spinal cord injury induction, each rat was administered a BMSC suspension through two injection sites selected on the gray and white matter boundary caudally and cephalically, seperately and near to injury site in the spinal cord. The model group received an equal volume of PBS through the identical injection sites.MAIN OUTCOME MEASURES: At 24 and 72 hours, as well as at 7 days, following spinal cord injury, the spinal cord at the T9 segment was removed. Eight rats were allocated to each time point in the BMSC transplantation and model

  8. Quantifying changes in the cellular thiol-disulfide status during differentiation of B cells into antibody-secreting plasma cells

    DEFF Research Database (Denmark)

    Hansen, Rosa Rebecca Erritzøe; Otsu, Mieko; Braakman, Ineke

    2013-01-01

    Plasma cells produce and secrete massive amounts of disulfide-containing antibodies. To accommodate this load on the secretory machinery, the differentiation of resting B cells into antibody-secreting plasma cells is accompanied by a preferential expansion of the secretory compartments of the cells...... and by an up-regulation of enzymes involved in redox regulation and protein folding. We have quantified the absolute levels of protein thiols, protein disulfides, and glutathionylated proteins in whole cells. The results show that while the global thiol-disulfide state is affected to some extent...... by the differentiation, steady-state levels of glutathionylated protein thiols are less than 0.3% of the total protein cysteines, even in fully differentiated cells, and the overall protein redox state is not affected until late in differentiation, when large-scale IgM production is ongoing. A general expansion...

  9. Diffusible Factors Secreted by Glioblastoma and Medulloblastoma Cells Induce Oxidative Stress in Bystander Neural Stem Progenitors.

    Science.gov (United States)

    Sharma, Neha; Colangelo, Nicholas W; de Toledo, Sonia M; Azzam, Edouard I

    2016-08-01

    Harmful effects that alter the homeostasis of neural stem or progenitor cells (NSPs) can affect regenerative processes in the central nervous system. We investigated the effect of soluble factors secreted by control or (137)Cs-γ-irradiated glioblastoma or medulloblastoma cells on redox-modulated endpoints in recipient human NSPs. Growth medium harvested from the nonirradiated brain tumor cells, following 24 h of growth, induced prominent oxidative stress in recipient NSPs as judged by overall increases in mitochondrial superoxide radical levels (p p21(Waf1) and p27(Kip1), and perturbations in cell cycle progression (p cells to radiation only slightly altered the induced oxidative changes in the bystander NSPs, except for medium from irradiated medulloblastoma cells that was more potent at inducing apoptosis in the NSPs than medium from nonirradiated cells (p cells is often used to support the growth of stem cells.

  10. Bone marrow stromal cell : mediated neuroprotection for spinal cord repair

    NARCIS (Netherlands)

    Ritfeld, Gaby Jane

    2014-01-01

    Currently, there is no treatment available that restores anatomy and function after spinal cord injury. This thesis explores transplantation of bone marrow-derived mesenchymal stem cells (bone marrow stromal cells; BMSCs) as a therapeutic approach for spinal cord repair. BMSCs secrete neurotrophic f

  11. In beta-cells, mitochondria integrate and generate metabolic signals controlling insulin secretion.

    Science.gov (United States)

    Maechler, Pierre; Carobbio, Stefania; Rubi, Blanca

    2006-01-01

    Pancreatic beta-cells are unique neuroendocrine cells displaying the peculiar feature of responding to nutrients, principally glucose, as primary stimulus. This requires translation of a metabolic substrate into intracellular messengers recognized by the exocytotic machinery. Central to this signal transduction mechanism, mitochondria integrate and generate metabolic signals, thereby coupling glucose recognition to insulin secretion. In response to a glucose rise, nucleotides and metabolites are generated by mitochondria and participate, together with cytosolic calcium, to the stimulation of insulin exocytosis. This review describes the mitochondrion-dependent pathways of regulated insulin secretion. In particular, importance of cataplerotic and anaplerotic processes is discussed, with special attention to the mitochondrial enzyme glutamate dehydrogenase. Mitochondrial defects, such as mutations and reactive oxygen species production, are presented in the context of beta-cell failure in the course of type 2 diabetes.

  12. Impact of fexofenadine, osthole and histamine on peripheral blood mononuclear cell proliferation and cytokine secretion.

    Science.gov (United States)

    Karolina Kordulewska, Natalia; Kostyra, Elżbieta; Matysiewicz, Michał; Cieślińska, Anna; Jarmołowska, Beata

    2015-08-15

    This paper compares results of peripheral blood mononuclear cell (PBMC) incubation with fexofenadine (FXF) and osthole. FXF is a third-generation antihistamine drug and osthole is assumed a natural antihistamine alternative. To our best knowledge, this is the first comparative study on FXF, osthole and histamine cytokine secretion and cytotoxicity in PBMC in vitro cultures using cell proliferation ELISA BrdU. The cultures were treated 12, 42, 48 and 72h with FXF and osthole at 150, 300 and 450ng/ml concentrations and histamine at 50, 100 and 200ng/ml. Our study results confirm that FXF, osthole and histamine exert no cytotoxic effect on PBMCs and that IL-6, IL-10 and TNF-α cytokine secretion following osthole cell stimulation was similar to that by FXF stimulation.This confirms our hypothesis that osthole is a natural histamine antagonist, and can therefore be beneficially applied in antihistamine treatment.

  13. Apoptotic neuron-secreted HN12 inhibits cell apoptosis in Hirschsprung’s disease

    Science.gov (United States)

    Du, Chunxia; Xie, Hua; Zang, Rujin; Shen, Ziyang; Li, Hongxing; Chen, Pingfa; Xu, Xiaoqun; Xia, Yankai; Tang, Weibing

    2016-01-01

    Perturbation in apoptosis can lead to Hirschsprung’s disease (HSCR), which is a genetic disorder of neural crest development. It is believed that long noncoding RNAs (lncRNAs) play a role in the progression of HSCR. This study shows that apoptotic neurons can suppress apoptosis of nonapoptotic cells by secreting exosomes that contain high levels of HN12 lncRNA. Elevated exogenous HN12 in nonapoptotic cells effectively inhibited cell apoptosis by maintaining the function of mitochondria, including the production of ATP and the release of cytochrome C. These results demonstrate that secreted lncRNAs may serve as signaling molecules mediating intercellular communication in HSCR. In addition, high HN12 levels in the circulation worked as a biomarker for predicting HSCR, providing a potential, novel, noninvasive diagnostic approach for early screening of HSCR. PMID:27853370

  14. Pericyte NF-κB activation enhances endothelial cell proliferation and proangiogenic cytokine secretion in vitro

    Science.gov (United States)

    LaBarbera, Katherine E; Hyldahl, Robert D; O'Fallon, Kevin S; Clarkson, Priscilla M; Witkowski, Sarah

    2015-01-01

    Pericytes are skeletal muscle resident, multipotent stem cells that are localized to the microvasculature. In vivo, studies have shown that they respond to damage through activation of nuclear-factor kappa-B (NF-κB), but the downstream effects of NF-κB activation on endothelial cell proliferation and cell–cell signaling during repair remain unknown. The purpose of this study was to examine pericyte NF-κB activation in a model of skeletal muscle damage; and use genetic manipulation to study the effects of changes in pericyte NF-κB activation on endothelial cell proliferation and cytokine secretion. We utilized scratch injury to C2C12 cells in coculture with human primary pericytes to assess NF-κB activation and monocyte chemoattractant protein-1 (MCP-1) secretion from pericytes and C2C12 cells. We also cocultured endothelial cells with pericytes that expressed genetically altered NF-κB activation levels, and then quantified endothelial cell proliferation and screened the conditioned media for secreted cytokines. Pericytes trended toward greater NF-κB activation in injured compared to control cocultures (P = 0.085) and in comparison to C2C12 cells (P = 0.079). Second, increased NF-κB activation in pericytes enhanced the proliferation of cocultured endothelial cells (1.3-fold, P = 0.002). Finally, we identified inflammatory signaling molecules, including MCP-1 and interleukin 8 (IL-8) that may mediate the crosstalk between pericytes and endothelial cells. The results of this study show that pericyte NF-κB activation may be an important mechanism in skeletal muscle repair with implications for the development of therapies for musculoskeletal and vascular diseases, including peripheral artery disease. PMID:25911453

  15. Trafficking through COPII stabilises cell polarity and drives secretion during Drosophila epidermal differentiation.

    Directory of Open Access Journals (Sweden)

    Michaela Norum

    Full Text Available BACKGROUND: The differentiation of an extracellular matrix (ECM at the apical side of epithelial cells implies massive polarised secretion and membrane trafficking. An epithelial cell is hence engaged in coordinating secretion and cell polarity for a correct and efficient ECM formation. PRINCIPAL FINDINGS: We are studying the molecular mechanisms that Drosophila tracheal and epidermal cells deploy to form their specific apical ECM during differentiation. In this work we demonstrate that the two genetically identified factors haunted and ghost are essential for polarity maintenance, membrane topology as well as for secretion of the tracheal luminal matrix and the cuticle. We show that they code for the Drosophila COPII vesicle-coating components Sec23 and Sec24, respectively, that organise vesicle transport from the ER to the Golgi apparatus. CONCLUSION: Taken together, epithelial differentiation during Drosophila embryogenesis is a concerted action of ECM formation, plasma membrane remodelling and maintenance of cell polarity that all three rely mainly, if not absolutely, on the canonical secretory pathway from the ER over the Golgi apparatus to the plasma membrane. Our results indicate that COPII vesicles constitute a central hub for these processes.

  16. Geniposide regulates glucose-stimulated insulin secretion possibly through controlling glucose metabolism in INS-1 cells.

    Directory of Open Access Journals (Sweden)

    Jianhui Liu

    Full Text Available Glucose-stimulated insulin secretion (GSIS is essential to the control of metabolic fuel homeostasis. The impairment of GSIS is a key element of β-cell failure and one of causes of type 2 diabetes mellitus (T2DM. Although the KATP channel-dependent mechanism of GSIS has been broadly accepted for several decades, it does not fully describe the effects of glucose on insulin secretion. Emerging evidence has suggested that other mechanisms are involved. The present study demonstrated that geniposide enhanced GSIS in response to the stimulation of low or moderately high concentrations of glucose, and promoted glucose uptake and intracellular ATP levels in INS-1 cells. However, in the presence of a high concentration of glucose, geniposide exerted a contrary role on both GSIS and glucose uptake and metabolism. Furthermore, geniposide improved the impairment of GSIS in INS-1 cells challenged with a high concentration of glucose. Further experiments showed that geniposide modulated pyruvate carboxylase expression and the production of intermediates of glucose metabolism. The data collectively suggest that geniposide has potential to prevent or improve the impairment of insulin secretion in β-cells challenged with high concentrations of glucose, likely through pyruvate carboxylase mediated glucose metabolism in β-cells.

  17. Differentiation-dependent secretion of proangiogenic factors by mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Allison I Hoch

    Full Text Available Mesenchymal stem cells (MSCs are a promising cell population for cell-based bone repair due to their proliferative potential, ability to differentiate into bone-forming osteoblasts, and their secretion of potent trophic factors that stimulate angiogenesis and neovascularization. To promote bone healing, autogenous or allogeneic MSCs are transplanted into bone defects after differentiation to varying degrees down the osteogenic lineage. However, the contribution of the stage of osteogenic differentiation upon angiogenic factor secretion is unclear. We hypothesized that the proangiogenic potential of MSCs was dependent upon their stage of osteogenic differentiation. After 7 days of culture, we observed the greatest osteogenic differentiation of MSCs when cells were cultured with dexamethasone (OM+. Conversely, VEGF protein secretion and upregulation of angiogenic genes were greatest in MSCs cultured in growth media (GM. Using conditioned media from MSCs in each culture condition, GM-conditioned media maximized proliferation and enhanced chemotactic migration and tubule formation of endothelial colony forming cells (ECFCs. The addition of a neutralizing VEGF(165/121 antibody to conditioned media attenuated ECFC proliferation and chemotactic migration. ECFCs seeded on microcarrier beads and co-cultured with MSCs previously cultured in GM in a fibrin gel exhibited superior sprouting compared to MSCs previously cultured in OM+. These results confirm that MSCs induced farther down the osteogenic lineage possess reduced proangiogenic potential, thereby providing important findings for consideration when using MSCs for bone repair.

  18. IL-8 secretion in primary cultures of prostate cells is associated with prostate cancer aggressiveness

    Directory of Open Access Journals (Sweden)

    Neveu B

    2014-05-01

    Full Text Available Bertrand Neveu*, Xavier Moreel*, Marie-Pier Deschênes-Rompré, Alain Bergeron, Hélène LaRue, Cherifa Ayari, Yves Fradet, Vincent FradetDepartment of Surgery, Laval University Cancer Research Centre, CHU de Quebec Research Centre, Quebec, QC, Canada *These authors contributed equally to this workBackground: Chronic inflammation is believed to be a major factor in prostate cancer initiation and promotion and has been studied using prostate cancer cells and immortalized cell lines. However, little is known about the contribution of normal cells to the prostatic microenvironment and inflammation. We aim to study the contribution of normal prostate epithelial cells to prostate inflammation and to link the inflammatory status of normal cells to prostate cancer aggressiveness.Materials and methods: Short-term primary cell cultures of normal epithelial prostate cells were derived from prostate biopsies from 25 men undergoing radical prostatectomy, cystoprostatectomy, or organ donation. Cells were treated with polyinosinic:polycytidylic acid, a mimic of double-stranded viral RNA and a potent inducer of the inflammatory response. Secretion of interleukin (IL-8 in the cell culture medium by untreated and treated cells was measured and we determined the association between IL-8 levels in these primary cell cultures and prostate cancer characteristics. The Fligner–Policello test was used to compare the groups.Results: Baseline and induced IL-8 secretion were highly variable between cultured cells from different patients. This variation was not related to drug use, past medical history, age, or preoperative prostate-specific antigen value. Nonetheless, an elevated secretion of IL-8 from normal cultured epithelial cells was associated with prostate cancer aggressiveness (P=0.0005.Conclusion: The baseline secretion of IL-8 from normal prostate epithelial cells in culture is strongly correlated with cancer aggressiveness and may drive prostate cancer

  19. Effect of spirulina on the secretion of cytokines from peripheral blood mononuclear cells.

    Science.gov (United States)

    Mao, T K; VAN DE Water, J; Gershwin, M E

    2000-01-01

    ABSTRACT The purpose of this study was to evaluate the immunomodulatory activity of Spirulina, a bluegreen alga used as a food supplement. The effects of Spirulina on the secretion of three cytokines from unstimulated and stimulated human peripheral blood mononuclear cells (PBMC) were examined. In resting PBMC, Spirulina stimulated secretion of interleukin (IL)-1beta, IL-4, and interferon (IFN)-gamma to nearly 2.0, 3.3, and 13.6 times basal levels, respectively. Spirulina induced levels of IFN-gamma (229 +/- 104 pg/ml) that were comparable to those seen after phytohemagglutinin (PHA) stimulation (476 +/- 121 pg/ml). However, it was much less mitogenic than PHA (13.1 +/- 6.9 pg/ml) with respect to the induction of IL-4 secretion (0.34 +/- 0.1 pg/ml). In PHA-stimulated cells, Spirulina enhanced secretion of IL-1beta, IL-4, and IFN-beta by 2.9, 4.0., and 1.6 times, respectively. Although Spirulina stimulates several cytokines, it is clearly more effective in the generation of a Thl-type response. This in vitro study offers additional data for consideration of the potential therapeutic benefits of Spirulina.

  20. Control of M. tuberculosis ESAT-6 secretion and specific T cell recognition by PhoP.

    Directory of Open Access Journals (Sweden)

    Wafa Frigui

    2008-02-01

    Full Text Available Analysis of mycobacterial strains that have lost their ability to cause disease is a powerful approach to identify yet unknown virulence determinants and pathways involved in tuberculosis pathogenesis. Two of the most widely used attenuated strains in the history of tuberculosis research are Mycobacterium bovis BCG (BCG and Mycobacterium tuberculosis H37Ra (H37Ra, which both lost their virulence during in vitro serial passage. Whereas the attenuation of BCG is due mainly to loss of the ESAT-6 secretion system, ESX-1, the reason why H37Ra is attenuated remained unknown. However, here we show that a point mutation (S219L in the predicted DNA binding region of the regulator PhoP is involved in the attenuation of H37Ra via a mechanism that impacts on the secretion of the major T cell antigen ESAT-6. Only H37Ra "knock-ins" that carried an integrated cosmid with the wild-type phoP gene from M. tuberculosis H37Rv showed changes in colony morphology, increased virulence, ESAT-6 secretion, and induction of specific T cell responses, whereas other H37Ra constructs did not. This finding established a link between the PhoP regulator and ESAT-6 secretion that opens exciting new perspectives for elucidating virulence regulation in M. tuberculosis.

  1. Combined effect of brain-derived neurotrophic factor and LINGO-1 fusion protein on long-term survival of retinal ganglion cells in chronic glaucoma.

    Science.gov (United States)

    Fu, Q-L; Li, X; Yip, H K; Shao, Z; Wu, W; Mi, S; So, K-F

    2009-08-18

    Glaucoma is a progressive neuropathy characterized by loss of vision as a result of retinal ganglion cell (RGC) death. There are no effective neuroprotectants to treat this disorder. Brain-derived neurotrophic factor (BDNF) is well known to transiently delay RGC death in ocular hypertensive eyes. The CNS-specific leucine-rich repeat protein LINGO-1 contributes to the negative regulation to some trophic pathways. We thereby examined whether BDNF combined with LINGO-1 antagonists can promote long-term RGC survival after ocular hypertension. In this study, intraocular pressure was elevated in adult rats using an argon laser to photocoagulate the episcleral and limbal veins. BDNF alone shows slight neuroprotection to RGCs after a long-term progress of 4 weeks following the induction of ocular hypertension. However, combination of BDNF and LINGO-1-Fc prevents RGC death in the same condition. We further identified that (1) LINGO-1 was co-expressed with BDNF receptor, TrkB in the RGCs, and (2) BDNF combined with LINGO-1-Fc activated more TrkB in the injured retina compared to BDNF alone. These results indicate that the combination of BDNF with LINGO-1 antagonist can provide long-term protection for RGCs in a chronic ocular hypertension model. TrkB may be the predominant mediator of this neuroprotection.

  2. Transport of Glial Cell Line-Derived Neurotrophic Factor into Liposomes across the Blood-Brain Barrier: In Vitro and in Vivo Studies

    Directory of Open Access Journals (Sweden)

    Shaoling Wu

    2014-02-01

    Full Text Available Glial cell line-derived neurotrophic factor (GDNF was encapsulated into liposomes in order to protect it from enzyme degradation in vivo and promote its permeability across the blood-brain barrier (BBB. In this study, GDNF conventional liposomes (GDNF-L and GDNF target sterically stabilized liposomes (GDNF-SSL-T were prepared. The average size of liposomes was below 90 nm. A primary model of BBB was established and evaluated by transendothelial electrical resistance (TEER and permeability. This BBB model was employed to study the permeability of GDNF liposomes in vitro. The results indicated that the liposomes could enhance transport of GDNF across the BBB and GDNF-SSL-T had achieved the best transport efficacy. The distribution of GDNF liposomes was studied in vivo. Free GDNF and GDNF-L were eliminated rapidly in the circulation. GDNF-SSL-T has a prolonged circulation time in the blood and favorable brain delivery. The values of the area under the curve (AUC(0–1 h in the brain of GDNF-SSL-T was 8.1 times and 6.8 times more than that of free GDNF and GDNF-L, respectively. These results showed that GDNF-SSL-T realized the aim of targeted delivery of therapeutic proteins to central nervous system.

  3. Glial cell line-derived neurotrophic factor (GDNF) is an endogenous protector in the mesolimbic system against excessive alcohol consumption and relapse.

    Science.gov (United States)

    Barak, Segev; Wang, Jun; Ahmadiantehrani, Somayeh; Ben Hamida, Sami; Kells, Adrian P; Forsayeth, John; Bankiewicz, Krystof S; Ron, Dorit

    2015-07-01

    Moderate social consumption of alcohol is common; however, only a small percentage of individuals transit from social to excessive, uncontrolled alcohol drinking. This suggests the existence of protective mechanisms that prevent the development of alcohol addiction. Here, we tested the hypothesis that the glial cell line-derived neurotrophic factor (GDNF) in the mesolimbic system [e.g. the nucleus accumbens (Acb) and ventral tegmental area (VTA)] is part of such a mechanism. We found that GDNF knockdown, by infecting rat Acb neurons with a small hairpin RNA (shRNA) targeting the GDNF gene, produced a rapid escalation to excessive alcohol consumption and enhanced relapse to alcohol drinking. Conversely, viral-mediated overexpression of the growth factor in the mesolimbic system blocked the escalation from moderate to excessive alcohol drinking. To access the mechanism underlying GDNF's actions, we measured the firing rate of dopaminergic (DAergic) neurons in the VTA after a history of excessive alcohol intake with or without elevating GDNF levels. We found that the spontaneous firing rate of DAergic neurons in the VTA was reduced during alcohol withdrawal and that GDNF reversed this alcohol-induced DA deficiency. Together, our results suggest that endogenous GDNF in the mesolimbic system controls the transition from moderate to excessive alcohol drinking and relapse via reversal of alcohol-dependent neuro-adaptations in DAergic VTA neurons.

  4. Transport of glial cell line-derived neurotrophic factor into liposomes across the blood-brain barrier: in vitro and in vivo studies.

    Science.gov (United States)

    Wu, Shaoling; Li, Guoqi; Li, Xiao; Lin, Caina; Yu, Ding; Luan, Shuo; Ma, Chao

    2014-02-27

    Glial cell line-derived neurotrophic factor (GDNF) was encapsulated into liposomes in order to protect it from enzyme degradation in vivo and promote its permeability across the blood-brain barrier (BBB). In this study, GDNF conventional liposomes (GDNF-L) and GDNF target sterically stabilized liposomes (GDNF-SSL-T) were prepared. The average size of liposomes was below 90 nm. A primary model of BBB was established and evaluated by transendothelial electrical resistance (TEER) and permeability. This BBB model was employed to study the permeability of GDNF liposomes in vitro. The results indicated that the liposomes could enhance transport of GDNF across the BBB and GDNF-SSL-T had achieved the best transport efficacy. The distribution of GDNF liposomes was studied in vivo. Free GDNF and GDNF-L were eliminated rapidly in the circulation. GDNF-SSL-T has a prolonged circulation time in the blood and favorable brain delivery. The values of the area under the curve (AUC(0-1 h)) in the brain of GDNF-SSL-T was 8.1 times and 6.8 times more than that of free GDNF and GDNF-L, respectively. These results showed that GDNF-SSL-T realized the aim of targeted delivery of therapeutic proteins to central nervous system.

  5. Harpagoside attenuates MPTP/MPP⁺ induced dopaminergic neurodegeneration and movement disorder via elevating glial cell line-derived neurotrophic factor.

    Science.gov (United States)

    Sun, Xiaoyu; Xiong, Zhongkui; Zhang, Yongfang; Meng, Ya; Xu, Gang; Xia, Zhiming; Li, Jiamei; Zhang, Rui; Ke, Zunji; Xia, Zongqin; Hu, Yaer

    2012-03-01

    Parkinson's disease is a chronic neurodegenerative movement disorder characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta. New therapeutic approaches aiming at delaying or reversing the neurodegenerative process are under active investigations. In this work, we found that harpagoside, an iridoid purified from the Chinese medicinal herb Scrophularia ningpoensis, could not only prevent but also rescue the dopaminergic neurodegeneration in MPTP/MPP(+) intoxication with promising efficacy. Firstly, in cultured mesencephalic neurons, harpagoside significantly attenuated the loss of TH-positive neuron numbers and the shortening of axonal length. Secondly, in a chronic MPTP mouse model, harpagoside dose-dependently improved the loco-motor ability (rotarod test), increased the TH-positive neuron numbers in the substantia nigra pars compacta (unbiased stereological counting) and increased the striatal DAT density ((125) I-FP-CIT autoradiography). Thirdly, harpagoside markedly elevated the GDNF mRNA and GDNF protein levels in MPTP/MPP(+) lesioned models. However, the protecting effect of harpagoside on the dopaminergic degeneration disappeared when the intrinsic GDNF action was blocked by either the Ret inhibitor PP1 or the neutralizing anti-GDNF antibody. Taken together, we conclude that harpagoside attenuates the dopaminergic neurodegeneration and movement disorder mainly through elevating glial cell line-derived neurotrophic factor.

  6. Tumor cell-macrophage interactions increase angiogenesis through secretion of EMMPRIN

    Directory of Open Access Journals (Sweden)

    Bat-Chen eAmit-Cohen

    2013-07-01

    Full Text Available Tumor macrophages are generally considered to be alternatively/M2 activated to induce secretion of pro-angiogenic factors such as VEGF and MMPs. EMMPRIN (CD147, basigin is overexpressed in many tumor types, and has been shown to induce fibroblasts and endothelial cell expression of MMPs and VEGF. We first show that tumor cell interactions with macrophages resulted in increased expression of EMMPRIN and induction of MMP-9 and VEGF. Human A498 renal carcinoma or MCF-7 breast carcinoma cell lines were co-cultured with the U937 monocytic-like cell line in the presence of TNFalpha (1 ng/ml. Membranal EMMPRIN expression was increased in the co-cultures (by 3-4 folds, p<0.01, as was the secretion of MMP-9 and VEGF (by 2-5 folds for both MMP-9 and VEGF, p<0.01, relative to the single cultures with TNFalpha. Investigating the regulatory mechanisms, we show that EMMPRIN was post-translationally regulated by miR-146a, as no change was observed in the tumoral expression of EMMPRIN mRNA during co-culture, expression of miR-146a was increased and its neutralization by its antagomir inhibited EMMPRIN expression. The secretion of EMMPRIN was also enhanced (by 2-3 folds, p<0.05, only in the A498 co-culture via shedding off of the membranal protein by a serine protease that is yet to be identified, as demonstrated by the use of wide range protease inhibitors. Finally, soluble EMMPRIN enhanced monocytic secretion of MMP-9 and VEGF, as inhibition of its expression levels by neutralizing anti-EMMPRIN or siRNA in the tumor cells lead to subsequent decreased induction of these two pro-angiogenic proteins. These results reveal a mechanism whereby tumor cell-macrophage interactions promote angiogenesis via an EMMPRIN-mediated pathway.

  7. Cytotoxic Activity and Antiproliferative Effects of Crude Skin Secretion from Physalaemus nattereri (Anura: Leptodactylidae on in vitro Melanoma Cells

    Directory of Open Access Journals (Sweden)

    Andréa Cruz e Carvalho

    2015-10-01

    Full Text Available Anuran secretions are rich sources of bioactive molecules, including antimicrobial and antitumoral compounds. The aims of this study were to investigate the therapeutic potential of Physalaemus nattereri skin secretion against skin cancer cells, and to assess its cytotoxic action mechanisms on the murine melanoma cell line B16F10. Our results demonstrated that the crude secretion reduced the viability of B16F10 cells, causing changes in cell morphology (e.g., round shape and structure shrinkage, reduction in mitochondrial membrane potential, increase in phosphatidylserine exposure, and cell cycle arrest in S-phase. Together, these changes suggest that tumor cells die by apoptosis. This skin secretion was also subjected to chromatographic fractioning using RP-HPLC, and eluted fractions were assayed for antiproliferative and antibacterial activities. Three active fractions showed molecular mass components in a range compatible with peptides. Although the specific mechanisms causing the reduced cell viability and cytotoxicity after the treatment with crude secretion are still unknown, it may be considered that molecules, such as the peptides found in the secretion, are effective against B16F10 tumor cells. Considering the growing need for new anticancer drugs, data presented in this study strongly reinforce the validity of P. nattereri crude secretion as a rich source of new anticancer molecules.

  8. Targeted taste cell-specific overexpression of brain-derived neurotrophic factor in adult taste buds elevates phosphorylated TrkB protein levels in taste cells, increases taste bud size, and promotes gustatory innervation.

    Science.gov (United States)

    Nosrat, Irina V; Margolskee, Robert F; Nosrat, Christopher A

    2012-05-11

    Brain-derived neurotrophic factor (BDNF) is the most potent neurotrophic factor in the peripheral taste system during embryonic development. It is also expressed in adult taste buds. There is a lack of understanding of the role of BDNF in the adult taste system. To address this, we generated novel transgenic mice in which transgene expression was driven by an α-gustducin promoter coupling BDNF expression to the postnatal expression of gustducin in taste cells. Immunohistochemistry revealed significantly stronger BDNF labeling in taste cells of high BDNF-expressing mouse lines compared with controls. We show that taste buds in these mice are significantly larger and have a larger number of taste cells compared with controls. To examine whether innervation was affected in Gust-BDNF mice, we used antibodies to neural cell adhesion molecule (NCAM) and ATP receptor P2X3. The total density of general innervation and specifically the gustatory innervation was markedly increased in high BDNF-expressing mice compared with controls. TrkB and NCAM gene expression in laser capture microdissected taste epithelia were significantly up-regulated in these mice. Up-regulation of TrkB transcripts in taste buds and elevated taste cell-specific TrkB phosphorylation in response to increased BDNF levels indicate that BDNF controls the expression and activation of its high affinity receptor in taste cells. This demonstrates a direct taste cell function for BDNF. BDNF also orchestrates and maintains taste bud innervation. We propose that the Gust-BDNF transgenic mouse models can be employed to further dissect the specific roles of BDNF in the adult taste system.

  9. Deconstructing the Iboga Alkaloid Skeleton: Potentiation of FGF2-induced Glial Cell Line-Derived Neurotrophic Factor Release by a Novel Compound.

    Science.gov (United States)

    Gassaway, Madalee M; Jacques, Teresa L; Kruegel, Andrew C; Karpowicz, Richard J; Li, Xiaoguang; Li, Shu; Myer, Yves; Sames, Dalibor

    2016-01-15

    Modulation of growth factor signaling pathways in the brain represents a new experimental approach to treating neuropsychiatric disorders such as depression, anxiety, and addiction. Neurotrophins and growth factors exert synaptic, neuronal, and circuit level effects on a wide temporal range, which suggests a possibility of rapid and lasting therapeutic effects. Consequently, identification of small molecules that can either enhance the release of growth factors or potentiate their respective pathways will provide a drug-like alternative to direct neurotrophin administration or viral gene delivery and thus represents an important frontier in chemical biology and drug design. Glial cell line-derived neurotrophic factor (GDNF), in particular, has been implicated in marked reduction of alcohol consumption in rodent addiction models, and the natural product ibogaine, a substance used traditionally in ritualistic ceremonies, has been suggested to increase the synthesis and release of GDNF in the dopaminergic system in rats. In this report, we describe a novel iboga analog, XL-008, created by unraveling the medium size ring of the ibogamine skeleton, and its ability to induce release of GDNF in C6 glioma cells. Additionally, XL-008 potentiates the release of GDNF induced by fibroblast growth factor 2 (FGF2), another neurotrophin implicated in major depressive disorder, increasing potency more than 2-fold (from 7.85 ± 2.59 ng/mL to 3.31 ± 0.98 ng/mL) and efficacy more than 3-fold. The GDNF release by both XL-008 and the FGF2/XL-008 mixture was found to be mediated through the MEK and PI3K signaling pathways but not through PLCγ in C6 glioma cells.

  10. Inhibition of TRPA1 channel activity in sensory neurons by the glial cell line-derived neurotrophic factor family member, artemin

    Directory of Open Access Journals (Sweden)

    Wang Shenglan

    2011-05-01

    Full Text Available Abstract Background The transient receptor potential (TRP channel subtype A1 (TRPA1 is known to be expressed on sensory neurons and respond to changes in temperature, pH and local application of certain noxious chemicals such as allyl isothiocyanate (AITC. Artemin is a neuronal survival and differentiation factor and belongs to the glial cell line-derived neurotrophic factor (GDNF family. Both TRPA1 and artemin have been reported to be involved in pathological pain initiation and maintenance. In the present study, using whole-cell patch clamp recording technique, in situ hybridization and behavioral analyses, we examined the functional interaction between TRPA1 and artemin. Results We found that 85.8 ± 1.9% of TRPA1-expressing neurons also expressed GDNF family receptor alpha 3 (GFR α3, and 87.5 ± 4.1% of GFRα3-expressing neurons were TRPA1-positive. In whole-cell patch clamp analysis, a short-term treatment of 100 ng/ml artemin significantly suppressed the AITC-induced TRPA1 currents. A concentration-response curve of AITC resulting from the effect of artemin showed that this inhibition did not change EC50 but did lower the AITC-induced maximum response. In addition, pre-treatment of artemin significantly suppressed the number of paw lifts induced by intraplantar injection of AITC, as well as the formalin-induced pain behaviors. Conclusions These findings that a short-term application of artemin inhibits the TRPA1 channel's activity and the sequential pain behaviors suggest a role of artemin in regulation of sensory neurons.

  11. Glial cell line-derived neurotrophic factor-induced mice liver defatting: A novel strategy to enable transplantation of steatotic livers.

    Science.gov (United States)

    Taba Taba Vakili, Sahar; Kailar, Roshni; Rahman, Khalidur; Nezami, Behtash Ghazi; Mwangi, Simon Musyoka; Anania, Frank A; Srinivasan, Shanthi

    2016-04-01

    Moderate macrovesicular steatosis (>30%), which is present in almost 50% of livers considered for transplantation, increases the risk of primary graft dysfunction. Our previously published data showed that glial cell line-derived neurotrophic factor (GDNF) is protective against high-fat diet (HFD)-induced hepatic steatosis in mice. Hence, we hypothesized that perfusion of steatotic livers with GDNF may reduce liver fat content before transplantation. Livers from 8 weeks of regular diet (RD) and of HFD-fed mice were perfused ex vivo for 4 hours with either vehicle, GDNF, or a previously described defatting cocktail. The liver's residual fat was quantified colorimetrically using a triglyceride (TG) assay kit and by Oil Red O (ORO) and Nile red/Hoechst staining. Liver tissue injury was assessed by using a lactate dehydrogenase (LDH) activity assay. In vitro induction of lipolysis in HepG2 cells was assessed by measuring glycerol and free fatty acid release. ORO staining showed significantly more steatosis in livers from HFD-fed mice compared with RD-fed mice (P defatting compared to the defatting cocktail; however, GDNF induces less liver damage than the defatting cocktail. These observations were consistent with data obtained from assessment of liver TG content. Assessment of liver injury revealed significant hepatocyte injury in livers perfused with the control defatting cocktail but no evidence of injury in livers perfused with either GDNF or vehicle. In vitro, GDNF reduced TG accumulation in HepG2 cells and stimulated increased TG lipolysis. In conclusion, GDNF can decrease mice liver fat content to an acceptable range and could be a potential defatting agent before liver transplantation.

  12. Effect of house dust mite immunotherapy on interleukin-10-secreting regulatory T cells in asthmatic children

    Institute of Scientific and Technical Information of China (English)

    WANG Wei; XIANG Li; LIU Yong-ge; WANG Yong-hong; SHEN Kun-ling

    2010-01-01

    Background Subcutaneous specific immunotherapy has been demonstrated to be capable of inducing T-cell regulatory response.Interleukin-10 (IL-10) plays a crucial role in inducing allergen-specific tolerance.However the reports of the changes of IL-10 in house dust mite (HDM)-specific immunotherapy were varied.The aim of this study was to evaluate the function of IL-10-secreting regulatory T cells in asthma children successfully treated with HDM immunotherapy.Methods Peripheral blood mononuclear cells (PBMCs) were isolated from 27 patients following 1.5--2 years of HDM-specific immunotherapy (SIT, SIT group) and from 27 matched treated asthmatic children allergic to HDM (asthmagroup).After 48 hours of in vitro stimulation with HDM extracts, IL-10-secreting regulatory T cells were measured by four colour flow cytometry.Sera were tested for allergen-specific IgG4 and IgE using the Immuno CAP 100 assay.Results PBMCs from children undergoing immunotherapy following HDM extracts stimuli produced significantly more IL-10 compared with the asthma group.The frequency of iTreg cells and aTreg cells increased in SIT group after HDM stimulation, while it was not affected in the asthma group.Among the iTreg cells and aTreg cells, the frequency of CD4+CD25-Foxp3-IL-10+ Treg cells increased the most which was 2 times higher than that in unstimulated cultures in SIT group.The levels of HDM-specific IgG4 of SIT group was significiently higher compared with asthma group, but there was no correlation of the levels of HDM-specific IgG4 and IL-10 secreting Treg cells.Conclusions HDM-specific immunotherapy can successfully upregulate the frequency of IL-10-secreting Treg cells.CD4+CD25-Foxp3-IL-10+ Treg cells may play a key role in inducing the immune tolerance in HDM-specific immunotherapy.

  13. Corticotropin-releasing factor secretion from dendritic cells stimulated by commensal bacteria

    Institute of Scientific and Technical Information of China (English)

    Mariko Hojo; Toshifumi Ohkusa; Harumi Tomeoku; Shigeo Koido; Daisuke Asaoka; Akihito Nagahara; Sumio Watanabe

    2011-01-01

    AIM: To study the production and secretion of corticotropin-releasing factor (CRF) by dendritic cells and the influence of commensal bacteria.METHODS: JAWSⅡ cells (ATCC CRL-11904), a mouse dendritic cell line, were seeded into 24-well culture plates and grown for 3 d. Commensal bacterial strains of Clostridium clostrodiiforme (JCM1291), Bacteroides vulgatus (B. vulgatus) (JCM5856), Escherichia coli (JCM1649), or Fusobacterium varium (F. varium) (ATCC8501) were added to the cells except for the control well, and incubated for 2 h. After incubation, we performed enzyme-linked immunosorbent assay for the cultured medium and reverse transcription polymerase chain reaction for the dendritic cells, and compared these values with controls.RESULTS: The level of CRF secretion by control dendritic cells was 40.4 ± 6.2 pg/mL. The CRF levels for cells incubated with F. varium and B. vulgatus were significantly higher than that of the control (P < 0.0001). CRF mRNA was present in the control sample without bacteria, and CRF mRNA levels in all samples treated with bacteria were above that of the control sample.F. varium caused the greatest increase in CRF mRNA expression. CONCLUSION: Our results suggest that dendritic cells produce CRF, a process augmented by commensal bacteria.

  14. Partial correction of defective Cl(-) secretion in cystic fibrosis epithelial cells by an analog of squalamine.

    Science.gov (United States)

    Jiang, C; Lee, E R; Lane, M B; Xiao, Y F; Harris, D J; Cheng, S H

    2001-11-01

    Defective cystic fibrosis (CF) transmembrane conductance regulator (CFTR)-mediated Cl(-) transport across the apical membrane of airway epithelial cells is implicated in the pathophysiology of CF lungs. A strategy to compensate for this loss is to augment Cl(-) transport through alternative pathways. We report here that partial correction of this defect could be attained through the incorporation of artificial anion channels into the CF cells. Introduction of GL-172, a synthetic analog of squalamine, into CFT1 cells increased cell membrane halide permeability. Furthermore, when a Cl(-) gradient was generated across polarized monolayers of primary human airway or Fischer rat thyroid cells in an Ussing chamber, addition of GL-172 caused an increase in the equivalent short-circuit current. The magnitude of this change in short-circuit current was ~30% of that attained when CFTR was maximally stimulated with cAMP agonists. Patch-clamp studies showed that addition of GL-172 to CFT1 cells also increased whole cell Cl(-) currents. These currents displayed a linear current-voltage relationship and no time dependence. Additionally, administration of GL-172 to the nasal epithelium of transgenic CF mice induced a hyperpolarization response to perfusion with a low-Cl(-) solution, indicating restoration of Cl(-) secretion. Together, these results demonstrate that in CF airway epithelial cells, administration of GL-172 is capable of partially correcting the defective Cl(-) secretion.

  15. Gene delivery of therapeutic polypeptides to brain capillary endothelial cells for protein secretion

    DEFF Research Database (Denmark)

    Larsen, Annette Burkhart; Thomsen, Louiza Bohn; Moos, Torben

    of the proteins. Morphological examination of the protein expression was determined using immunofluorescence detecting FLAG. Additionally, the transfection efficiency were determined by Flow cytometry. Perspective: Our study opens for knowledge on how non-viral gene therapy to BCECs can lead to protein secretion......Background: The potential for treatment of chronic disorders affecting the CNS is complicated by the inability of several drugs to cross the blood-brain barrier (BBB). None-viral gene therapy applied to brain capillary endothelial cells (BCECs) denotes a novel approach to overcome the restraints....... Results: mRNA expression of proteins with neuroprotective potential in RBEC were enabled. Their expression patters were compared with those of RBE4 and HeLa cells using RT-qPCR analyzes. The evidence for protein synthesis and secretion was obtained by detection of FLAG-tagged to the C-terminal of any...

  16. Effects of saturated, mono-, and polyunsaturated fatty acids on the secretion of apo B containing lipoproteins by Caco-2 cells

    NARCIS (Netherlands)

    van Greevenbroek, M.M.J.; van Meer, G.; Erkelens, D.W.; de Bruin, T.W.A.

    1996-01-01

    We studied the effects of addition of physiological concentrations (0.5 mM) of fatty acids i.e., palmitic (16:0), stearic (18:0), oleic (18:1) and linoleic acid (18:2) on lipoprotein secretion by polarized Caco-2 cells. With saturated fatty acids, secreted lipoproteins were at IDL/LDL density, 1.009

  17. A 3D cell culture system: separation distance between INS-1 cell and endothelial cell monolayers co-cultured in fibrin influences INS-1 cells insulin secretion.

    Science.gov (United States)

    Sabra, Georges; Vermette, Patrick

    2013-02-01

    The aim of this study was to develop an in vitro cell culture system allowing studying the effect of separation distance between monolayers of rat insulinoma cells (INS-1) and human umbilical vein endothelial cells (HUVEC) co-cultured in fibrin over INS-1 cell insulin secretion. For this purpose, a three-dimensional (3D) cell culture chamber was designed, built using micro-fabrication techniques and validated. The co-culture was successfully carried out and the effect on INS-1 cell insulin secretion was investigated. After 48 and 72 h, INS-1 cells co-cultured with HUVEC separated by a distance of 100 µm revealed enhanced insulin secretion compared to INS-1 cells cultured alone or co-cultured with HUVEC monolayers separated by a distance of 200 µm. These results illustrate the importance of the separation distance between two cell niches for cell culture design and the possibility to further enhance the endocrine function of beta cells when this factor is considered.

  18. Transforming growth factor-beta, but not ciliary neurotrophic factor, inhibits DNA synthesis of adrenal medullary cells in vitro

    DEFF Research Database (Denmark)

    Wolf, N; Krohn, K; Bieger, S;

    1999-01-01

    by the neuroendocrine chromaffin cells, which also express the transforming growth factor-beta receptor type II. In contrast to the developmentally related sympathetic neurons, chromaffin cells continue to proliferate throughout postnatal life. Using 5-bromo-2'-deoxyuridine pulse labeling and tyrosine hydroxylase...... regulator of chromaffin cell division.......Transforming growth factor-betas are members of a superfamily of multifunctional cytokines regulating cell growth and differentiation. Their functions in neural and endocrine cells are not well understood. We show here that transforming growth factor-betas are synthesized, stored and released...

  19. Lipid synthesis and secretion in HepG2 cells is not affected by ACTH

    Directory of Open Access Journals (Sweden)

    Nilsson-Ehle Peter

    2010-05-01

    Full Text Available Abstract Apolipoprotein B (apoB containing lipoproteins, i.e. VLDL, LDL and Lp(a, are consequently lowered by ACTH treatment in humans. This is also seen as reduced plasma apoB by 20-30% and total cholesterol by 30-40%, mostly accounted for by a decrease in LDL-cholesterol. Studies in hepatic cell line (HepG2 cells showed that apoB mRNA expression is reduced in response to ACTH incubation and is followed by a reduced apoB secretion, which may hypothesize that ACTH lowering apoB containing lipoproteins in humans may be mediated by the inhibition of hepatic apoB synthesis. This was recently confirmed in vivo in a human postprandial study, where ACTH reduced transient apoB48 elevation from the small intestine, however, the exogenic lipid turnover seemed unimpaired. In the present study we investigated if lipid synthesis and/or secretion in HepG2 cells were also affected by pharmacological levels of ACTH to accompany the reduced apoB output. HepG2 cells were incubated with radiolabelled precursors ([14C]acetate and [3H]glycerol either before or during ACTH stimuli. Cellular and secreted lipids were extracted with chloroform:methanol and separated by the thin layer chromatography (TLC, and [14C]labelled cholesterol and cholesteryl ester and [3H]labelled triglycerides and phospholipids were quantitated by the liquid scintillation counting. It demonstrated that ACTH administration did not result in any significant change in neither synthesis nor secretion of the studied lipids, this regardless of presence or absence of oleic acid, which is known to stabilize apoB and enhance apoB production. The present study suggests that ACTH lowers plasma lipids in humans mainly mediated by the inhibition of apoB synthesis and did not via the reduced lipid synthesis.

  20. Conazole fungicides inhibit Leydig cell testosterone secretion and androgen receptor activation in vitro

    Directory of Open Access Journals (Sweden)

    Maarke J.E. Roelofs

    2014-01-01

    Full Text Available Conazole fungicides are widely used in agriculture despite their suspected endocrine disrupting properties. In this study, the potential (anti-androgenic effects of ten conazoles were assessed and mutually compared with existing data. Effects of cyproconazole (CYPRO, fluconazole (FLUC, flusilazole (FLUS, hexaconazole (HEXA, myconazole (MYC, penconazole (PEN, prochloraz (PRO, tebuconazole (TEBU, triadimefon (TRIA, and triticonazole (TRIT were examined using murine Leydig (MA-10 cells and human T47D-ARE cells stably transfected with an androgen responsive element and a firefly luciferase reporter gene. Six conazoles caused a decrease in basal testosterone (T secretion by MA-10 cells varying from 61% up to 12% compared to vehicle-treated control. T secretion was concentration-dependently inhibited after exposure of MA-10 cells to several concentrations of FLUS (IC50 = 12.4 μM or TEBU (IC50 = 2.4 μM in combination with LH. The expression of steroidogenic and cholesterol biosynthesis genes was not changed by conazole exposure. Also, there were no changes in reactive oxygen species (ROS formation that could explain the altered T secretion after exposure to conazoles. Nine conazoles decreased T-induced AR activation (IC50s ranging from 10.7 to 71.5 μM and effect potencies (REPs were calculated relative to the known AR antagonist flutamide (FLUT. FLUC had no effect on AR activation by T. FLUS was the most potent (REP = 3.61 and MYC the least potent (REP = 0.03 AR antagonist. All other conazoles had a comparable REP from 0.12 to 0.38. Our results show distinct in vitro anti-androgenic effects of several conazole fungicides arising from two mechanisms: inhibition of T secretion and AR antagonism, suggesting potential testicular toxic effects. These effects warrant further mechanistic investigation and clearly show the need for accurate exposure data in order to perform proper (human risk assessment of this class of compounds.

  1. BDNF-secreting capsule exerts neuroprotective effects on epilepsy model of rats.

    Science.gov (United States)

    Kuramoto, Satoshi; Yasuhara, Takao; Agari, Takashi; Kondo, Akihiko; Jing, Meng; Kikuchi, Yoichiro; Shinko, Aiko; Wakamori, Takaaki; Kameda, Masahiro; Wang, Feifei; Kin, Kyohei; Edahiro, Satoru; Miyoshi, Yasuyuki; Date, Isao

    2011-01-12

    Brain-derived neurotrophic factor (BDNF) is a well neurotrophic factor with neuroprotective potentials for various diseases in the central nervous system. However several previous studies demonstrated that BDNF might deteriorate symptoms for epilepsy model of animals by progression of abnormal neurogenesis. We hypothesized that continuous administration of BDNF at low dose might be more effective for epilepsy model of animals because high dose of BDNF was used in many studies. BDNF-secreting cells were genetically made and encapsulated for transplantation. Rats receiving BDNF capsule showed significant amelioration of seizure stage and reduction of the number of abnormal spikes at 7 days after kainic acid administration, compared to those of control group. The number of BrdU and BrdU/doublecortin positive cells in the hippocampus of BDNF group significantly increased, compared to that of control group. NeuN positive cells in the CA1 and CA3 of BDNF group were significantly preserved, compared to control group. In conclusion, low dose administration using encapsulated BDNF-secreting cells exerted neuroprotective effects with enhanced neurogenesis on epilepsy model of rats. These results might suggest the importance of the dose and administrative way of this neurotrophic factor to the epilepsy model of animals.

  2. Isocitrate-to-SENP1 signaling amplifies insulin secretion and rescues dysfunctional β cells.

    Science.gov (United States)

    Ferdaoussi, Mourad; Dai, Xiaoqing; Jensen, Mette V; Wang, Runsheng; Peterson, Brett S; Huang, Chao; Ilkayeva, Olga; Smith, Nancy; Miller, Nathanael; Hajmrle, Catherine; Spigelman, Aliya F; Wright, Robert C; Plummer, Gregory; Suzuki, Kunimasa; Mackay, James P; van de Bunt, Martijn; Gloyn, Anna L; Ryan, Terence E; Norquay, Lisa D; Brosnan, M Julia; Trimmer, Jeff K; Rolph, Timothy P; Kibbey, Richard G; Manning Fox, Jocelyn E; Colmers, William F; Shirihai, Orian S; Neufer, P Darrell; Yeh, Edward T H; Newgard, Christopher B; MacDonald, Patrick E

    2015-10-01

    Insulin secretion from β cells of the pancreatic islets of Langerhans controls metabolic homeostasis and is impaired in individuals with type 2 diabetes (T2D). Increases in blood glucose trigger insulin release by closing ATP-sensitive K+ channels, depolarizing β cells, and opening voltage-dependent Ca2+ channels to elicit insulin exocytosis. However, one or more additional pathway(s) amplify the secretory response, likely at the distal exocytotic site. The mitochondrial export of isocitrate and engagement with cytosolic isocitrate dehydrogenase (ICDc) may be one key pathway, but the mechanism linking this to insulin secretion and its role in T2D have not been defined. Here, we show that the ICDc-dependent generation of NADPH and subsequent glutathione (GSH) reduction contribute to the amplification of insulin exocytosis via sentrin/SUMO-specific protease-1 (SENP1). In human T2D and an in vitro model of human islet dysfunction, the glucose-dependent amplification of exocytosis was impaired and could be rescued by introduction of signaling intermediates from this pathway. Moreover, islet-specific Senp1 deletion in mice caused impaired glucose tolerance by reducing the amplification of insulin exocytosis. Together, our results identify a pathway that links glucose metabolism to the amplification of insulin secretion and demonstrate that restoration of this axis rescues β cell function in T2D.

  3. Physiological and clinical significance of enterochromaffin-like cell activation in the regulation of gastric acid secretion

    Institute of Scientific and Technical Information of China (English)

    Guanglin Cui; Helge L Waldum

    2007-01-01

    Gastric acid plays an important role in digesting food (especially protein), iron absorption, and destroying swallowed micro-organisms. H+ is secreted by the oxyntic parietal cells and its secretion is regulated by endocrine, neurocrine and paracrine mechanisms.Gastrin released from the antral G cell is the principal physiological stimulus of gastric acid secretion. Activation of the enterochromaffin-like (ECL) cell is accepted as the main source of histamine participating in the regulation of acid secretion and is functionally and trophically controlled by gastrin, which is mediated by gastrin/CCK-2 receptors expressed on the ECL cell. However, longterm hypergastrinemia will induce ECL cell hyperplasia and probably carcinoids. Clinically, potent inhibitors of acid secretion have been prescribed widely to patients with acid-related disorders. Long-term potent acid inhibition evokes a marked increase in plasma gastrin levels,leading to enlargement of oxyntic mucosa with ECL cell hyperplasia. Accordingly, the induction of ECL cell hyperplasia and carcinoids remains a topic of considerable concern, especially in long-term use. In addition, the activation of ECL cells also induces another clinical concern, i.e., rebound acid hypersecretion after acid inhibition. Recent experimental and clinical findings indicate that the activation of ECL cells plays a critical role both physiologically and clinically in the regulation of gastric acid secretion.

  4. Intrathecal injection of lentivirus-mediated glial cell line-derived neurotrophic factor RNA interference relieves bone cancer-induced pain in rats.

    Science.gov (United States)

    Meng, Fu-Fen; Xu, Yang; Dan, Qi-Qin; Wei, La; Deng, Ying-Jie; Liu, Jia; He, Mu; Liu, Wei; Xia, Qing-Jie; Zhou, Fiona H; Wang, Ting-Hua; Wang, Xi-Yan

    2015-04-01

    Bone cancer pain is a common symptom in cancer patients with bone metastases and the underlying mechanisms are largely unknown. The aim of this study is to explore the endogenous analgesic mechanisms to develop new therapeutic strategies for bone-cancer induced pain (BCIP) as a result of metastases. MRMT-1 tumor cells were injected into bilateral tibia of rats and X-rays showed that the area suffered from bone destruction, accompanied by an increase in osteoclast numbers. In addition, rats with bone cancer showed apparent mechanical and thermal hyperalgesia at day 28 after intratibial MRMT-1 inoculation. However, intrathecal injection of morphine or lentivirus-mediated glial cell line-derived neurotrophic factor RNAi (Lvs-siGDNF) significantly attenuated mechanical and thermal hyperalgesia, as shown by increases in paw withdrawal thresholds and tail-flick latencies, respectively. Furthermore, Lvs-siGDNF interference not only substantially downregulated GDNF protein levels, but also reduced substance P immunoreactivity and downregulated the ratio of pERK/ERK, where its activation is crucial for pain signaling, in the spinal dorsal horn of this model of bone-cancer induced pain. In this study, Lvs-siGDNF gene therapy appeared to be a beneficial method for the treatment of bone cancer pain. As the effect of Lvs-siGDNF to relieve pain was similar to morphine, but it is not a narcotic, the use of GDNF RNA interference may be considered as a new therapeutic strategy for the treatment of bone cancer pain in the future.

  5. Activation of secretion and surface alteration of cytolytic T-lymphocytes interacting with target cells.

    Science.gov (United States)

    Bykovskaya, S N; Shevelev, A A; Kupriyanova, T A

    1988-01-01

    Cells obtained in mixed lymphocyte culture (MLC) and memory cells adsorbed on the surface of target cells (TC) were examined using scanning and transmission electron microscopy depending on the time of interaction with TC. Three types of lymphocytes were revealed: type I - cells of spherical shape with a smooth surface or an insignificant amount of microvilli; predominantly small and medium-sized lymphocytes contacting TC with non significant involvement of their surface or by several microvilli; type II - oval or round-shaped lymphocytes evenly covered with microvilli with considerably enlarged region of contact; type III cells - predominantly large lymphocytes and lymphoblasts flattened (spread) on TC, with multiple microvilli, ridge-like projections, and ruffles on their surface. TEM revealed activation of the secretory apparatus in the cytoplasm of such lymphocytes. With increased time of interaction, type III cells increase in number (from 8.6% after 10 min to 90.2% after 60 min of incubation). Memory cells show no morphologic signs of secretion in correlation with the absence of lysis of TC on which they are adsorbed. The surface of the lymphocytes adsorbed on the substrate with poly-L-lysin is not noticeably altered. It is suggested that 3 morphological types of lymphocytes correspond to 3 stages of secretion activation. Lymphocyte contact with TC surface is evidently a specific stimulus for activating secretory apparatus of CTL. SEM can be used for quantitation of activated lymphocytes.

  6. Human umbilical cord-derived mesenchymal stem cells can secrete insulin in vitro and in vivo.

    Science.gov (United States)

    Boroujeni, Zahra Niki; Aleyasin, Ahmad

    2014-01-01

    Diabetes mellitus is characterized by autoimmune destruction of pancreatic beta cells, leading to decreased insulin production. Differentiation of mesenchymal stem cells (MSCs) into insulin-producing cells offers novel ways of diabetes treatment. MSCs can be isolated from the human umbilical cord tissue and differentiate into insulin-secreting cells. Human umbilical cord-derived stem cells (hUDSCs) were obtained after birth, selected by plastic adhesion, and characterized by flow cytometric analysis. hUDSCs were transduced with nonintegrated lentivirus harboring PDX1 (nonintegrated LV-PDX1) and was cultured in differentiation medium in 21 days. Pancreatic duodenum homeobox protein-1 (PDX1) is a transcription factor in pancreatic development. Significant expressions of PDX1, neurogenin3 (Ngn3), glucagon, glucose transporter2 (Glut2), and somatostatin were detected by quantitative RT-PCR (P insulin proteins were shown by immunocytochemistry analysis. Insulin secretion of hUDSCs(PDX1+) in the high-glucose medium was 1.8 μU/mL. They were used for treatment of diabetic rats and could decrease the blood glucose level from 400 mg/dL to a normal level in 4 days. In conclusion, our results demonstrated that hUDSCs are able to differentiate into insulin-producing cells by transduction with nonintegrated LV-PDX1. These hUDSCs(PDX1+) have the potential to be used as a viable resource in cell-based gene therapy of type 1 diabetes.

  7. Global impact of Salmonella type III secretion effector SteA on host cells

    Energy Technology Data Exchange (ETDEWEB)

    Cardenal-Muñoz, Elena, E-mail: e_cardenal@us.es; Gutiérrez, Gabriel, E-mail: ggpozo@us.es; Ramos-Morales, Francisco, E-mail: framos@us.es

    2014-07-11

    Highlights: • We analyzed HeLa cells transcriptome in response to Salmonella SteA. • Significant differential expression was detected for 58 human genes. • They are involved in ECM organization and regulation of some signaling pathways. • Cell death, cell adhesion and cell migration were decreased in SteA-expressing cells. • These results contribute to understand the role of SteA during infections. - Abstract: Salmonella enterica is a Gram-negative bacterium that causes gastroenteritis, bacteremia and typhoid fever in several animal species including humans. Its virulence is greatly dependent on two type III secretion systems, encoded in pathogenicity islands 1 and 2. These systems translocate proteins called effectors into eukaryotic host cell. Effectors interfere with host signal transduction pathways to allow the internalization of pathogens and their survival and proliferation inside vacuoles. SteA is one of the few Salmonella effectors that are substrates of both type III secretion systems. Here, we used gene arrays and bioinformatics analysis to study the genetic response of human epithelial cells to SteA. We found that constitutive synthesis of SteA in HeLa cells leads to induction of genes related to extracellular matrix organization and regulation of cell proliferation and serine/threonine kinase signaling pathways. SteA also causes repression of genes related to immune processes and regulation of purine nucleotide synthesis and pathway-restricted SMAD protein phosphorylation. In addition, a cell biology approach revealed that epithelial cells expressing steA show altered cell morphology, and decreased cytotoxicity, cell–cell adhesion and migration.

  8. Exosomes secreted from human colon cancer cells influence the adhesion of neighboring metastatic cells: Role of microRNA-210

    Science.gov (United States)

    Bigagli, Elisabetta; Luceri, Cristina; Guasti, Daniele; Cinci, Lorenzo

    2016-01-01

    ABSTRACT Cancer-secreted exosomes influence tumor microenvironment and support cancer growth and metastasis. MiR-210 is frequently up-regulated in colorectal cancer tissues and correlates with metastatic disease. We investigated whether exosomes are actively released by HCT-8 colon cancer cells, the role of exosomal miR-210 in the cross-talk between primary cancer cells and neighboring metastatic cells and its contribution in regulating epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET). After 7 d of culture, a subpopulation of viable HCT-8 cells detached the monolayer and started to grow in suspension, suggesting anoikis resistance and a metastatic potential. The expression of key proteins of EMT revealed that these cells were E-cadherin negative and vimentin positive further confirming their metastatic phenotype and the acquisition of anoikis resistance. Metastatic cells, in the presence of adherently growing HCT-8, continued to grow in suspension whereas only if seeded in cell-free wells, were able to adhere again and to form E-cadherin positive and vimentin negative new colonies, suggesting the occurrence of MET. The chemosensitivity to 5 fluorouracil and to FOLFOX-like treatment of metastatic cells was significantly diminished compared to adherent HCT-8 cells. Of note, adherent new colonies undergoing MET, were insensitive to both chemotherapeutic strategies. Electron microscopy analysis demonstrated that adherently growing HCT-8, actually secreted exosomes and that exosomes in turn were taken up by metastatic cells. When exosomes secreted by adherently growing HCT-8 were administered to metastatic cells, MET was significantly inhibited. miR-210 was significantly upregulated in exosomes compared to its intracellular levels in adherently growing HCT-8 cells and correlated to anoikis resistance and EMT markers. Exosomes containing miR-210 might be considered as EMT promoting signals that preserve the local cancer

  9. Role of adipose secreted factors and kisspeptin in the metabolic control of gonadotropin secretion and puberty

    Science.gov (United States)

    Factors secreted by adipose tissue continue to be discovered. Evidence indicates a strong link between neural influences and adipocyte expression and secretion of a wide array of cytokines, neurotrophic factors, growth factors, binding proteins, and neuropeptides. These “adipokines” are linked to im...

  10. Perinatal undernutrition modifies cell proliferation and brain-derived neurotrophic factor levels during critical time-windows for hypothalamic and hippocampal development in the male rat.

    Science.gov (United States)

    Coupé, B; Dutriez-Casteloot, I; Breton, C; Lefèvre, F; Mairesse, J; Dickes-Coopman, A; Silhol, M; Tapia-Arancibia, L; Lesage, J; Vieau, D

    2009-01-01

    Maternal perinatal undernutrition (MPU) modifies the activity of the hypothalamic-pituitary-adrenal axis and sensitises to the development of metabolic and cognitive adult diseases. Because the hypothalamus and hippocampus are involved in the regulation of neuroendocrine activity, energy metabolism and cognition, we hypothesised that a maternal 50% food restriction (FR50) from day 14 of pregnancy (E14) until postnatal day 21 (P21) would affect the development of these structures in male rat offspring. Protein and mRNA levels of brain-derived neurotrophic factor (BDNF) and cell proliferation [analysed by 5-bromodeoxyuridine (BrdU) incorporation] were compared in both control and FR50 rats from E21 to P22. Although the pattern of the evolution of BDNF concentration and cell proliferation throughout development was not strikingly different between groups, several disturbances at specific developmental stages were observed. FR50 rats exhibited a delayed increase of hippocampal BDNF content whereas, in the hypothalamus, BDNF level was augmented from E21 to P14 and associated, at this latter stage, with an increased mRNA expression of TRkB-T2. In both groups, a correlation between BDNF content and the number of BrdU positive cells was noted in the dentate gyrus, whereas opposite variations were observed in CA1, CA2 and CA3 layers, and in the arcuate and ventromedial nuclei. In the hippocampus, P15-FR50 rats showed an increased number of BrdU positive cells in all regions, whereas, at P22, a decrease was observed in the CA2. In the hypothalamus, between E21 and P8, MPU increases the number of BrdU positive cells in all regions analysed and, until P15, marked differences were noticed in the median eminence, the paraventricular nucleus and the arcuate nucleus. Taken together, the results obtained in the present study show that MPU changes the time course of production of BDNF and cell proliferation in specific hippocampal and hypothalamic areas during sensitive

  11. Directed evolution of brain-derived neurotrophic factor for improved folding and expression in Saccharomyces cerevisiae.

    Science.gov (United States)

    Burns, Michael L; Malott, Thomas M; Metcalf, Kevin J; Hackel, Benjamin J; Chan, Jonah R; Shusta, Eric V

    2014-09-01

    Brain-derived neurotrophic factor (BDNF) plays an important role in nervous system function and has therapeutic potential. Microbial production of BDNF has resulted in a low-fidelity protein product, often in the form of large, insoluble aggregates incapable of binding to cognate TrkB or p75 receptors. In this study, employing Saccharomyces cerevisiae display and secretion systems, it was found that BDNF was poorly expressed and partially inactive on the yeast surface and that BDNF was secreted at low levels in the form of disulfide-bonded aggregates. Thus, for the purpose of increasing the compatibility of yeast as an expression host for BDNF, directed-evolution approaches were employed to improve BDNF folding and expression levels. Yeast surface display was combined with two rounds of directed evolution employing random mutagenesis and shuffling to identify BDNF mutants that had 5-fold improvements in expression, 4-fold increases in specific TrkB binding activity, and restored p75 binding activity, both as displayed proteins and as secreted proteins. Secreted BDNF mutants were found largely in the form of soluble homodimers that could stimulate TrkB phosphorylation in transfected PC12 cells. Site-directed mutagenesis studies indicated that a particularly important mutational class involved the introduction of cysteines proximal to the native cysteines that participate in the BDNF cysteine knot architecture. Taken together, these findings show that yeast is now a viable alternative for both the production and the engineering of BDNF.

  12. Identification of chromosomal genes in Yersinia pestis that influence type III secretion and delivery of Yops into target cells.

    Directory of Open Access Journals (Sweden)

    Andrew S Houppert

    Full Text Available Pathogenic Yersinia species possess a type III secretion system, which is required for the delivery of effector Yop proteins into target cells during infection. Genes encoding the type III secretion machinery, its substrates, and several regulatory proteins all reside on a 70-Kb virulence plasmid. Genes encoded in the chromosome of yersiniae are thought to play important roles in bacterial perception of host environments and in the coordinated activation of the type III secretion pathway. Here, we investigate the contribution of chromosomal genes to the complex regulatory process controlling type III secretion in Yersinia pestis. Using transposon mutagenesis, we identified five chromosomal genes required for expression or secretion of Yops in laboratory media. Four out of the five chromosomal mutants were defective to various extents at injecting Yops into tissue culture cells. Interestingly, we found one mutant that was not able to secrete in vitro but was fully competent for injecting Yops into host cells, suggesting independent mechanisms for activation of the secretion apparatus. When tested in a mouse model of plague disease, three mutants were avirulent, whereas two strains were severely attenuated. Together these results demonstrate the importance of Y. pestis chromosomal genes in the proper function of type III secretion and in the pathogenesis of plague.

  13. Cytokine expression and secretion by skeletal muscle cells: regulatory mechanisms and exercise effects.

    Science.gov (United States)

    Peake, Jonathan M; Della Gatta, Paul; Suzuki, Katsuhiko; Nieman, David C

    2015-01-01

    Cytokines are important mediators of various aspects of health and disease, including appetite, glucose and lipid metabolism, insulin sensitivity, skeletal muscle hypertrophy and atrophy. Over the past decade or so, considerable attention has focused on the potential for regular exercise to counteract a range of disease states by modulating cytokine production. Exercise stimulates moderate to large increases in the circulating concentrations of interleukin (IL)-6, IL-8, IL- 10, IL-1 receptor antagonist, granulocyte-colony stimulating factor, and smaller increases in tumor necrosis factor-α, monocyte chemotactic protein-1, IL-1β, brain-derived neurotrophic factor, IL-12p35/p40 and IL-15. Although many of these cytokines are also expressed in skeletal muscle, not all are released from skeletal muscle into the circulation during exercise. Conversely, some cytokines that are present in the circulation are not expressed in skeletal muscle after exercise. The reasons for these discrepant cytokine responses to exercise are unclear. In this review, we address these uncertainties by summarizing the capacity of skeletal muscle cells to produce cytokines, analyzing other potential cellular sources of circulating cytokines during exercise, and discussing the soluble factors and intracellular signaling pathways that regulate cytokine synthesis (e.g., RNA-binding proteins, microRNAs, suppressor of cytokine signaling proteins, soluble receptors).

  14. Depletion of polyamines prevents the neurotrophic activity of the GABA-agonist THIP in cultured rat cerebellar granule cells

    DEFF Research Database (Denmark)

    Abraham, J H; Hansen, Gert Helge; Seiler, N

    1993-01-01

    Effects of polyamine depletion by alpha-difluoromethylornithine (DFMO) were studied on the GABA-agonist mediated enhancement of the morphological development of cultured rat cerebellar granule cells. An increase in the number of neurite extending cells and in the cytoplasmic density of organelles...... endoplasmic reticulum, Golgi apparatus and different types of vesicles was prevented by the exposure to DFMO....

  15. Hypoxic tumor cell modulates its microenvironment to enhance angiogenic and metastatic potential by secretion of proteins and exosomes.

    Science.gov (United States)

    Park, Jung Eun; Tan, Hon Sen; Datta, Arnab; Lai, Ruenn Chai; Zhang, Huoming; Meng, Wei; Lim, Sai Kiang; Sze, Siu Kwan

    2010-06-01

    Under hypoxia, tumor cells produce a secretion that modulates their microenvironment to facilitate tumor angiogenesis and metastasis. Here, we observed that hypoxic or reoxygenated A431 carcinoma cells exhibited enhanced angiogenic and metastatic potential such as reduced cell-cell and cell-extracellular matrix adhesion, increased invasiveness, and production of a secretion with increased chorioallantoic membrane angiogenic activity. Consistent with these observations, quantitative proteomics revealed that under hypoxia the tumor cells secreted proteins involved in angiogenesis, focal adhesion, extracellular matrix-receptor interaction, and immune cell recruitment. Unexpectedly, the secreted proteins were predominantly cytoplasmic and membrane proteins. Ultracentrifugation at 100,000 x g precipitated 54% of the secreted proteins and enriched for many exosome-associated proteins such as the tetraspanins and Alix and also proteins with the potential to facilitate angiogenesis and metastasis. Two tetraspanins, CD9 and CD81, co-immunoprecipitated. Together, these data suggested that tumor cells secrete proteins and exosomes with the potential to modulate their microenvironment and facilitate angiogenesis and metastasis.

  16. Ghrelin but not obestatin regulates insulin secretion from INS1 beta cell line via UCP2-dependent mechanism.

    Science.gov (United States)

    Chmielewska, J; Szczepankiewicz, D; Skrzypski, M; Kregielska, D; Strowski, M Z; Nowak, K W

    2010-01-01

    The mitochondrial UCP2 mediates glucose-stimulated insulin secretion by decreasing intracellular ATP/ADP ratio. Insulin secretion is a tightly regulated process. Ghrelin, as well as obestatin, were intensively studied to determine their ability to modify insulin secretion. Ghrelin is considered to be an inhibitor of insulin release from pancreatic islets, however little is known about the effects of obestatin. In our study we demonstrate the stimulating effects of both peptides on insulin secretion in INS1 cells. Furthermore, we investigate the potential role of UCP2 in mediating the effects of both peptides on insulin secretion. UCP2 mRNA expression was down-regulated by ghrelin in the presence of 26.4 mM glucose, however it was unchanged after obestatin treatment. Our results confirm that UCP2 could be involved in the stimulating effect of ghrelin on insulin release from INS1 cells.

  17. Genetic engineering of cell lines using lentiviral vectors to achieve antibody secretion following encapsulated implantation.

    Science.gov (United States)

    Lathuilière, Aurélien; Bohrmann, Bernd; Kopetzki, Erhard; Schweitzer, Christoph; Jacobsen, Helmut; Moniatte, Marc; Aebischer, Patrick; Schneider, Bernard L

    2014-01-01

    The controlled delivery of antibodies by immunoisolated bioimplants containing genetically engineered cells is an attractive and safe approach for chronic treatments. To reach therapeutic antibody levels there is a need to generate renewable cell lines, which can long-term survive in macroencapsulation devices while maintaining high antibody specific productivity. Here we have developed a dual lentiviral vector strategy for the genetic engineering of cell lines compatible with macroencapsulation, using separate vectors encoding IgG light and heavy chains. We show that IgG expression level can be maximized as a function of vector dose and transgene ratio. This approach allows for the generation of stable populations of IgG-expressing C2C12 mouse myoblasts, and for the subsequent isolation of clones stably secreting high IgG levels. Moreover, we demonstrate that cell transduction using this lentiviral system leads to the production of a functional glycosylated antibody by myogenic cells. Subsequent implantation of antibody-secreting cells in a high-capacity macroencapsulation device enables continuous delivery of recombinant antibodies in the mouse subcutaneous tissue, leading to substantial levels of therapeutic IgG detectable in the plasma.

  18. Prostaglandin E2 regulates macrophage colony stimulating factor secretion by human bone marrow stromal cells.

    Science.gov (United States)

    Besse, A; Trimoreau, F; Faucher, J L; Praloran, V; Denizot, Y

    1999-07-08

    Bone marrow stromal cells regulate marrow haematopoiesis by secreting growth factors such as macrophage colony stimulating factor (M-CSF) that regulates the proliferation, differentiation and several functions of cells of the mononuclear-phagocytic lineage. By using a specific ELISA we found that their constitutive secretion of M-CSF is enhanced by tumour necrosis factor-alpha (TNF-alpha). The lipid mediator prostaglandin E2 (PGE2) markedly reduces in a time- and dose-dependent manner the constitutive and TNF-alpha-induced M-CSF synthesis by bone marrow stromal cells. In contrast, other lipid mediators such as 12-HETE, 15-HETE, leukotriene B4, leukotriene C4 and lipoxin A4 have no effect. EP2/EP4 selective agonists (11-deoxy PGE1 and 1-OH PGE1) and EP2 agonist (19-OH PGE2) inhibit M-CSF synthesis by bone marrow stromal cells while an EP1/EP3 agonist (sulprostone) has no effect. Stimulation with PGE2 induces an increase of intracellular cAMP levels in bone marrow stromal cells. cAMP elevating agents (forskolin and cholera toxin) mimic the PGE2-induced inhibition of M-CSF production. In conclusion, PGE2 is a potent regulator of M-CSF production by human bone marrow stromal cells, its effects being mediated via cAMP and PGE receptor EP2/EP4 subtypes.

  19. CD109 is a component of exosome secreted from cultured cells.

    Science.gov (United States)

    Sakakura, Hiroki; Mii, Shinji; Hagiwara, Sumitaka; Kato, Takuya; Yamamoto, Noriyuki; Hibi, Hideharu; Takahashi, Masahide; Murakumo, Yoshiki

    2016-01-22

    Exosomes are 50-100-nm-diameter membrane vesicles released from various types of cells. Exosomes retain proteins, mRNAs and miRNAs, which can be transported to surrounding cells. CD109 is a glycosylphosphatidylinositol-anchored glycoprotein, and is released from the cell surface to the culture medium in vitro. Recently, it was reported that secreted CD109 from the cell surface downregulates transforming growth factor-β signaling in human keratinocytes. In this study, we revealed that CD109 is a component of the exosome in conditioned medium. FLAG-tagged human CD109 (FLAG-CD109) in conditioned medium secreted from HEK293 cells expressing FLAG-CD109 (293/FLAG-CD109) was immunoprecipitated with anti-FLAG affinity gel, and the co-precipitated proteins were analyzed by mass spectrometry and western blotting. Exosomal proteins were associated with CD109. We revealed the presence of CD109 in exosome fractions from conditioned medium of 293/FLAG-CD109. Moreover, the localization of CD109 in the exosome was demonstrated using immuno-electron microscopy. When we used HEK293 cells expressing FLAG-tagged truncated CD109, which does not contain the C-terminal region, the association of truncated CD109 with exosomes was not detected in conditioned medium. These findings indicate that CD109 is an exosomal protein and that the C-terminal region of CD109 is required for its presence in the exosome.

  20. Transcriptional profiling of mouse B cell terminal differentiation defines a signature for antibody-secreting plasma cells.

    Science.gov (United States)

    Shi, Wei; Liao, Yang; Willis, Simon N; Taubenheim, Nadine; Inouye, Michael; Tarlinton, David M; Smyth, Gordon K; Hodgkin, Philip D; Nutt, Stephen L; Corcoran, Lynn M

    2015-06-01

    When B cells encounter an antigen, they alter their physiological state and anatomical localization and initiate a differentiation process that ultimately produces antibody-secreting cells (ASCs). We have defined the transcriptomes of many mature B cell populations and stages of plasma cell differentiation in mice. We provide a molecular signature of ASCs that highlights the stark transcriptional divide between B cells and plasma cells and enables the demarcation of ASCs on the basis of location and maturity. Changes in gene expression correlated with cell-division history and the acquisition of permissive histone modifications, and they included many regulators that had not been previously implicated in B cell differentiation. These findings both highlight and expand the core program that guides B cell terminal differentiation and the production of antibodies.

  1. Mathematical Beta Cell Model for Insulin Secretion following IVGTT and OGTT

    DEFF Research Database (Denmark)

    Madsen, Henrik; Henriksen, Jan Erik; Karlsson, Mats

    2006-01-01

    Evaluation of beta cell function is conducted by a variety of glucose tolerance tests and evaluated by a number of different models with less than perfect consistency among results obtained from different tests. We formulated a new approximation of the distributed threshold model for insulin...... secretion in order to approach a model for quantifying beta cell function, not only for one, but for several different experiments. Data was obtained from 40 subjects that had both an oral glucose tolerance test (OGTT) and an intravenous tolerance test (IVGTT) performed. Parameter estimates from the two...

  2. Effects of dimethylsulfoxide (DMSO), nocodazole, and taxol on mast cell histamine secretion

    DEFF Research Database (Denmark)

    Nielsen, E H; Johansen, Torben

    1986-01-01

    Nocodazole depolymerized microtubules and increased the number of microfilaments, and dimethylsulfoxide increased the number of microfilaments. Both drugs inhibited compound 48/80-induced histamine release from rat mast cells. Taxol, which increased the number of microtubules, had no effect...... on histamine release. These observations support the view that microtubules may not be directly involved in secretion, but apparently an increased number of microfilaments is associated with a decreased capacity of the mast cells for histamine release. We suggest that microfilaments have to be depolymerized...

  3. Probiotics inhibit TNF-α-induced interleukin-8 secretion of HT29 cells

    Institute of Scientific and Technical Information of China (English)

    Ai-Ping Bai; Qin Ouyang; Wen Zhang; Chun-Hui Wang; Sheng-Fu Li

    2004-01-01

    AIM: To study the effect of probiotics on interleukin-8 secretion in intestinal epithelia when stimulated by METHODS: Colonic adenocarcinoma HT29 cells were cultured and divided into four groups: control, TNF-α (group T in short),bifidobacterium (group B), lactobacillus (group L). B. Longum and L. bulgaricus were suspended in culture medium with a concentration of 1x108 cfu/ml and added into 24 wells respectively. One hour later TNF-α (10 ng/ml) was added into each well of groups T, B, L. The supernatants were collected and measured for IL-8 after 3 hours, nucfear factorκB (NF-κB) p65 was also examined by Western blotting.RESULTS: There was less interleukin-8 secretion in HT29 cells when preincubated with B. Longum or L. bulgaricus compared with group T. Less p65 appeared in nuclei in groups B and L compared with group T, as detected by Western blot.CONCLUSION: Probiotics can suppress interleukin-8 secretion in intestinal epithelia when stimulated by proinflammatory cytokines, which is most likely mediated by NF-κB.

  4. Effect of PKC on Glucose-Mediated Insulin Secretion in HIT-T15 Cells

    Directory of Open Access Journals (Sweden)

    Akiyoshi H

    2000-09-01

    Full Text Available OBJECTIVE: To clarify the regulation of protein kinase C on glucose-mediated insulin secretion. MAIN OUTCOME MEASURES: We examined the effect of protein kinase C on the cytosolic free Ca(2+ concentration ([Ca(2+]i and the activity of Ca(2+-activated K(+ channels (K(Ca-channel in the insulinoma cell line, HIT-T15. RESULTS: Glucose at a concentration of 10 mmol/L increased the secretion of insulin. This increase was partly inhibited by 1 nmol/L staurosporine, a protein kinase C inhibitor. Staurosporine (1 nmol/L also attenuated the glucose-induced elevations in [Ca(2+]i. On the contrary, glibenclamide (100 nmol/L specifically blocked ATP-sensitive K(+ channels, and increased both [Ca(2+]i and insulin secretion, but staurosporine had no effect on them. Patch clamp studies showed that 10 mmol/L glucose almost completely blocked K(Ca channel activity, an effect that was reversed by 1 nmol/L staurosporine. Phorbol 12-myristate 13-acetate (1 mmol/L, a protein kinase C activator, also decreased K(Ca channel activity. CONCLUSIONS: These results indicate that the activation of protein kinase C is involved in the glucose-induced release of insulin by modulating K(+ channel function in HIT-T15 cells.

  5. Vectorial secretion of interleukin-8 mediates autocrine signalling in intestinal epithelial cells via apically located CXCR1

    NARCIS (Netherlands)

    Rossi, Oriana; Karczewski, Jurgen; Stolte, Ellen H; Brummer, Robert J M; van Nieuwenhoven, Michiel A; Meijerink, Marjolein; van Neerven, Joost R J; van Ijzendoorn, Sven C D; van Baarlen, Peter; Wells, Jerry M

    2013-01-01

    BACKGROUND: In the intestinal mucosa, several adaptations of TLR signalling have evolved to avoid chronic inflammatory responses to the presence of commensal microbes. Here we investigated whether polarized monolayers of intestinal epithelial cells might regulate inflammatory responses by secreting

  6. Direct visualization of secretion from single bovine adrenal chromaffin cells by laser-induced native fluorescence imaging microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Tong, W.; Yeung, E.S. [Ames Laboratory---USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 50011 (United States)

    1998-03-01

    Direct visualization of the secretion process of individual bovine adrenal chromaffin cells was achieved with laser-induced native fluorescence imaging microscopy. By monitoring the native fluorescence of catecholamines excited by the 275 nm laser line with an intensified charge-coupled-device (CCD) camera, we obtained good temporal and spatial resolution simultaneously without using additional fluorescent probes. Large variations were found among individual cells in terms of the amounts of catecholamines secreted and the rates of secretion. Different regions of a cell also behave differently during the secretion process. However, the degree of this local heterogeneity is smaller than in neurons and neuralgia. The influence of deep-ultraviolet (UV) laser excitation on cells is also discussed. This quantitative imaging technique provides a useful noninvasive approach for the study of dynamic cellular changes and the understanding of the molecular mechanisms of secretory processes. {copyright} {ital 1998} {ital Society for Applied Spectroscopy}

  7. In vitro culture of hybridoma cells in agarose beads producing antibody secretion for two weeks.

    Science.gov (United States)

    Cadic, C; Dupuy, B; Pianet, I; Merle, M; Margerin, C; Bezian, J H

    1992-01-05

    A new process for embedding cells in agarose is described. Beads were obtained by extruding an ultralow gelling temperature agarose solution in a capillary containing a hydrophobic medium flowthrough. The toxicity of the procedure has been evaluated by monitoring the energy status of agarose-embedded C(6) glioma cells with (31)P nuclear magnetic resonance (NMR). Suspension and microbead cultures of hybridoma cell line were compared. In suspension culture the number of cells and the antibody concentrations increased for 5 days before the stationary phase began, when the cultures were stopped. In agarose bead cultures, the gel provided an enormous support surface area (50 m(2)/ mL of gel). It was possible to seed 20-fold more cells. The gel pressure modified the proliferative process and antibody pattern secretion. In particular, the antibodies could be harvested for two weeks.

  8. Glial cell-line derived neurotrophic factor (GDNF) replacement attenuates motor impairments and nigrostriatal dopamine deficits in 12-month-old mice with a partial deletion of GDNF.

    Science.gov (United States)

    Littrell, Ofelia M; Granholm, Ann-Charlotte; Gerhardt, Greg A; Boger, Heather A

    2013-03-01

    Glial cell-line derived neurotrophic factor (GDNF) has been established as a growth factor for the survival and maintenance of dopamine (DA) neurons. In phase I clinical trials, GDNF treatment in Parkinson's disease patients led to improved motor function and GDNF has been found to be down regulated in Parkinson's disease patients. Studies using GDNF heterozygous (Gdnf(+/-)) mice have demonstrated that a partial reduction of GDNF leads to an age-related accelerated decline in nigrostriatal DA system- and motor-function and increased neuro-inflammation and oxidative stress in the substantia nigra (SN). Therefore, the purpose of the current studies was to determine if GDNF replacement restores motor function and functional markers within the nigrostriatal DA system in middle-aged Gdnf(+/-) mice. At 11months of age, male Gdnf(+/-) and wildtype (WT) mice underwent bilateral intra-striatal injections of GDNF (10μg) or vehicle. Locomotor activity was assessed weekly 1-4weeks after treatment. Four weeks after treatment, their brains were processed for analysis of GDNF levels and various DAergic and oxidative stress markers. An intrastriatal injection of GDNF increased motor activity in Gdnf(+/-) mice to levels comparable to WT mice (1week after injection) and this effect was maintained through the 4-week time point. This increase in locomotion was accompanied by a 40% increase in striatal GDNF protein levels and SN GDNF expression in Gdnf(+/-) mice. Additionally, GDNF treatment significantly increased the number of tyrosine hydroxylase (TH)-positive neurons in the SN of middle-aged Gdnf(+/-) mice, but not WT mice, which was coupled with reduced oxidative stress in the SN. These studies further support that long-term changes related to the dysfunction of the nigrostriatal pathway are influenced by GDNF expression and add that this dysfunction appears to be responsive to GDNF treatment. Additionally, these studies suggest that long-term GDNF depletion alters the biological

  9. Brain-derived neurotrophic factor gene delivery in an animal model of multiple sclerosis using bone marrow stem cells as a vehicle.

    Science.gov (United States)

    Makar, Tapas K; Bever, Christopher T; Singh, Ishwar S; Royal, Walter; Sahu, Surasri Nandan; Sura, Tushar P; Sultana, Shireen; Sura, Karna T; Patel, Niraj; Dhib-Jalbut, Suhayl; Trisler, David

    2009-05-29

    Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, is neuroprotective in animal models of neurodegenerative diseases. However, BDNF has a short half-life and its efficacy in the central nervous system (CNS), when delivered peripherally, is limited due to the blood-brain barrier (BBB). We have developed a means of delivering BDNF into the CNS using genetically engineered bone marrow stem cells (BMSCs) as a vehicle, and have explored the clinical effects of BDNF on outcomes in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). BDNF-engineered-BMSCs were transplanted (i.v.) into irradiated 2-week-old SJL/J female mice. Eight weeks after transplantation, mice were immunized with a peptide of proteolipid protein (PLP(139-151)). Mice, which had received BDNFengineered BMSCs, showed a significant delay in EAE onset and a reduction in overall clinical severity compared to mice receiving BMSC transfected with an empty vector lacking the BDNF gene. In addition, pathological examination showed that BDNF delivery reduced demyelination and increased remyelination. Inhibition of pro-inflammatory cytokines TNF-alpha and IFN-gamma and enhanced expression of the antiinflammatory cytokines IL-4, IL-10, and IL-11 were found in the CNS tissues of the BDNF transplanted group. These results support the use of BMSCs as vehicles to deliver BDNF into the CNS of EAE animals. This is a potentially novel therapeutic approach that might be used to deliver BDNF gene or genes for other therapeutic proteins into the CNS in MS or in other diseases of the CNS in which accessibility of therapeutic proteins is limited due to the BBB.

  10. Relationship Between Chronic Tinnitus and Glial Cell Line-Derived Neurotrophic Factor Gene rs3812047, rs1110149, and rs884344 Polymorphisms in a Turkish Population.

    Science.gov (United States)

    Orenay-Boyacioglu, Seda; Coskunoglu, Aysun; Caki, Zerrin; Cam, Fethi Sirri

    2016-08-01

    Glial cell line-derived neurotrophic factor (GDNF) plays a key role in early development of central auditory pathway and the inner ear. However, the auditory pathway studies of GDNF gene polymorphisms are scarce in the literature, and the studies especially associated with tinnitus are limited. Our study aimed to identify whether GDNF gene polymorphisms play any roles in the pathophysiology of tinnitus by investigating the relationship between tinnitus and GDNF polymorphisms. A total of 52 patients with chronic tinnitus and ages ranging from 18 to 55 were admitted to the Ear-Nose-Throat outpatient clinic of Celal Bayar University Medical Faculty Hospital of Manisa, Turkey and constituted the study group. Another 42 patients of the same age range, without tinnitus symptoms and lacking any systemic disease, were also admitted to the clinic and formed the control group. The tympanometric, audiological, and psychoacoustic assessments of the subjects were performed. Deoxyribonucleic acid samples obtained using venous blood taken for routine inspections were used to investigate GDNF gene polymorphisms (rs884344, rs3812047, and rs1110149) by polymerase chain reaction-based restriction fragment length polymorphism method. No correlation could be detected between GDNF rs884344 and rs3812047 polymorphisms and subjects with tinnitus (p > 0.05). Heterozygosity was significantly lower for GDNF rs1110149 polymorphism in tinnitus subjects compared to the controls (p tinnitus and control groups (p > 0.05). Failure to detect correlations between tinnitus and GDNF gene polymorphisms suggests this may be due to the fact that the GDNF gene has a variable expression pattern in different tissues and pathologies. Therefore, the study should be improved and its scope should be expanded by including a larger group of patients and different tissues to investigate the expression pattern of GDNF.

  11. Diet-induced obesity has neuroprotective effects in murine gastric enteric nervous system: involvement of leptin and glial cell line-derived neurotrophic factor.

    Science.gov (United States)

    Baudry, Charlotte; Reichardt, François; Marchix, Justine; Bado, André; Schemann, Michael; des Varannes, Stanislas Bruley; Neunlist, Michel; Moriez, Raphaël

    2012-02-01

    Nutritional factors can induce profound neuroplastic changes in the enteric nervous system (ENS), responsible for changes in gastrointestinal (GI) motility. However, long-term effects of a nutritional imbalance leading to obesity, such as Western diet (WD), upon ENS phenotype and control of GI motility remain unknown. Therefore, we investigated the effects of WD-induced obesity (DIO) on ENS phenotype and function as well as factors involved in functional plasticity. Mice were fed with normal diet (ND) or WD for 12 weeks. GI motility was assessed in vivo and ex vivo. Myenteric neurons and glia were analysed with immunohistochemical methods using antibodies against Hu, neuronal nitric oxide synthase (nNOS), Sox-10 and with calcium imaging techniques. Leptin and glial cell line-derived neurotrophic factor (GDNF) were studied using immunohistochemical, biochemical or PCR methods in mice and primary culture of ENS. DIO prevented the age-associated decrease in antral nitrergic neurons observed in ND mice. Nerve stimulation evoked a stronger neuronal Ca(2+) response in WD compared to ND mice. DIO induced an NO-dependent increase in gastric emptying and neuromuscular transmission in the antrum without any change in small intestinal transit. During WD but not ND, a time-dependent increase in leptin and GDNF occurred in the antrum. Finally, we showed that leptin increased GDNF production in the ENS and induced neuroprotective effects mediated in part by GDNF. These results demonstrate that DIO induces neuroplastic changes in the antrum leading to an NO-dependent acceleration of gastric emptying. In addition, DIO induced neuroplasticity in the ENS is likely to involve leptin and GDNF.

  12. Functional insights into the Shigella type III needle tip IpaD in secretion control and cell contact.

    Science.gov (United States)

    Schiavolin, Lionel; Meghraoui, Alaeddine; Cherradi, Youness; Biskri, Latéfa; Botteaux, Anne; Allaoui, Abdelmounaaïm

    2013-04-01

    Type III secretion apparatus (T3SA) are complex nanomachines that insert a translocation pore into the host cell membrane through which effector proteins are injected into the cytosol. In Shigella, the pore is inserted by a needle tip complex that also controls secretion. IpaD is the key protein that rules the composition of the tip complex before and upon cell contact or Congo red (CR) induction. However, how IpaD is involved in secretion control and translocon insertion remains not fully understood. Here, we report the phenotypic analysis of 20 10-amino acids deletion variants all along the coiled-coil and the central domains of IpaD (residues 131-332). Our results highlight three classes of T3S phenotype; (i) wild-type secretion, (ii) constitutive secretion of all classes of effectors, and (iii) constitutive secretion of translocators and early effectors, but not of late effectors. Our data also suggest that the composition of the tip complex defines both the T3SA inducibility state and late effectors secretion. Finally, we shed light on a new aspect regarding the contact of the needle tip with cell membrane by uncoupling the Shigella abilities to escape macrophage vacuole, and to insert the translocation pore or to invade non-phagocytic cells.

  13. IFN type I and II induce BAFF secretion from human decidual stromal cells.

    Science.gov (United States)

    Lundell, Anna-Carin; Nordström, Inger; Andersson, Kerstin; Lundqvist, Christina; Telemo, Esbjörn; Nava, Silvia; Kaipe, Helen; Rudin, Anna

    2017-01-06

    B cell activating factor (BAFF) is a critical cytokine for maturation of immature B cells. In murine lymph nodes, BAFF is mainly produced by podoplanin-expressing stromal cells. We have previously shown that circulating BAFF levels are maximal at birth, and that farmers' children exhibit higher BAFF levels in cord blood than non-farmers' children. Here, we sought to investigate whether maternal-derived decidual stromal cells from placenta secrete BAFF and examine what factors could stimulate this production. We found that podoplanin is expressed in decidua basalis and in the underlying villous tissue as well as on isolated maternal-derived decidual stromal cells. Decidual stromal cells produced BAFF when stimulated with IFN-γ and IFN-α, and NK cells and NK-T-like cells competent of IFN-γ production were isolated from the decidua. Finally, B cells at different maturational stages are present in decidua and all expressed BAFF-R, while stromal cells did not. These findings suggest that decidual stromal cells are a cellular source of BAFF for B cells present in decidua during pregnancy.

  14. Development of Gonadotropin-Releasing Hormone-Secreting Neurons from Human Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Carina Lund

    2016-08-01

    Full Text Available Gonadotropin-releasing hormone (GnRH neurons regulate human puberty and reproduction. Modeling their development and function in vitro would be of interest for both basic research and clinical translation. Here, we report a three-step protocol to differentiate human pluripotent stem cells (hPSCs into GnRH-secreting neurons. Firstly, hPSCs were differentiated to FOXG1, EMX2, and PAX6 expressing anterior neural progenitor cells (NPCs by dual SMAD inhibition. Secondly, NPCs were treated for 10 days with FGF8, which is a key ligand implicated in GnRH neuron ontogeny, and finally, the cells were matured with Notch inhibitor to bipolar TUJ1-positive neurons that robustly expressed GNRH1 and secreted GnRH decapeptide into the culture medium. The protocol was reproducible both in human embryonic stem cells and induced pluripotent stem cells, and thus provides a translational tool for investigating the mechanisms of human puberty and its disorders.

  15. Flavonoids stimulate cholecystokinin peptide secretion from the enteroendocrine STC-1 cells.

    Science.gov (United States)

    Al Shukor, Nadin; Ravallec, Rozenn; Van Camp, John; Raes, Katleen; Smagghe, Guy

    2016-09-01

    Animal experiments showed that flavonoids might have the potential for an anti-obesity effect by reducing weight and food intake. However, the exact mechanisms that could be involved in these proposed effects are still under investigation. The complex process of food intake is partially regulated by gastrointestinal hormones. Cholecystokinin (CCK) is the best known gastrointestinal hormone to induce satiety signal that plays a key role in food intake regulation. It is released from the endocrine cells (I cell) in response to the ingestion of nutrients into the small intestine. In this study, we investigated the possible effects of flavonoids (quercetin, kaempferol, apigenin, rutin and baicalein) on stimulation of CCK release in vitro using enteroendocrine STC-1 cells. In comparison with the control, quercetin, kaempferol and apigenin resulted in a significant increase in CCK secretion with quercetin showing the highest activity. On the other hand, no significant effect was seen by rutin and baicalein. To our knowledge, this is the first report to study the stimulation of CCK peptide hormone secretion from STC-1 cells by quercetin and kaempferol, rutin, apigenin and baicalein. Based on the cell-based results in this work, it can be suggested that the reported activity of flavonoids against food intake and weight could be mediated by stimulation of CCK signal which in turn is responsible for food intake reduction, but future animal and human studies are needed to confirm this conclusion at organism level.

  16. P2Y₁ receptor-dependent diacylglycerol signaling microdomains in β cells promote insulin secretion.

    Science.gov (United States)

    Wuttke, Anne; Idevall-Hagren, Olof; Tengholm, Anders

    2013-04-01

    Diacylglycerol (DAG) controls numerous cell functions by regulating the localization of C1-domain-containing proteins, including protein kinase C (PKC), but little is known about the spatiotemporal dynamics of the lipid. Here, we explored plasma membrane DAG dynamics in pancreatic β cells and determined whether DAG signaling is involved in secretagogue-induced pulsatile release of insulin. Single MIN6 cells, primary mouse β cells, and human β cells within intact islets were transfected with translocation biosensors for DAG, PKC activity, or insulin secretion and imaged with total internal reflection fluorescence microscopy. Muscarinic receptor stimulation triggered stable, homogenous DAG elevations, whereas glucose induced short-lived (7.1 ± 0.4 s) but high-amplitude elevations (up to 109 ± 10% fluorescence increase) in spatially confined membrane regions. The spiking was mimicked by membrane depolarization and suppressed after inhibition of exocytosis or of purinergic P2Y₁, but not P2X receptors, reflecting involvement of autocrine purinoceptor activation after exocytotic release of ATP. Each DAG spike caused local PKC activation with resulting dissociation of its substrate protein MARCKS from the plasma membrane. Inhibition of spiking reduced glucose-induced pulsatile insulin secretion. Thus, stimulus-specific DAG signaling patterns appear in the plasma membrane, including distinct microdomains, which have implications for the kinetic control of exocytosis and other membrane-associated processes.

  17. Combination of hydrogel nanoparticles and proteomics to reveal secreted proteins associated with decidualization of human uterine stromal cells

    Directory of Open Access Journals (Sweden)

    Stephens Andrew N

    2011-09-01

    Full Text Available Abstract Background Identification of secreted proteins of low abundance is often limited by abundant and high molecular weight (MW proteins. We have optimised a procedure to overcome this limitation. Results Low MW proteins in the conditioned media of cultured cells were first captured using dual-size exclusion/affinity hydrogel nanoparticles and their identities were then revealed by proteomics. Conclusions This technique enables the analysis of secreted proteins of cultured cells low MW and low abundance.

  18. Role of histamine H3 receptor in glucagon-secreting αTC1.6 cells.

    Science.gov (United States)

    Nakamura, Tadaho; Yoshikawa, Takeo; Naganuma, Fumito; Mohsen, Attayeb; Iida, Tomomitsu; Miura, Yamato; Sugawara, Akira; Yanai, Kazuhiko

    2015-01-01

    Pancreatic α-cells secrete glucagon to maintain energy homeostasis. Although histamine has an important role in energy homeostasis, the expression and function of histamine receptors in pancreatic α-cells remains unknown. We found that the histamine H3 receptor (H3R) was expressed in mouse pancreatic α-cells and αTC1.6 cells, a mouse pancreatic α-cell line. H3R inhibited glucagon secretion from αTC1.6 cells by inhibiting an increase in intracellular Ca(2+) concentration. We also found that immepip, a selective H3R agonist, decreased serum glucagon concentration in rats. These results suggest that H3R modulates glucagon secretion from pancreatic α-cells.

  19. Human pituitary and placental hormones control human insulin-like growth factor II secretion in human granulosa cells

    Energy Technology Data Exchange (ETDEWEB)

    Ramasharma, K.; Li, C.H.

    1987-05-01

    Human granulosa cells cultured with calf serum actively proliferated for 18-20 generations and secreted progesterone into the medium; progesterone levels appeared to decline with increase in generation number. Cells cultured under serum-free conditions secreted significant amounts of progesterone and insulin-like growth factor II (IGF-II). The progesterone secretion was enhanced by the addition of human follitropin, lutropin, and chorionic gonadotropin but not by growth hormone. These cells, when challenged to varying concentrations of human growth hormone, human chorionic somatomammotropin, human prolactin, chorionic gonadotropin, follitropin, and lutropin, secreted IGF-II into the medium as measured by specific IGF-II RIA. Among these human hormones, chorionic gonadotropin, follitropin, and lutropin were most effective in inducing IGF-II secretion from these cells. When synthetic lutropin-releasing hormone and ..cap alpha..-inhibin-92 were tested, only lutropin-releasing hormone was effective in releasing IGF-II. The results described suggest that cultured human granulosa cells can proliferate and actively secrete progesterone and IGF-II into the medium. IGF-II production in human granulosa cells was influenced by a multi-hormonal complex including human growth hormone, human chorionic somatomammotropin, and prolactin.

  20. The Secreted Protein Rv1860 of Mycobacterium tuberculosis Stimulates Human Polyfunctional CD8+ T Cells.

    Science.gov (United States)

    Satchidanandam, Vijaya; Kumar, Naveen; Biswas, Sunetra; Jumani, Rajiv S; Jain, Chandni; Rani, Rajni; Aggarwal, Bharti; Singh, Jaya; Kotnur, Mohan Rao; Sridharan, Anand

    2016-04-01

    We previously reported that Rv1860 protein from Mycobacterium tuberculosis stimulated CD4(+)and CD8(+)T cells secreting gamma interferon (IFN-γ) in healthy purified protein derivative (PPD)-positive individuals and protected guinea pigs immunized with a DNA vaccine and a recombinant poxvirus expressing Rv1860 from a challenge with virulent M. tuberculosis We now show Rv1860-specific polyfunctional T (PFT) cell responses in the blood of healthy latently M. tuberculosis-infected individuals dominated by CD8(+) T cells, using a panel of 32 overlapping peptides spanning the length of Rv1860. Multiple subsets of CD8(+) PFT cells were significantly more numerous in healthy latently infected volunteers (HV) than in tuberculosis (TB) patients (PAT). The responses of peripheral blood mononuclear cells (PBMC) from PAT to the peptides of Rv1860 were dominated by tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10) secretions, the former coming predominantly from non-T cell sources. Notably, the pattern of the T cell response to Rv1860 was distinctly different from those of the widely studied M. tuberculosis antigens ESAT-6, CFP-10, Ag85A, and Ag85B, which elicited CD4(+) T cell-dominated responses as previously reported in other cohorts. We further identified a peptide spanning amino acids 21 to 39 of the Rv1860 protein with the potential to distinguish latent TB infection from disease due to its ability to stimulate differential cytokine signatures in HV and PAT. We suggest that a TB vaccine carrying these and other CD8(+) T-cell-stimulating antigens has the potential to prevent progression of latent M. tuberculosis infection to TB disease.

  1. Schizandra arisanensis extract attenuates cytokine-mediated cytotoxicity in insulin-secreting cells

    Institute of Scientific and Technical Information of China (English)

    Yi-Shin Hsu; Yao-Haur Kuo; Hui-Ling Cheng; Peter R Flatt; Hui-Kang Liu

    2012-01-01

    AIM:To explore the bioactivity of an ethanolic extract of Schizandra arisanensis (SA-Et) and isolated constituents against interleukin-1β and interferon-γ-mediated β cell death and abolition of insulin secretion.METHODS:By employing BRIN-BD11 cells,the effects of SA-Et administration on cytokine-mediated cell death and abolition of insulin secretion were evaluated by a viability assay,cell cycle analysis,and insulin assay.The associated gene and protein expressions were also measured.In addition,the bioactivities of several peak compounds collected from the SA-Et were tested against cytokine-mediated β cell death.RESULTS:Our results revealed that SA-Et dose-dependently ameliorated cytokine-mediated β cell death and apoptosis.Instead of suppressing inducible nitric oxide synthase/nitric oxide cascade or p38MAPK activity,suppression of stress-activated protein kinase/c-Jun NH2-terminal kinase activity appeared to be the target for SA-Et against the cytokine mix.In addition,SA-Et provided some insulinotropic effects which re-activated the abolished insulin exocytosis in cytokine-treated BRIN-BD11 cells.Finally,schiarisanrin A and B isolated from the SA-Et showed a dose-dependent protective effect against cytokine-mediated β cell death.CONCLUSION:This is the first report on SA-Et ameliorating cytokine-mediated β cell death and dysfunction via anti-apoptotic and insulinotropic actions.

  2. Oxytocin and dopamine stimulate ghrelin secretion by the ghrelin-producing cell line, MGN3-1 in vitro.

    Science.gov (United States)

    Iwakura, Hiroshi; Ariyasu, Hiroyuki; Hosoda, Hiroshi; Yamada, Go; Hosoda, Kiminori; Nakao, Kazuwa; Kangawa, Kenji; Akamizu, Takashi

    2011-07-01

    To understand the physiological role of ghrelin, it is crucial to study both the actions of ghrelin and the regulation of ghrelin secretion. Although ghrelin actions have been extensively revealed, the direct factors regulating ghrelin secretion by ghrelin-producing cells (X/A-like cells), however, is not fully understood. In this study, we examined the effects of peptide hormones and neurotransmitters on in vitro ghrelin secretion by the recently developed ghrelin-producing cell line MGN3-1. Oxytocin and vasopressin significantly stimulated ghrelin secretion by MGN3-1 cells. Because MGN3-1 cells express only oxytocin receptor mRNA, not vasopressin receptor mRNA, oxytocin is the likely regulator, with the effect of vasopressin mediated by a cross-reaction. We also discovered that dopamine stimulates ghrelin secretion from MGN3-1 cells in a similar manner to the previously known ghrelin stimulators, epinephrine and norepinephrine. MGN3-1 cells expressed mRNA encoding dopamine receptors D1a and D2. The dopamine receptor D1 agonist fenoldopam stimulated ghrelin secretion, whereas the D2, D3 agonist bromocriptine did not. Furthermore, the D1 receptor antagonist SKF83566 attenuated the stimulatory effect of dopamine. These results indicate that the stimulatory effect of dopamine on ghrelin secretion is mediated by the D1a receptor. In conclusion, we identified two direct regulators of ghrelin, oxytocin and dopamine. These findings will provide new direction for further studies seeking to further understand the regulation of ghrelin secretion, which will in turn lead to greater understanding of the physiological role of ghrelin.

  3. Drosophila adiponectin receptor in insulin producing cells regulates glucose and lipid metabolism by controlling insulin secretion.

    Directory of Open Access Journals (Sweden)

    Su-Jin Kwak

    Full Text Available Adipokines secreted from adipose tissue are key regulators of metabolism in animals. Adiponectin, one of the adipokines, modulates pancreatic beta cell function to maintain energy homeostasis. Recently, significant conservation between Drosophila melanogaster and mammalian metabolism has been discovered. Drosophila insulin like peptides (Dilps regulate energy metabolism similarly to mammalian insulin. However, in Drosophila, the regulatory mechanism of insulin producing cells (IPCs by adipokine signaling is largely unknown. Here, we describe the discovery of the Drosophila adiponectin receptor and its function in IPCs. Drosophila adiponectin receptor (dAdipoR has high homology with the human adiponectin receptor 1. The dAdipoR antibody staining revealed that dAdipoR was expressed in IPCs of larval and adult brains. IPC- specific dAdipoR inhibition (Dilp2>dAdipoR-Ri showed the increased sugar level in the hemolymph and the elevated triglyceride level in whole body. Dilps mRNA levels in the Dilp2>dAdipoR-Ri flies were similar with those of controls. However, in the Dilp2>dAdipoR-Ri flies, Dilp2 protein was accumulated in IPCs, the level of circulating Dilp2 was decreased, and insulin signaling was reduced in the fat body. In ex vivo fly brain culture with the human adiponectin, Dilp2 was secreted from IPCs. These results indicate that adiponectin receptor in insulin producing cells regulates insulin secretion and controls glucose and lipid metabolism in Drosophila melanogaster. This study demonstrates a new adipokine signaling in Drosophila and provides insights for the mammalian adiponectin receptor function in pancreatic beta cells, which could be useful for therapeutic application.

  4. Plant cell wall-degrading enzymes and their secretion in plant-pathogenic fungi.

    Science.gov (United States)

    Kubicek, Christian P; Starr, Trevor L; Glass, N Louise

    2014-01-01

    Approximately a tenth of all described fungal species can cause diseases in plants. A common feature of this process is the necessity to pass through the plant cell wall, an important barrier against pathogen attack. To this end, fungi possess a diverse array of secreted enzymes to depolymerize the main structural polysaccharide components of the plant cell wall, i.e., cellulose, hemicellulose, and pectin. Recent advances in genomic and systems-level studies have begun to unravel this diversity and have pinpointed cell wall-degrading enzyme (CWDE) families that are specifically present or enhanced in plant-pathogenic fungi. In this review, we discuss differences between the CWDE arsenal of plant-pathogenic and non-plant-pathogenic fungi, highlight the importance of individual enzyme families for pathogenesis, illustrate the secretory pathway that transports CWDEs out of the fungal cell, and report the transcriptional regulation of expression of CWDE genes in both saprophytic and phytopathogenic fungi.

  5. Engineering the supply chain for protein production/secretion in yeasts and mammalian cells.

    Science.gov (United States)

    Klein, Tobias; Niklas, Jens; Heinzle, Elmar

    2015-03-01

    Metabolic bottlenecks play an increasing role in yeasts and mammalian cells applied for high-performance production of proteins, particularly of pharmaceutical ones that require complex posttranslational modifications. We review the present status and developments focusing on the rational metabolic engineering of such cells to optimize the supply chain for building blocks and energy. Methods comprise selection of beneficial genetic modifications, rational design of media and feeding strategies. Design of better producer cells based on whole genome-wide metabolic network analysis becomes increasingly possible. High-resolution methods of metabolic flux analysis for the complex networks in these compartmented cells are increasingly available. We discuss phenomena that are common to both types of organisms but also those that are different with respect to the supply chain for the production and secretion of pharmaceutical proteins.

  6. Activation of PPARd and RXRa stimulates fatty acid oxidatin and insulin secretion inpancreatic beta-cells

    DEFF Research Database (Denmark)

    Børgesen, Michael; Ravnskjær, Kim; Frigerio, Francesca;

    ACTIVATION OF PPARd AND RXRa STIMULATES FATTY ACID OXIDATION AND INSULIN SECRETION IN PANCREATIC b-CELLS Michael Boergesen1, Kim Ravnskjaer2, Francesca Frigerio3, Allan E. Karlsen4, Pierre Maechler3 and Susanne Mandrup1 1 Department of Biochemistry and Molecular Biology, University of Southern...... oxidation and dissipation of lipids particularly in skeletal muscle. Here we show that PPARd at the RNA as well as protein level is the most abundant PPAR subtype in the rat pancreatic ß-cell line INS-1E and in isolated rat pancreatic islets. In keeping with that, a large number of PPAR target genes...... involved in fatty acid uptake and oxidation. This correlates with a 5-fold induction of 14C-Oleate ß-oxidation when INS-1E cells are exposed to PPARd and RXR agonists. Notably, culture of INS-1E cells with oleate and other unsaturated fatty acids in the presence of an RXR agonist induces the same subset...

  7. Microvesicles secreted by macrophages shuttle invasion-potentiating microRNAs into breast cancer cells

    Directory of Open Access Journals (Sweden)

    Lin Ling

    2011-09-01

    Full Text Available Abstract Background Tumor-associated macrophages (TAMs are alternatively activated cells induced by interleukin-4 (IL-4-releasing CD4+ T cells. TAMs promote breast cancer invasion and metastasis; however, the mechanisms underlying these interactions between macrophages and tumor cells that lead to cancer metastasis remain elusive. Previous studies have found microRNAs (miRNAs circulating in the peripheral blood and have identified microvesicles, or exosomes, as mediators of cell-cell communication. Therefore, one alternative mechanism for the promotion of breast cancer cell invasion by TAMs may be through macrophage-secreted exosomes, which would deliver invasion-potentiating miRNAs to breast cancer cells. Results We utilized a co-culture system with IL-4-activated macrophages and breast cancer cells to verify that miRNAs are transported from macrophages to breast cancer cells. The shuttling of fluorescently-labeled exogenous miRNAs from IL-4-activated macrophages to co-cultivated breast cancer cells without direct cell-cell contact was observed. miR-223, a miRNA specific for IL-4-activated macrophages, was detected within the exosomes released by macrophages and was significantly elevated in the co-cultivated SKBR3 and MDA-MB-231 cells. The invasiveness of the co-cultivated breast cancer cells decreased when the IL-4-activated macrophages were treated with a miR-223 antisense oligonucleotide (ASO that would inhibit miR-223 expression. Furthermore, results from a functional assay revealed that miR-223 promoted the invasion of breast cancer cells via the Mef2c-β-catenin pathway. Conclusions We conclude that macrophages regulate the invasiveness of breast cancer cells through exosome-mediated delivery of oncogenic miRNAs. Our data provide insight into the mechanisms underlying the metastasis-promoting interactions between macrophages and breast cancer cells.

  8. Influence of endogenous ciliary neurotrophic factor on neural differentiation of adult rat hippocampal progenitors

    Institute of Scientific and Technical Information of China (English)

    Jun Ding; Zhili He; Juan Ruan; Ying Liu; Chengxin Gong; Shenggang Sun; Honghui Chen

    2013-01-01

    Ciliary neurotrophic factor is the only known neurotrophic factor that can promote differentiation of hippocampal neural progenitor cells to glial cells and neurons in adult rats. This process is similar to spontaneous differentiation. Therefore, ciliary neurotrophic factor may be involved in spontaneous differentiation of neural stem cells. To verify this hypothesis, the present study isolated neural progenitor cells from adult male rats and cultured them in vitro. Results showed that when neural progenitor cells were cultured in the absence of mitogen fibroblast growth factor-2 or epidermal growth factor, they underwent spontaneous differentiation into neurons and glial cells. Western blot and immunocytochemical staining showed that exogenous ciliary neurotrophic factor strongly induced adult hippocampal progenitor cells to differentiate into neurons and glial cells. Moreover, passage 4 adult hippocampal progenitor cells expressed high levels of endogenous ciliary neurotrophic factor, and a neutralizing antibody against ciliary neurotrophic factor prevented the spontaneous neuronal and glial differentiation of adult hippocampal progenitor cells. These results suggest that the spontaneous differentiation of adult hippocampal progenitor cells is mediated partially by endogenous ciliary neurotrophic factor.

  9. Composition of culture media for steroid hormone secretion by murine adrenal tumor cells, Y-1 clone.

    Directory of Open Access Journals (Sweden)

    Ichikawa,Yoshiko

    1989-04-01

    Full Text Available Murine adrenal tumor cells (Y-1 clone were stimulated by adrenocorticotropic hormone (ACTH and cyclic adenosine 3',5'-monophosphate (cyclic AMP to produce steroid hormone (delta 4, 3-keto steroids. The steroids were secreted into the medium immediately after synthesis. The optimum concentrations of ACTH and cyclic AMP for stimulation of steroid production were 10(-2 U/ml and 1.0 mM, respectively. In serum-free medium, ACTH and cyclic AMP stimulated steroidogenesis in Y-1 cells, but the amount of steroid hormone in the culture medium was low. However, a high level of steroid production was maintained with medium containing 10 mg/ml bovine serum albumin (BSA. In culture medium containing a higher concentration of BSA, Y-1 cells did not become spherical as is usually the case when steroid production is stimulated by ACTH or cyclic AMP. The morphological changes did not always correlate with steroid secretion by Y-1 cells.

  10. The pre-synaptic blocker toosendanin does not inhibit secretion in exocrine cells

    Institute of Scientific and Technical Information of China (English)

    Zong-Jie Cui; Xue-Hui He

    2002-01-01

    AIM: Toosendanin is a pre-synaptic blocker at theneuromuscular junction and its inhibitory effect is dividedinto an initial facilitative/stimulatory phase followed by aprolonged inhibitory phase. The present study investigatedwhether the subsequent inhibitory phase was due toexhaustion of the secretory machinery as a result of extensivestimulation during the initial facilitative phase. Morespecifically, this paper examined whether toosendanin coulddirectly inhibit the secretory machinery in exocrine cells.METHODS: Rat pancreatic acinar cells were isolated bycollagenase digestion. Secretion was assessed by measuringthe amount of amylase released into the extracellular mediumas a percentage of the total present in the cells beforestimulation. Cholecystokinin (CCK)-induced increases inintracellular calcium in single cells were measured with fura-2 microfluorometry.RESULTS: Effects of toosendanin on CCK-induced amylasesecretion and calcium oscillations were investigated.Toosendanin of 87-870 tM had no effect on 10 pM-100 nMCCK-stimulated amylase secretion, nor did 8.7-870 μMtoosendanin inhibit 5 pM CCK-induced calcium oscillations.In contrast, 10 nM CCK1 receptor antagonist FK 480 completelyblocked 5 pM CCK-induced calcium oscillations.CONCLUSION: The pre-synaptic "blocker" toosendanin is aselective activator of the voltage-dependent calcium channels,but does not interfere with the secretory machinery itself.

  11. Effects of glial cell line-derived neurotrophic factor, fibroblast growth factor 2 and epidermal growth factor on proliferation and the expression of some genes in buffalo (Bubalus bubalis) spermatogonial cells.

    Science.gov (United States)

    Kadam, Prashant H; Kala, Sushila; Agrawal, Himanshu; Singh, Karn P; Singh, Manoj K; Chauhan, Manmohan S; Palta, Prabhat; Singla, Suresh K; Manik, Radhay S

    2013-01-01

    The present study evaluated the effects of glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor (FGF) 2 and epidermal growth factor (EGF) on proliferation and the expression of some genes in spermatogonial cells. Spermatogonial cells were isolated from prepubertal buffalo testes and enriched by double enzyme treatment, filtration through 80- and 60-μm nylon mesh filters, differential plating on lectin-coated dishes and Percoll density gradient centrifugation. Cells were then cultured on a buffalo Sertoli cell feeder layer and formed colonies within 15-18 days. The colonies were found to predominantly contain undifferentiated Type A spermatogonia because they bound Dolichos biflorus agglutinin and did not express c-kit. The colonies expressed alkaline phosphatase, NANOG, octamer-binding transcription factor (OCT)-4 and tumour rejection antigen (TRA)-1-60. Cells were subcultured for 15 days, with or without growth factor supplementation. After 15 days, colony area and the relative mRNA abundance of PLZF were higher (Pgrowth factor supplementation. In the Sertoli cell feeder layer, EGF and FGF2 decreased (Pgrowth factors was developed for the short-term culture of buffalo spermatogonia.

  12. Changes in brain-derived neurotrophic factor expression after transplanting microencapsulated sciatic nerve cells of rabbits into injured spinal cord of rats

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    BACKGROUND: Changes of brain-derived neurotrophic factor (BDNF) expression reflect function of nerve cells; meanwhile, they play a significant role in researching interventions on plerosis of nerve injury.OBJECTIVE: To observe and compare the effects on changes of BDNF expression in rats with spinal cord injury between microencapsulated sciatic nerve cells of rabbits and only transplanting sciatic nerve cells of rabbits.DESIGN: Randomized controlled animal study.SETTING: Medical School of Jiujiang College.MATERIALS: The experiment was carried out in the Medical Science Researching Center, Jiujiang College from May 2004 to May 2006. A total of 90 healthy adult SD rats, weighing 250 - 300 g, of either gender; and 10 rabbits, weighing 2.0 - 2.5 kg, of either gender, were provided by Jiangxi Experimental Animal Center.METHODS: Sciatic nerve tissue of rabbits was separated to make cell suspension. After centrifugation,suspension was mixed with 15 g/L alginate saline solution and ejaculated to 20 mmol/L barium chloride saline solution by double-cavity ejaculator. The obtained cell microcapsules were suspended in saline. Rats were randomly divided into microencapsulated group, only suspension group, and only injured group with 30 animals in each group. After anesthesia, T10 spinous process and vertebra lamina of rats in the former two groups were exposed. Spinal cord tissue in 2-mm length was removed from rats by spinal cord right hemi-section. The gelatin sponges with the size of 2 mm × 2 mm × 2 mm were grafted as filing cage,and absorbed 10 μμ L microencapsulated sciatic nerve cells of rabbit in the microencapsulated group and 10 μ L sciatic nerve cells of rabbits in the only suspension group; respectively. No graft was placed in the only injured group.MAIN OUTCOME MEASURES: On the 1st, 3rd, 7th, 14th and 28th days after operation,immunohistochemistry (SABC technique) was used to detect distribution and amount of positive-reactive neurons in BDNF of spinal cord

  13. A histamine H2 receptor antagonist, roxatidine, stimulates mucus secretion and synthesis by cultured rabbit gastric mucosal cells.

    Science.gov (United States)

    Takahashi, S; Okabe, S

    1995-12-01

    We examined the effects of the known antisecretory and mucosal protective drug, roxatidine, on the secretion and synthesis of mucus by cultured rabbit gastric mucosal cells. The amounts of secreted and synthesized mucus were determined by the [3H] glucosamine labelling method. Exposure of the cells to roxatidine for 8 hr caused increases in the secretion and synthesis of mucus in a dose-related manner. The increase in mucus synthesis was maximally induced 4 hr after the addition of roxatidine, while mucus secretion was maximally enhanced a further 4 hr later. However, other H2 antagonists such as cimetidine, rantidine and famotidine failed to stimulate the secretion and synthesis of gastric mucus. In addition, neither indomethacin nor NG-nitro-L-arginine methyl ester affected the roxatidine-induced increases in mucus secretion and synthesis. We conclude that roxatidine directly acts on gastric mucosal cells, inducing increases in both the secretion and synthesis of mucus, and that an unknown regulatory pathway might be involved in these stimulatory actions of roxatidine.

  14. Preproorexin and orexin receptors are expressed in cortisol-secreting adrenocortical adenomas, and orexins stimulate in vitro cortisol secretion and growth of tumor cells.

    Science.gov (United States)

    Spinazzi, R; Rucinski, M; Neri, G; Malendowicz, L K; Nussdorfer, G G

    2005-06-01

    Orexins A and B are hypothalamic peptides that originate from the proteolytic cleavage of preproorexin and act through two subtypes of receptors, named OX1-R and OX2-R. OX1-R almost exclusively binds orexin-A, whereas OX2-R is nonselective for both orexins. We previously found that orexin-A, via the OX1-R, stimulates cortisol secretion from dispersed human adrenocortical cells. In this study, we demonstrate that six of eight cortisol-secreting adenomas expressed preproorexin mRNA, and seven of 10 adenomas contained measurable amounts of orexin-A but not orexin-B. Normal adrenal cortexes neither expressed preproorexin nor contained orexins. All adenomas expressed OX1-R and OX2-R mRNAs, and real-time PCR showed that the expression of both receptors was up-regulated in adenomas, compared with normal adrenal cortex. Orexin-A concentration-dependently raised basal cortisol secretion from freshly dispersed normal and adenomatous cells, minimal and maximal effective concentrations being 10(-10) and 10(-8) m, and the peptide efficacy (percent increase elicited by 10(-8) m orexin-A) was significantly higher in adenomas than in the normal adrenal cortex. Orexin-B was ineffective, thereby indicating that orexin secretagogue action is mediated by the OX1-R. In contrast, both orexins (10(-8) m) raised the proliferative activity of cultured normal and adenomatous cells, suggesting that this effect is mediated by OX2-R or both receptor subtypes. Collectively, our findings allow us to conclude that the orexin system is overexpressed in cortisol-secreting adenomas and suggest that orexin-A may act as an autocrine-paracrine regulator of the secretory activity and growth of some of these adrenal tumors.

  15. Vitamin D modulates different IL-17-secreting T cell subsets in multiple sclerosis patients.

    Science.gov (United States)

    da Costa, Denise S M Medrado; Hygino, Joana; Ferreira, Thais B; Kasahara, Taissa M; Barros, Priscila O; Monteiro, Clarice; Oliveira, Aleida; Tavares, Felipe; Vasconcelos, Claudia Cristina; Alvarenga, Regina; Bento, Cleonice A M

    2016-10-15

    Vitamin D deficiency is an environmental risk factor for MS, a Th17 cell-mediated autoimmune disease that results in demyelination in the CNS. Therefore, we aimed to evaluate the ability of in vitro 1,25(OH)2D in modulating different Th17 cell subsets in MS patients in remission phase. In the present study, the production of Th17-related cytokines (IL-1β, IL-6, IL-17, IL-22), as well as GM-CSF, was significantly higher in cell cultures from MS patients than in healthy subjects (HS). The 1,25(OH)2D reduced all pro-inflammatory cytokines essayed, mainly those released from HS cell cultures. The proportion of both IL-17(+)IFN-γ(+) (CD4(+) and CD8(+)) T cells and IL-17(+)IFN-γ(-)CD8(+) T cells was positively related with neurological disorders, determined by EDSS score. The addition of 1,25(OH)2D reduced not only these pathogenic T cell subsets but elevated the percentage of IL-10-secreting conventional (FoxP3(+)CD25(+)CD127(-)CD4(+)) and non-conventional (IL-17(+)) regulatory-like T cells. Taken together, the results indicate that the active form of vitamin D should benefit MS patients by attenuating the percentage of pathogenic T cells. This effect could be direct and/or indirect, by enhancing classical and non-classical regulatory T cells.

  16. Microelectrode and Impedance Analysis of Anion Secretion in Calu-3 Cells

    Directory of Open Access Journals (Sweden)

    Tamada T

    2001-07-01

    Full Text Available Calu-3 cells secrete HCO(3(- in response to cAMP agonists but can be stimulated to secrete Cl(- with K(+ channel activating agonists. Microelectrode and impedance analysis experiments were performed to obtain a better understanding of the conductances and driving forces involved in these different modes of anion secretion in Calu-3 cells. Microelectrode studies revealed apical and basolateral membrane depolarizations upon the addition of forskolin (V(ap -52 mV vs. -21 mV; V(bl -60 mV vs. -44 mV that paralleled the hyperpolarization of the mucosal negative transepithelial voltage (V(T -8 mV vs. -23 mV. These changes were accompanied by a decrease in the apical membrane fractional resistance (F(Rap from approximately 0.50 to 0.08, consistent with the activation of an apical membrane conductance. The subsequent addition of 1-ethyl-2-benzimidazolinone (1-EBIO, a K(+ channel activator, hyperpolarized V(ap to -27 mV, V(bl to -60 mV and V(T to -33 mV. Impedance analysis revealed the apical membrane resistance (R(ap of the forskolin-stimulated cells was less than 20 ohm cm(2, indeed in most monolayers R(ap fell to less than 5 ohm cm(2. The impedance derived estimate of the basolateral membrane resistance (R(bl was approximately 170 ohm cm(2 in forskolin treated cells and fell to 50 ohm cm(2 with the addition of 1-EBIO. Using these values for the R(bl and the F(Rap value of 0.08 yields a R(ap of approximately 14 ohm cm(2 in the presence of forskolin and 4 ohm cm(2 in the presence of forskolin plus 1-EBIO. Thus, by two independent methods, forskolin-stimulated Calu-3 cells are seen to have a very high apical membrane conductance of 50 to 200 mS/cm(2. Therefore, we would assert that even at one-tenth the anion selectivity for Cl(-, this high conductance could support the conductive exit of HCO(3(- across the apical membrane. We further propose that this high apical membrane conductance serves to clamp the apical membrane potential near the equilibrium

  17. Hippocampal neurogenesis, neurotrophic factors and depression: possible therapeutic targets?

    Science.gov (United States)

    Serafini, Gianluca; Hayley, Shawn; Pompili, Maurizio; Dwivedi, Yogesh; Brahmachari, Goutam; Girardi, Paolo; Amore, Mario

    2014-01-01

    Major depression is one of the leading causes of disability and psychosocial impairment worldwide. Although many advances have been made in the neurobiology of this complex disorder, the pathophysiological mechanisms are still unclear. Among the proposed theories, impaired neuroplasticity and hippocampal neurogenesis have received considerable attention. The possible association between hippocampal neurogenesis, neurotrophic factors, major depression, and antidepressant responses was critically analyzed using a comprehensive search of articles/book chapters in English language between 1980 and 2014. One common emerging theme was that chronic stress and major depression are associated with structural brain changes such as a loss of dendritic spines and synapses, as well as reduced dendritic arborisation, together with diminished glial cells in the hippocampus. Both central monoamines and neurotrophic factors were associated with a modulation of hippocampal progenitor proliferation and cell survival. Accordingly, antidepressants are generally suggested to reverse stress-induced structural changes augmenting dendritic arborisation and synaptogenesis. Such antidepressant consequences are supposed to stem from their stimulatory effects on neurotrophic factors, and possibly modulation of glial cells. Of course, accumulating evidence also suggested that glutamatergic systems are implicated in not only basic neuroplastic processes, but also in the core features of depression. Hence, it is critical that antidepressant strategies focus on links between the various neurotransmitter systems, neurotrophic processes of hippocampal neurogenesis, and neurotrophic factors with regards to depressive symptomology. The identification of novel alternative antidepressant medications that target these systems is discussed in this review.

  18. Induction of mucin secretion from human bronchial tissue and epithelial cells by rhinovirus and lipopolysaccharide

    Institute of Scientific and Technical Information of China (English)

    Shao-heng HE; Jian ZHENG; Ming-ke DUAN

    2004-01-01

    AIM: To examine the effects of rhinovirus and lipopolysaccharide (LPS) on mucin secretion from bronchial tissue and epithelial cells in vitro. METHODS: Human small bronchial tissue fragments (HSBTF) and human bronchial epithelial cells (HBEC) were cultured with rhinovirus 16 and LPS, respectively and culture supernatants were collected for mucin measurement. To determine mucin levels in the culture supernatants, a MUC5AC enzyme linked immunosorbent assay and an enzyme linked lectin assay procedure with dolichos bifiorus agglutinin (DBA)were developed, and mucin release was expressed as percentage increased (or decreased) secretion over baseline level. RESULTS: A concentration-dependent release of DBA mucin and MUC5AC mucin were observed when HSBTF were infected with various concentrations of rhinovirus 16 at 37 ℃. The maximum-induced DBA mucin and MUC5AC mucin release were approximately 258 % and 83 % over baseline. The response of HSBTF to rhinovirus was completely abolished by metabolic inhibitors. Rhinovirus was also able to induce a concentrationdependent release of DBA mucin and MUC5AC mucin from primarily cultured HBEC. LPS 100 mg/L was able to provoke up to approximately 19 % and 54 % increase in DBA and MUC5AC mucin release over baseline, respectively from HSBTF, and 3.1% and 57 % increase from HBEC at 20 h. Soybean trypsin inhibitor (SBTI) 30 mg/L was able to inhibit LPS-induced mucin release from HSBTF and HBEC. CONCLUSION: Rhinovirus is able to induce mucin secretion from human bronchial tissue and bronchial epithelial cells in vitro. LPS can induce MUC5AC mucin release from HSBTF and HBEC.

  19. Beta-cell specific deletion of Dicer1 leads to defective insulin secretion and diabetes mellitus

    DEFF Research Database (Denmark)

    Kalis, Martins; Bolmeson, Caroline; Esguerra, Jonathan L.S.;

    2011-01-01

    Mature microRNAs (miRNAs), derived through cleavage of pre-miRNAs by the Dicer1 enzyme, regulate protein expression in many cell-types including cells in the pancreatic islets of Langerhans. To investigate the importance of miRNAs in mouse insulin secreting ß-cells, we have generated mice with a ...

  20. Secreted cerberus1 as a marker for quantification of definitive endoderm differentiation of the pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Hidefumi Iwashita

    Full Text Available To date, CXCR4 and E-cadherin double-positive cells detected by flow cytometry have been used to identify the differentiation of embryonic stem (ES cells or induced pluripotent stem (iPS cells into definitive endoderm (DE lineages. Quantification of DE differentiation from ES/iPS cells by using flow cytometry is a multi-step procedure including dissociation of the cells, antibody reaction, and flow cytometry analysis. To establish a quick assay method for quantification of ES/iPS cell differentiation into the DE without dissociating the cells, we examined whether secreted Cerberus1 (Cer1 protein could be used as a marker. Cer1 is a secreted protein expressed first in the anterior visceral endoderm and then in the DE. The amount of Cer1 secreted correlated with the proportion of CXCR4+/E-Cadherin+ cells that differentiated from mouse ES cells. In addition, we found that human iPS cell-derived DE also expressed the secreted CER1 and that the expression level correlated with the proportion of SOX17+/FOXA2+ cells present. Taken together, these results show that Cer1 (or CER1 serves as a good marker for quantification of DE differentiation of mouse and human ES/iPS cells.

  1. Distribution of kappa and lambda light chain isotypes among human blood immunoglobulin-secreting cells after vaccination with pneumococcal polysaccharides

    DEFF Research Database (Denmark)

    Heilmann, C; Barington, T

    1989-01-01

    The light chain isotype of immunoglobulin-secreting blood cells was investigated by means of monolayer plaque-forming cell assays allowing direct immunofluorescence staining for cytoplasmic kappa and lambda light chains in centre cells. The study revealed that cultured, polyclonally activated pok...

  2. Phloem sugar flux and jasmonic acid-responsive cell wall invertase control extrafloral nectar secretion in Ricinus communis.

    Science.gov (United States)

    Millán-Cañongo, Cynthia; Orona-Tamayo, Domancar; Heil, Martin

    2014-07-01

    Plants secrete extrafloral nectar (EFN) that attracts predators. The efficiency of the resulting anti-herbivore defense depends on the quantity and spatial distribution of EFN. Thus, according to the optimal defense hypothesis (ODH), plants should secrete EFN on the most valuable organs and when herbivore pressure is high. Ricinus communis plants secreted most EFN on the youngest (i.e., most valuable) leaves and after the simulation of herbivory via the application of jasmonic acid (JA). Here, we investigated the physiological mechanisms that might produce these seemingly adaptive spatiotemporal patterns. Cell wall invertase (CWIN; EC 3.2.1.26) was most active in the hours before peak EFN secretion, its decrease preceded the decrease in EFN secretion, and CWIN activity was inducible by JA. Thus, CWIN appears to be a central player in EFN secretion: its activation by JA is likely to cause the induction of EFN secretion after herbivory. Shading individual leaves decreased EFN secretion locally on these leaves with no effect on CWIN activity in the nectaries, which is likely to be because it decreased the content of sucrose, the substrate of CWIN, in the phloem. Our results demonstrate how the interplay of two physiological processes can cause ecologically relevant spatiotemporal patterns in a plant defense trait.

  3. Galectin-9 enhances cytokine secretion, but suppresses survival and degranulation, in human mast cell line.

    Directory of Open Access Journals (Sweden)

    Reiji Kojima

    Full Text Available Galectin-9 (Gal-9, a lectin having a β-galactoside-binding domain, can induce apoptosis of Th1 cells by binding to TIM-3. In addition, Gal-9 inhibits IgE/Ag-mediated degranulation of mast cell/basophilic cell lines by binding to IgE, thus blocking IgE/Ag complex formation. However, the role of Gal-9 in mast cell function in the absence of IgE is not fully understood. Here, we found that recombinant Gal-9 directly induced phosphorylation of Erk1/2 but not p38 MAPK in a human mast cell line, HMC-1, which does not express FcεRI. Gal-9 induced apoptosis and inhibited PMA/ionomycin-mediated degranulation of HMC-1 cells. On the other hand, Gal-9 induced cytokine and/or chemokine production by HMC-1 cells, dependent on activation of ERK1/2 but not p38 MAPK. In addition, the lectin activity of Gal-9 was required for Gal-9-mediated cytokine secretion by HMC-1 cells. These observations suggest that Gal-9 has dual properties as both a regulator and an activator of mast cells.

  4. Macroautophagy and Cell Responses Related to Mitochondrial Dysfunction, Lipid Metabolism and Unconventional Secretion of Proteins

    Science.gov (United States)

    Demine, Stéphane; Michel, Sébastien; Vannuvel, Kayleen; Wanet, Anaïs; Renard, Patricia; Arnould, Thierry

    2012-01-01

    Macroautophagy has important physiological roles and its cytoprotective or detrimental function is compromised in various diseases such as many cancers and metabolic diseases. However, the importance of autophagy for cell responses has also been demonstrated in many other physiological and pathological situations. In this review, we discuss some of the recently discovered mechanisms involved in specific and unspecific autophagy related to mitochondrial dysfunction and organelle degradation, lipid metabolism and lipophagy as well as recent findings and evidence that link autophagy to unconventional protein secretion. PMID:24710422

  5. Macroautophagy and Cell Responses Related to Mitochondrial Dysfunction, Lipid Metabolism and Unconventional Secretion of Proteins

    Directory of Open Access Journals (Sweden)

    Thierry Arnould

    2012-06-01

    Full Text Available Macroautophagy has important physiological roles and its cytoprotective or detrimental function is compromised in various diseases such as many cancers and metabolic diseases. However, the importance of autophagy for cell responses has also been demonstrated in many other physiological and pathological situations. In this review, we discuss some of the recently discovered mechanisms involved in specific and unspecific autophagy related to mitochondrial dysfunction and organelle degradation, lipid metabolism and lipophagy as well as recent findings and evidence that link autophagy to unconventional protein secretion.

  6. Quantitative analysis of the secretion of the MCP family of chemokines by muscle cells

    DEFF Research Database (Denmark)

    Henningsen, Jeanette; Pedersen, Bente Klarlund; Kratchmarova, Irina

    2011-01-01

    by Amino acids in Cell culture (SILAC) method for quantitative analysis resulted in the identification and generation of quantitative profiles of 59 growth factors and cytokines, including 9 classical chemokines. The members of the CC chemokine family of proteins such as monocyte chemotactic proteins 1, 2......, and 3 (MCP-1/CCL2, MCP-2/CCL8, and MCP-3/CCL7) showed a distinct pattern of secretion during differentiation. Further analysis using combinatorial RNA and protein approaches demonstrated that the MCPs are regulated via both post-transcriptional and post-translational mechanisms. Analyses...

  7. Synthesis and secretion of transferrin by a bovine trabecular meshwork cell line

    Directory of Open Access Journals (Sweden)

    R. Bertazolli-Filho

    2007-10-01

    Full Text Available The trabecular meshwork (TM is the main outflow pathway in the mammalian eye. Oxidative damage to TM cells has been suggested to be an important cause of impairment of TM functions, leading to deficient drainage of aqueous humor, with deleterious consequences to the eye. Transferrin, a metalloprotein involved in iron transport, has been characterized as an intrinsic eye protein. Since transferrin is implicated in the control of oxidative stress, the objective of the present study was to determine if a bovine TM cell line (CTOB synthesizes and secretes transferrin. The CTOB cell line was cultured in the presence of 35S-methionine and the incubation medium was submitted to immunoprecipitation. Total RNAs from CTOB and isolated bovine TM (freshly isolated, incubated or not were subjected to the reverse transcription-polymerase chain reaction and the amplification products were sequenced. Also, both CTOB and histological TM preparations were processed for transferrin immunolocalization. A labeled peptide of about 80 kDa, the expected size for transferrin, was immunopurified from CTOB samples obtained from the incubation assays. The reverse transcription-polymerase chain reaction and sequencing experiments detected the presence of transferrin mRNA in CTOB and isolated bovine TM. Reactivity to antibodies against transferrin was observed both in CTOB and TM. The results obtained in all of these experiments indicated that the TM is capable of synthesizing and secreting transferrin. The possible implications for the physiology of the eye are discussed.

  8. A Pseudomonas aeruginosa type VI secretion phospholipase D effector targets both prokaryotic and eukaryotic cells.

    Science.gov (United States)

    Jiang, Feng; Waterfield, Nicholas R; Yang, Jian; Yang, Guowei; Jin, Qi

    2014-05-14

    Widely found in animal and plant-associated proteobacteria, type VI secretion systems (T6SSs) are potentially capable of facilitating diverse interactions with eukaryotes and/or other bacteria. Pseudomonas aeruginosa encodes three distinct T6SS haemolysin coregulated protein (Hcp) secretion islands (H1, H2, and H3-T6SS), each involved in different aspects of the bacterium's interaction with other organisms. Here we describe the characterization of a P. aeruginosa H3-T6SS-dependent phospholipase D effector, PldB, and its three tightly linked cognate immunity proteins. PldB targets the periplasm of prokaryotic cells and exerts an antibacterial activity. Surprisingly, PldB also facilitates intracellular invasion of host eukaryotic cells by activation of the PI3K/Akt pathway, revealing it to be a trans-kingdom effector. Our findings imply a potentially widespread T6SS-mediated mechanism, which deploys a single phospholipase effector to influence both prokaryotic cells and eukaryotic hosts.

  9. Apoptotic neuron-secreted HN12 inhibits cell apoptosis in Hirschsprung's disease

    Directory of Open Access Journals (Sweden)

    Du C

    2016-11-01

    Full Text Available Chunxia Du,1,2,* Hua Xie,1,2,* Rujin Zang,1,2,* Ziyang Shen,1,2 Hongxing Li,1,2 Pingfa Chen,1,2 Xiaoqun Xu,1,2 Yankai Xia,2,3 Weibing Tang1,2 1Department of Pediatric Surgery, Nanjing Children’s Hospital Affiliated to Nanjing Medical University, 2State Key Laboratory of Reproductive Medicine, Institute of Toxicology, School of Public Health, 3Key Laboratory of Modern Toxicology, Ministry of Education, Nanjing Medical University, Nanjing, People’s Republic of China *These authors contributed equally to this work Abstract: Perturbation in apoptosis can lead to Hirschsprung’s disease (HSCR, which is a genetic disorder of neural crest development. It is believed that long noncoding RNAs (lncRNAs play a role in the progression of HSCR. This study shows that apoptotic neurons can suppress apoptosis of nonapoptotic cells by secreting exosomes that contain high levels of HN12 lncRNA. Elevated exogenous HN12 in nonapoptotic cells effectively inhibited cell apoptosis by maintaining the function of mitochondria, including the production of ATP and the release of cytochrome C. These results demonstrate that secreted lncRNAs may serve as signaling molecules mediating intercellular communication in HSCR. In addition, high HN12 levels in the circulation worked as a biomarker for predicting HSCR, providing a potential, novel, noninvasive diagnostic approach for early screening of HSCR. Keywords: Hirschsprung’s disease, neuronal development, exosomal long noncoding RNA, intercellular communication, apoptosis, mitochondria

  10. The expression and putative role of brain-derived neurotrophic factor and its receptor in bovine sperm.

    Science.gov (United States)

    Li, C; Li, C; Zhu, X; Wang, C; Liu, Zhuo; Li, W; Lu, Chen; Zhou, Xu

    2012-02-01

    The neurotrophin family of proteins promote the survival and differentiation of nerve cells and are thought to play an important role in development of reproductive tissues. The objective of the present study was to detect the presence of Brain-derived neurotrophic factor (BDNF) and its receptor TrkB in bovine sperm, and explore the potential role of BDNF in sperm function. We demonstrated that both the neorotrophin BDNF and the tyrosine kinase receptor protein TrkB were expressed in ejaculated bovine sperm. Furthermore, BDNF per se was secreted by sperm. Insulin and leptin secretion by bovine sperm were increased (P BDNF, whereas insulin was decreased by K252a. Therefore, we inferred that BDNF could be a regulator of sperm secretion of insulin and leptin through the TrkB receptor. Sperm viability and mitochondrial activity were both decreased (P BDNF/TrkB signaling pathway was blocked with K252a. Furthermore, BDNF promoted apoptosis of bovine sperm through TrkB binding (P BDNF secreted by bovine sperm was important in regulation of insulin and leptin secretion in ejaculated bovine sperm. Furthermore, BDNF may affect sperm mitochondrial activity and apoptosis, as well as their viability.

  11. PAI-1 secretion of endometrial and endometriotic cells is Smad2/3- and ERK1/2-dependent and influences cell adhesion.

    Science.gov (United States)

    Sui, Cong; Mecha, Ezekiel; Omwandho, Charles Oa; Starzinski-Powitz, Anna; Stammler, Angelika; Tinneberg, Hans-Rudolf; Konrad, Lutz

    2016-01-01

    In the endometrium transforming growth factor-betas (TGF-βs) are involved mainly in menstruation and endometriosis. After binding of the ligands to the high-affinity receptors, TGF-β receptors (TBR1 and TBR2), TGF-βs activate Smad signaling to modulate gene expression and cellular functions. However, recently also Smad-independent pathways have been studied in more details. To evaluate both pathways, we have analyzed TGF-β signaling in human endometrial and endometriotic cells. Although endometrial and endometriotic cells secrete TGF-β1, secretion by stromal cells was higher compared to epithelial cells. In contrast, secretion of TGF-β2 was higher in endometriotic stromal and endometriotic epithelial cells compared to normal endometrial cells. Treatment of endometrial and endometriotic stromal and epithelial cells with TGF-β1 or TGF-β2 increased Smad-dependent secretion of plasminogen activator inhibitor-1 (PAI-1) dramatically in all three cell lines. Of note, endometriotic cells secreted clearly higher levels of PAI-1 compared to endometrial cells. Whereas a TBR1 kinase inhibitor completely blocked the TGF-β1 or TGF-β2-induced PAI-1 secretion, an ERK1/2 inhibitor only partially reduced PAI-1 secretion. This inhibition was not dependent on epidermal growth factor receptor (EGFR) activation by phosphorylation but on kinase activity of the TBR1. Finally, treatment of endometrial and endometriotic cell lines with recombinant PAI-1 showed reduced cell adhesion, especially of the endometrial cells. In summary, our results demonstrate that both Smad-dependent and TBR1-dependent ERK1/2 pathways are necessary for TGF-β-dependent high level secretion of PAI-1, which might increase cellular deadhesion.

  12. Engineering human cells for in vivo secretion of antibody and non-antibody therapeutic proteins.

    Science.gov (United States)

    Sánchez-Martín, David; Sanz, Laura; Álvarez-Vallina, Luis

    2011-12-01

    Purified proteins such as antibodies are widely used as therapeutic agents in clinical medicine. However, clinical-grade proteins for therapeutic use require sophisticated technologies and are extremely expensive to produce. In vivo secretion of therapeutic proteins by genetically engineered human cells may advantageously replace injection of highly purified proteins. The use of gene transfer methods circumvents problems related to large-scale production and purification and offers additional benefits by achieving sustained concentrations of therapeutic protein with a syngenic glycosylation pattern that make the protein potentially less immunogenic. The feasibility of the in vivo production of therapeutic proteins by diverse cells/tissues has now been demonstrated using different techniques, such as ex vivo genetically modified cells and in vivo gene transfer mediated by viral vectors.

  13. Differentiating of banked human umbilical cord blood-derived mesenchymal stem cells into insulin-secreting cells.

    Science.gov (United States)

    Phuc, Pham Van; Nhung, Truong Hai; Loan, Dang Thi Tung; Chung, Doan Chinh; Ngoc, Phan Kim

    2011-01-01

    Umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) are multipotent cells. They are able to differentiate into functional cells from not only mesoderm but also endoderm. Many researches showed that cells derived from fresh human UCB could transdifferentiate into insulin-secreting cells. In this study, transdifferentiating potential of cryopreserved human UCB-derived MSCs into insulin-secreting cell was investigated. Fresh human UCB was enriched the mononuclear cells by Ficoll-Paque centrifugation. The mononuclear cell population was cryopreserved in cryo-medium containing Iscove's modified Dulbecco's media (IMDM) with 10% DMSO at -196°C for 1 yr. After thawing, mononuclear cells were cultured to isolate MSCs in medium IMDM with 20% FBS supplemented with growth factors. At the fifth passages, MSCs were confirmed by flow cytometry about expression of CD13, CD14, CD34, CD45, CD166, and HLA-DR markers; after that, they were induced to differentiate into adipocytes and osteoblasts. After inducing with specific medium for islet differentiation, there were many clusters of cell like islet at day 14-28. Using real-time reverse transcription polymerase chain reaction (RT-PCR) to analyze the expression of functional genes, the result showed that Nestin, Pdx-1, Ngn3, Ils-1, Pax6, Pax4, Nkx2.2, Nkx6.1, Glut-2, Insulin genes expressed. The results showed that MSCs derived from banked cord blood can differentiate into functional pancreatic islet-like cells in vitro. If human MSCs, especially MSCs from banked cord blood of diabetes patients themselves can be isolated, proliferated, differentiated into functional pancreatic islet-like cells, and transplanted back into them (autologous transplantation), their high-proliferation potency and rejection avoidance will provide one promising therapy for diabetes.

  14. Glucagon-like peptide 1 receptor playsa critical role in geniposide-regulated insulin secretion in INS-1 cells

    Institute of Scientific and Technical Information of China (English)

    Li-xia GUO; Zhi-ning XIA; Xue GAO; Fei YIN; Jian-hui LIU

    2012-01-01

    Aim:To explore the role of the glucagon-like peptide 1 receptor (GLP-1R) in geniposide regulated insulin secretion in rat INS-1 insulinoma cells.Methods:Rat INS-1 insulinoma cells were cultured.The content of insulin in the culture medium was measured with ELISA assay.GLP-1R gene in INS-1 cells was knocked down with shRNA interference.The level of GLP-1R protein in INS-1 cells was measured with Western blotting.Results:Geniposide (0.01-100 μmol/L) increased insulin secretion from INS-1 cells in a concentration-dependent manner.Geniposide (10 μmol/L) enhanced acute insulin secretion in response to both the low (5.5 mmol/L) and moderately high levels (11 mmol/L) of glucose.Blockade of GLP-1R with the GLP-1R antagonist exendin (9-39) (200 nmol/L) or knock-down of GLP-1R with shRNA interference in INS-1 cells decreased the effect of geniposide (10 μmol/L) on insulin secretion stimulated by glucose (5.5 mmol/L).Conclusion:Geniposide increases insulin secretion through glucagon-like peptide 1 receptors in rat INS-1 insulinoma cells.

  15. Zinc Up-Regulates Insulin Secretion from β Cell-Like Cells Derived from Stem Cells from Human Exfoliated Deciduous Tooth (SHED)

    Science.gov (United States)

    Kim, Gyuyoup; Shin, Ki-Hyuk; Pae, Eung-Kwon

    2016-01-01

    Stem cells from human exfoliated deciduous tooth (SHED) offer several advantages over other stem cell sources. Using SHED, we examined the roles of zinc and the zinc uptake transporter ZIP8 (Zrt- and irt-like protein 8) while inducing SHED into insulin secreting β cell-like stem cells (i.e., SHED-β cells). We observed that ZIP8 expression increased as SHED differentiated into SHED-β cells, and that zinc supplementation at day 10 increased the levels of most pancreatic β cell markers—particularly Insulin and glucose transporter 2 (GLUT2). We confirmed that SHED-β cells produce insulin successfully. In addition, we note that zinc supplementation significantly increases insulin secretion with a significant elevation of ZIP8 transporters in SHED-β cells. We conclude that SHED can be converted into insulin-secreting β cell-like cells as zinc concentration in the cytosol is elevated. Insulin production by SHED-β cells can be regulated via modulation of zinc concentration in the media as ZIP8 expression in the SHED-β cells increases. PMID:27983594

  16. Zinc Up-Regulates Insulin Secretion from β Cell-Like Cells Derived from Stem Cells from Human Exfoliated Deciduous Tooth (SHED

    Directory of Open Access Journals (Sweden)

    Gyuyoup Kim

    2016-12-01

    Full Text Available Stem cells from human exfoliated deciduous tooth (SHED offer several advantages over other stem cell sources. Using SHED, we examined the roles of zinc and the zinc uptake transporter ZIP8 (Zrt- and irt-like protein 8 while inducing SHED into insulin secreting β cell-like stem cells (i.e., SHED-β cells. We observed that ZIP8 expression increased as SHED differentiated into SHED-β cells, and that zinc supplementation at day 10 increased the levels of most pancreatic β cell markers—particularly Insulin and glucose transporter 2 (GLUT2. We confirmed that SHED-β cells produce insulin successfully. In addition, we note that zinc supplementation significantly increases insulin secretion with a significant elevation of ZIP8 transporters in SHED-β cells. We conclude that SHED can be converted into insulin-secreting β cell-like cells as zinc concentration in the cytosol is elevated. Insulin production by SHED-β cells can be regulated via modulation of zinc concentration in the media as ZIP8 expression in the SHED-β cells increases.

  17. Brain Ciliary Neurotrophic Factor (CNTF and hypothalamic control of energy homeostasis

    Directory of Open Access Journals (Sweden)

    Vacher Claire-Marie

    2011-09-01

    Full Text Available Cytokines play an important role in energy-balance regulation. Notably leptin, an adipocyte-secreted cytokine, regulates the activity of hypothalamic neurons that are involved in the modulation of appetite. Leptin decreases appetite and stimulates weight loss in rodents. Unfortunately, numerous forms of obesity in humans seem to be resistant to leptin action. The ciliary neurotrophic factor (CNTF is a neurocytokine that belongs to the same family as leptin and that was originally characterized as a neurotrophic factor that promotes the survival of a broad spectrum of neuronal cell types and that enhances neurogenesis in adult rodents. It presents the advantage of stimulating weight loss in humans, despite the leptin resistance. Moreover, the weight loss persists several weeks after the cessation of treatment. Hence, CNTF has been considered as a promising therapeutic tool for the treatment of obesity and has prompted intense research aimed at identifying the cellular and molecular mechanisms underlying its potent anorexigenic properties. It has been found that CNTF shares signaling pathways with leptin and is expressed in the arcuate nucleus (ARC, a key hypothalamic region controlling food intake. Endogenous CNTF may also participate in the control of energy balance. Indeed, its expression in the ARC is inversely correlated to body weight in rats fed a high-sucrose diet. Thus hypothalamic CNTF may act, in some individuals, as a protective factor against weight gain during hypercaloric diet and could account for individual differences in the susceptibility to obesity.

  18. Modulation of the NMDA Receptor Through Secreted Soluble Factors.

    Science.gov (United States)

    Cerpa, Waldo; Ramos-Fernández, Eva; Inestrosa, Nibaldo C

    2016-01-01

    Synaptic activity is a critical determinant in the formation and development of excitatory synapses in the central nervous system (CNS). The excitatory current is produced and regulated by several ionotropic receptors, including those that respond to glutamate. These channels are in turn regulated through several secreted factors that function as synaptic organizers. Specifically, Wnt, brain-derived neurotrophic factor (BDNF), fibroblast growth factor (FGF), and transforming growth factor (TGF) particularly regulate the N-methyl-D-aspartate receptor (NMDAR) glutamatergic channel. These factors likely regulate early embryonic development and directly control key proteins in the function of important glutamatergic channels. Here, we review the secreted molecules that participate in synaptic organization and discuss the cell signaling behind of this fine regulation. Additionally, we discuss how these factors are dysregulated in some neuropathologies associated with glutamatergic synaptic transmission in the CNS.

  19. FAM20: an evolutionarily conserved family of secreted proteins expressed in hematopoietic cells

    Directory of Open Access Journals (Sweden)

    Cobos Everardo

    2005-01-01

    Full Text Available Abstract Background Hematopoiesis is a complex developmental process controlled by a large number of factors that regulate stem cell renewal, lineage commitment and differentiation. Secreted proteins, including the hematopoietic growth factors, play critical roles in these processes and have important biological and clinical significance. We have employed representational difference analysis to identify genes that are differentially expressed during experimentally induced myeloid differentiation in the murine EML hematopoietic stem cell line. Results One identified clone encoded a previously unidentified protein of 541 amino acids that contains an amino terminal signal sequence but no other characterized domains. This protein is a member of family of related proteins that has been named family with sequence similarity 20 (FAM20 with three members (FAM20A, FAM20B and FAM20C in mammals. Evolutionary comparisons revealed the existence of a single FAM20 gene in the simple vertebrate Ciona intestinalis and the invertebrate worm Caenorhabditis elegans and two genes in two insect species, Drosophila melanogaster and Anopheles gambiae. Six FAM20 family members were identified in the genome of the pufferfish, Fugu rubripes and five members in the zebrafish, Danio rerio. The mouse Fam20a protein was ectopically expressed in a mammalian cell line and found to be a bona fide secreted protein and efficient secretion was dependent on the integrity of the signal sequence. Expression analysis revealed that the Fam20a gene was indeed differentially expressed during hematopoietic differentiation and that the other two family members (Fam20b and Fam20c were also expressed during hematcpoiesis but that their mRNA levels did not vary significantly. Likewise FAM20A was expressed in more limited set of human tissues than the other two family members. Conclusions The FAM20 family represents a new family of secreted proteins with potential functions in regulating

  20. Genetic deletion of Rab27B in pancreatic acinar cells affects granules size and has inhibitory effects on amylase secretion.

    Science.gov (United States)

    Hou, Yanan; Ernst, Stephen A; Lentz, Stephen I; Williams, John A

    2016-03-18

    Small G protein Rab27B is expressed in various secretory cell types and plays a role in mediating secretion. In pancreatic acinar cells, Rab27B was found to be expressed on the zymogen granule membrane and by overexpression to regulate the secretion of zymogen granules. However, the effect of Rab27B deletion on the physiology of pancreatic acinar cells is unknown. In the current study, we utilized the Rab27B KO mouse model to better understand the role of Rab27B in the secretion of pancreatic acinar cells. Our data show that Rab27B deficiency had no obvious effects on the expression of major digestive enzymes and other closely related proteins, e.g. similar small G proteins, such as Rab3D and Rab27A, and putative downstream effectors. The overall morphology of acinar cells was not changed in the knockout pancreas. However, the size of zymogen granules was decreased in KO acinar cells, suggesting a role of Rab27B in regulating the maturation of secretory granules. The secretion of digestive enzymes was moderately decreased in KO acini, compared with the WT control. These data indicate that Rab27B is involved at a different steps of zymogen granule maturation and secretion, which is distinct from that of Rab3D.

  1. Mycobacterial secretion systems ESX-1 and ESX-5 play distinct roles in host cell death and inflammasome activation

    KAUST Repository

    Abdallah, Abdallah

    2011-09-28

    During infection of humans and animals, pathogenic mycobacteria manipulate the host cell causing severe diseases such as tuberculosis and leprosy. To understand the basis of mycobacterial pathogenicity, it is crucial to identify the molecular virulence mechanisms. In this study, we address the contribution of ESX-1 and ESX-5 - two homologous type VII secretion systems of mycobacteria that secrete distinct sets of immune modulators - during the macrophage infection cycle. Using wild-type, ESX-1- and ESX-5-deficient mycobacterial strains, we demonstrate that these secretion systems differentially affect subcellular localization and macrophage cell responses. We show that in contrast to ESX-1, the effector proteins secreted by ESX-5 are not required for the translocation of Mycobacterium tuberculosis or Mycobacterium marinum to the cytosol of host cells. However, the M. marinum ESX-5 mutant does not induce inflammasome activation and IL-1b activation. The ESX-5 system also induces a caspase-independent cell death after translocation has taken place. Importantly, by means of inhibitory agents and small interfering RNA experiments, we reveal that cathepsin B is involved in both the induction of cell death and inflammasome activation upon infection with wild-type mycobacteria. These results reveal distinct roles for two different type VII secretion systems during infection and shed light on how virulent mycobacteria manipulate the host cell in various ways to replicate and spread. Copyright © 2011 by The American Association of Immunologists, Inc.

  2. Protective effect of liposome-mediated glial cell line-derived neurotrophic factor gene transfer in vivo on motoneurons following spinal cord injury in rats

    Institute of Scientific and Technical Information of China (English)

    鲁凯伍; 陈哲宇; 侯铁胜

    2004-01-01

    Objective:To investigate the effect of liposomemediated glial cell line-derived neurotrophic factor (GDNF) gene transfer in vivo on spinal cord motoneurons after spinal cord injury (SCI) in adult rats.Methods: Sixty male Sprague-Dawley rats were divided equally into two groups: GDNF group and control group. The SCI model was established according to the method of Nystrom, and then the DC-Chol liposomes and recombinant plasmid pEGFP-GDNF cDNA complexes were injected into the injured spinal cord. The expression of GDNF cDNA 1 week after injection was detected by RTPCR and fluorescence microscope. We observed the remaining motoneurons in the anterior horn and the changes of cholinesterase (CHE) and acid phosphatase (ACP) activity using Nissl and enzyme histochemistry staining. The locomotion function of hind limbs of rats was evaluated using inclined plane test and BBB locomotor scale.Results: RT-PCR and fluorescence observation confirmed the presence of expression of GDNF cDNA 1week and 4 weeks after injection. At 1, 2, 4 weeks after SCI, the number of motoneurons in the anterior horn in GDNF group (20.4±3.2, 21.7±3.6, 22.5±3.4) was more than that in control group ( 16.8±2.8, 17.3 ± 2.7,18.2±3.2, P<0.05). At 1, 2 weeks after SCI, the mean gray of the CHE-stained spinal motoneurons in GDNF group (74.2± 25.8, 98.7± 31.6 was less than that in control group (98.5 ±32.2, 134.6 ±45.2, P<0.01), and the mean gray of ACP in GDNF group (84.5±32.6, 79.5±28.4) was more than that in control group (61.2±24.9,52.6±19.9, P<0.01). The locomotion functional scales in GDNF group were higher than that in control group within 1 to 4 weeks after SCI (P<0.05).Conclusions: GDNF gene transfer in vivo can protect motoneurons from death and degeneration induced by incompleted spinal cord injury as well as enhance locomotion functional restoration of hind limbs. These results suggest that liposome-mediated delivery of GDNF cDNA might be a practical method for treating

  3. beta-Estradiol induces synaptogenesis in the hippocampus by enhancing brain-derived neurotrophic factor release from dentate gyrus granule cells.

    Science.gov (United States)

    Sato, Kaoru; Akaishi, Tatsuhiro; Matsuki, Norio; Ohno, Yasuo; Nakazawa, Ken

    2007-05-30

    We investigated the effect of beta-estradiol (E2) on synaptogenesis in the hippocampus using organotypic hippocampal slice cultures and subregional hippocampal neuron cultures. E2 increased the expression of PSD95, a postsynaptic marker, specifically in stratum lucidum of Cornu Ammonis 3 (CA3SL) in cultured hippocampal slices. E2 also increased the spine density at the proximal site of CA3 apical dendrites in CA3SL and PSD95 was clustered on these spine heads. The effects of E2 on the expression of PSD95 and the spine density disappeared when the dentate gyrus (DG) had been excised at 1 day in vitro (DIV). FM1-43 analysis of subregional hippocampal neuron cultures which were comprised of Ammon's horn neurons, DG neurons, or a mixture of these neurons, revealed that E2 increased the number of presynaptic sites in the cultures that contained DG neurons. K252a, a potent inhibitor of the high affinity receptor of brain-derived neurotrophic factor (BDNF), and function-blocking antibody to BDNF (BDNFAB) completely inhibited the effects of E2 in hippocampal slice cultures and subregional neuron cultures, whereas ICI182,780 (ICI), a strong antagonist of nuclear estrogen receptors (nERs), did not. Expression of BDNF in DG neurons was markedly higher than that in Ammon's horn neurons and E2 did not affect these expression levels. E2 significantly increased the BDNF release from DG neurons. KT5720, a specific inhibitor of 3'-5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA), and Rp-adenosine 3', 5'-cyclic monophosphorothioate triethylammonium salt (Rp-cAMP), a non-hydrolyzable diastereoisomer and a potent inhibitor of PKA, completely suppressed the E2-induced increase in BDNF release, whereas ICI and U0126, a potent inhibitor of MAP kinase kinase (MEK), did not. These results suggest that E2 induces synaptogenesis between mossy fibers and CA3 neurons by enhancing BDNF release from DG granule cells in a nER-independent and PKA-dependent manner.

  4. 酪氨酸激酶受体B-脑源性神经营养因子信号传导通路对神经母细胞瘤细胞分泌血管内皮生长因子及基质金属蛋白酶-9的影响%Effects of tyrosine kinase receptor B-brain-derived neurotrophic factor signal pathway on the secretion of vascular endothelial growth factor and matrix metalloproteinase-9 of neuroblastoma

    Institute of Scientific and Technical Information of China (English)

    刘建英; 高惠敏; 李爱敏; 蔡维艳; 初清

    2013-01-01

    .%Objective To study the effects of tyrosine kinase receptor B-brain-derived neurotrophic factor (TrkB-BDNF) signal pathway on the secretion of vascular endothelial growth factor (VEGF) and matrix metalloproteinases-9(MMP-9) of neuroblastoma.Methods We used all-trans retinoic acid (ATRA) to induce the high expression of TrkB in the SH-SY5Y cell line,and then added the ectogenid BDNF to activate the TrkB-BDNF and its three downstream signal pathways.TrkB-BDNF signal pathway was inhibited by specific tyrosine kinase inhibitor K252a.The three downstream signal pathway was respectively inhibited by LY294002 (the phosphatidylinositol 3-hydroxy kinase (PI3 K) pathway inhibitor)、U73122 (the phospholipase C pathway inhibitor) 、U0126(the mitogen activated protein kinase pathway inhibitor).Enzyme linked immunosorbent assay was used to detect the concentration of VEGF and MMP-9 protein in the SY5Y cell culture supernatants.Results VEGF [(485.89 ± 109.99) pg/ml] and MMP-9 [(15.73 ± 1.72) pg/ml] protein levels in neuroblastoma cells cultured in serum-free media in the group of ATRA + BDNF were significantly higher than that of the control group and ATRA alone group(P <0.05).VEGF [(272.42 ±86.33) pg/ml]and MMP-9 [(5.25 ± 1.44) pg/ml] protein levels in the group of ATRA + BDNF + K252a were significantly lower than those of the ATRA + BDNF group(P < 0.05) and had no significant difference compared with the control group and the ATRA alone group(P >0.05).VEGF [(314.12 ±24.68) pg/ml] and MMP-9 [(4.91 ± 1.08) pg/ml] protein levels in the group of ATRA + BDNF + LY294002 were significantly lower than those of the ATRA + BDNF group(P < 0.05) and had no significant difference compared with the control group and the ATRA alone group(P >0.05).VEGF [(444.08 ±64.49) pg/ml] and MMP-9 [(13.28 ±3.38) pg/ml] protein levels in neuroblastoma cells cultured in serum-free media in the group of ATRA +BDNF + U73122 had no significant difference compared with the ATRA + BDNF group(P > 0

  5. Sulindac modulates secreted protein expression from LIM1215 colon carcinoma cells prior to apoptosis.

    Science.gov (United States)

    Greening, David W; Ji, Hong; Kapp, Eugene A; Simpson, Richard J

    2013-11-01

    Colorectal cancer (CRC) is a major cause of mortality in Western populations. Growing evidence from human and rodent studies indicate that nonsteroidal anti-inflammatory drugs (NSAIDs) cause regression of existing colon tumors and act as effective chemopreventive agents in sporadic colon tumor formation. Although much is known about the action of the NSAID sulindac, especially its role in inducing apoptosis, mechanisms underlying these effects is poorly understood. In previous secretome-based proteomic studies using 2D-DIGE/MS and cytokine arrays we identified over 150 proteins released from the CRC cell line LIM1215 whose expression levels were dysregulated by treatment with 1mM sulindac over 16h; many of these proteins are implicated in molecular and cellular functions such as cell proliferation, differentiation, adhesion, angiogenesis and apoptosis (Ji et al., Proteomics Clin. Appl. 2009, 3, 433-451). We have extended these studies and describe here an improved protein/peptide separation strategy that facilitated the identification of 987 proteins and peptides released from LIM1215 cells following 1mM sulindac treatment for 8h preceding the onset of apoptosis. This peptidome separation strategy involved fractional centrifugal ultrafiltration of concentrated cell culture media (CM) using nominal molecular weight membrane filters (NMWL 30K, 3K and 1K). Proteins isolated in the >30K and 3-30K fractions were electrophoretically separated by SDS-PAGE and endogenous peptides in the 1-3K membrane filter were fractioned by RP-HPLC; isolated proteins and peptides were identified by nanoLC-MS-MS. Collectively, our data show that LIM1215 cells treated with 1mM sulindac for 8h secrete decreased levels of proteins associated with extracellular matrix remodeling (e.g., collagens, perlecan, syndecans, filamins, dyneins, metalloproteinases and endopeptidases), cell adhesion (e.g., cadherins, integrins, laminins) and mucosal maintenance (e.g., glycoprotein 340 and mucins 5AC, 6

  6. Exosomes secreted by nematode parasites transfer small RNAs to mammalian cells and modulate innate immunity.

    Science.gov (United States)

    Buck, Amy H; Coakley, Gillian; Simbari, Fabio; McSorley, Henry J; Quintana, Juan F; Le Bihan, Thierry; Kumar, Sujai; Abreu-Goodger, Cei; Lear, Marissa; Harcus, Yvonne; Ceroni, Alessandro; Babayan, Simon A; Blaxter, Mark; Ivens, Alasdair; Maizels, Rick M

    2014-11-25

    In mammalian systems RNA can move between cells via vesicles. Here we demonstrate that the gastrointestinal nematode Heligmosomoides polygyrus, which infects mice, secretes vesicles containing microRNAs (miRNAs) and Y RNAs as well as a nematode Argonaute protein. These vesicles are of intestinal origin and are enriched for homologues of mammalian exosome proteins. Administration of the nematode exosomes to mice suppresses Type 2 innate responses and eosinophilia induced by the allergen Alternaria. Microarray analysis of mouse cells incubated with nematode exosomes in vitro identifies Il33r and Dusp1 as suppressed genes, and Dusp1 can be repressed by nematode miRNAs based on a reporter assay. We further identify miRNAs from the filarial nematode Litomosoides sigmodontis in the serum of infected mice, suggesting that miRNA secretion into host tissues is conserved among parasitic nematodes. These results reveal exosomes as another mechanism by which helminths manipulate their hosts and provide a mechanistic framework for RNA transfer between animal species.

  7. Wnt secretion is required to maintain high levels of Wnt activity in colon cancer cells.

    Science.gov (United States)

    Voloshanenko, Oksana; Erdmann, Gerrit; Dubash, Taronish D; Augustin, Iris; Metzig, Marie; Moffa, Giusi; Hundsrucker, Christian; Kerr, Grainne; Sandmann, Thomas; Anchang, Benedikt; Demir, Kubilay; Boehm, Christina; Leible, Svenja; Ball, Claudia R; Glimm, Hanno; Spang, Rainer; Boutros, Michael

    2013-01-01

    Aberrant regulation of the Wnt/β-catenin pathway has an important role during the onset and progression of colorectal cancer, with over 90% of cases of sporadic colon cancer featuring mutations in APC or β-catenin. However, it has remained a point of controversy whether these mutations are sufficient to activate the pathway or require additional upstream signals. Here we show that colorectal tumours express elevated levels of Wnt3 and Evi/Wls/GPR177. We found that in colon cancer cells, even in the presence of mutations in APC or β-catenin, downstream signalling remains responsive to Wnt ligands and receptor proximal signalling. Furthermore, we demonstrate that truncated APC proteins bind β-catenin and key components of the destruction complex. These results indicate that cells with mutations in APC or β-catenin depend on Wnt ligands and their secretion for a sufficient level of β-catenin signalling, which potentially opens new avenues for therapeutic interventions by targeting Wnt secretion via Evi/Wls.

  8. Human dendritic cell maturation and cytokine secretion upon stimulation with Bordetella pertussis filamentous haemagglutinin.

    Science.gov (United States)

    Dirix, Violette; Mielcarek, Nathalie; Debrie, Anne-Sophie; Willery, Eve; Alonso, Sylvie; Versheure, Virginie; Mascart, Françoise; Locht, Camille

    2014-07-01

    In addition to antibodies, Th1-type T cell responses are also important for long-lasting protection against pertussis. However, upon immunization with the current acellular vaccines, many children fail to induce Th1-type responses, potentially due to immunomodulatory effects of some vaccine antigens, such as filamentous haemagglutinin (FHA). We therefore analysed the ability of FHA to modulate immune functions of human monocyte-derived dendritic cells (MDDC). FHA was purified from pertussis toxin (PTX)-deficient or from PTX- and adenylate cyclase-deficient Bordetella pertussis strains, and residual endotoxin was neutralized with polymyxin B. FHA from both strains induced phenotypic maturation of human MDDC and cytokine secretion (IL-10, IL-12p40, IL-12p70, IL-23 and IL-6). To identify the FHA domains responsible for MDDC immunomodulation, MDDC were stimulated with FHA containing a Gly→Ala substitution at its RGD site (FHA-RAD) or with an 80-kDa N-terminal moiety of FHA (Fha44), containing its heparin-binding site. Whereas FHA-RAD induced maturation and cytokine production comparable to those of FHA, Fha44 did not induce IL-10 production, but maturated MDDC at least partially. Nevertheless, Fha44 induced the secretion of IL-12p40, IL-12p70, IL-23 and IL-6 by MDDC, albeit at lower levels than FHA. Thus, FHA can modulate MDDC responses in multiple ways, and IL-10 induction can be dissociated from the induction of other cytokines.

  9. Factors secreted from dental pulp stem cells show multifaceted benefits for treating experimental rheumatoid arthritis.

    Science.gov (United States)

    Ishikawa, Jun; Takahashi, Nobunori; Matsumoto, Takuya; Yoshioka, Yutaka; Yamamoto, Noriyuki; Nishikawa, Masaya; Hibi, Hideharu; Ishigro, Naoki; Ueda, Minoru; Furukawa, Koichi; Yamamoto, Akihito

    2016-02-01

    Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovial hyperplasia and chronic inflammation, which lead to the progressive destruction of cartilage and bone in the joints. Numerous studies have reported that administrations of various types of MSCs improve arthritis symptoms in animal models, by paracrine mechanisms. However, the therapeutic effects of the secreted factors alone, without the cell graft, have been uncertain. Here, we show that a single intravenous administration of serum-free conditioned medium (CM) from human deciduous dental pulp stem cells (SHED-CM) into anti-collagen type II antibody-induced arthritis (CAIA), a mouse model of rheumatoid arthritis (RA), markedly improved the arthritis symptoms and joint destruction. The therapeutic efficacy of SHED-CM was associated with an induction of anti-inflammatory M2 macrophages in the CAIA joints and the abrogation of RANKL expression. SHED-CM specifically depleted of an M2 macrophage inducer, the secreted ectodomain of sialic acid-binding Ig-like lectin-9 (ED-Siglec-9), exhibited a reduced ability to induce M2-related gene expression and attenuate CAIA. SHED-CM also inhibited the RANKL-induced osteoclastogenesis in vitro. Collectively, our findings suggest that SHED-CM provides multifaceted therapeutic effects for treating CAIA, including the ED-Siglec-9-dependent induction of M2 macrophage polarization and inhibition of osteoclastogenesis. Thus, SHED-CM may represent a novel anti-inflammatory and reparative therapy for RA.

  10. Inhibition of myocardial ischemia/reperfusion injury by exosomes secreted from mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    ZHANG Heng; XIANG Meng; MENG Dan; SUN Ning; CHEN Si-feng

    2016-01-01

    Exosomes secreted by mesenchymal stem cells have shown great therapeutic potential in regenerative medicine .In this study, we performed meta-analysis to assess the clinical effectiveness of using exosomes in ischemia /reperfusion injury based on the reports pub-lished between January 2000 and September 2015 and indexed in the PubMed and Web of Science databases .The effect of exosomes on heart function was evaluated according to the following parameters:the area at risk as a percentage of the left ventricle , infarct size as a percentage of the area at risk , infarct size as a percentage of the left ventricle , left ventricular ejection fraction , left ventricular frac-tion shortening , end-diastolic volume , and end-systolic volume .Our analysis indicated that the currently available evidence confirmed the therapeutic potential of mesenchymal stem cell-secreted exosomes in the improvement of heart function .However , further mechanis-tic studies, therapeutic safety and clinical trials are required for optimization and validation of this approach to cardiac regeneration after ischemia/reperfusion injury .

  11. Modulation of pathogen-induced CCL20 secretion from HT-29 human intestinal epithelial cells by commensal bacteria.

    LENUS (Irish Health Repository)

    Sibartie, Shomik

    2009-01-01

    BACKGROUND: Human intestinal epithelial cells (IECs) secrete the chemokine CCL20 in response to infection by various enteropathogenic bacteria or exposure to bacterial flagellin. CCL20 recruits immature dendritic cells and lymphocytes to target sites. Here we investigated IEC responses to various pathogenic and commensal bacteria as well as the modulatory effects of commensal bacteria on pathogen-induced CCL20 secretion. HT-29 human IECs were incubated with commensal bacteria (Bifidobacterium infantis or Lactobacillus salivarius), or with Salmonella typhimurium, its flagellin, Clostridium difficile, Mycobacterium paratuberculosis, or Mycobacterium smegmatis for varying times. In some studies, HT-29 cells were pre-treated with a commensal strain for 2 hr prior to infection or flagellin stimulation. CCL20 and interleukin (IL)-8 secretion and nuclear factor (NF)-kappaB activation were measured using enzyme-linked immunosorbent assays. RESULTS: Compared to untreated cells, S. typhimurium, C. difficile, M. paratuberculosis, and flagellin activated NF-kappaB and stimulated significant secretion of CCL20 and IL-8 by HT-29 cells. Conversely, B. infantis, L. salivarius or M. smegmatis did not activate NF-kappaB or augment CCL20 or IL-8 production. Treatment with B. infantis, but not L. salivarius, dose-dependently inhibited the baseline secretion of CCL20. In cells pre-treated with B. infantis, C. difficile-, S. typhimurium-, and flagellin-induced CCL20 were significantly attenuated. B. infantis did not limit M. Paratuberculosis-induced CCL20 secretion. CONCLUSION: This study is the first to demonstrate that a commensal strain can attenuate CCL20 secretion in HT-29 IECs. Collectively, the data indicate that M. paratuberculosis may mediate mucosal damage and that B. infantis can exert immunomodulatory effects on IECs that mediate host responses to flagellin and flagellated enteric pathogens.

  12. Effect of substrate rigidity in tissue culture on the function of insulin-secreting INS-1E cells.

    Science.gov (United States)

    Naujok, O; Bandou, Y; Shikama, Y; Funaki, M; Lenzen, S

    2017-01-01

    Insulin-secreting INS-1E cells are a useful tool in diabetes research. However, during permanent culture the cells tend to lose their β cell phenotype, with resultant loss of insulin-secretory responsiveness. This can be at least partially attributed to inappropriate cell culture conditions. One of the important causative factors is the rigidity of the extracellular matrix. We have therefore systematically studied the performance of INS-1E insulin-secreting cells cultured on polyacrylamide gels of different stiffnesses and analysed changes in insulin content and secretion, glucokinase enzyme activity, gene expression of β cell transcription factors and cell death and proliferation rates. INS-1E cells were cultured on polyacrylamide gels with a wide range of rigidities, including the one that simulates the stiffness of the pancreas. We detected changes in insulin content and the insulin-secretory response to glucose stimulation in parallel to the increasing stiffness of the polyacrylamide gels in the range 1700-111 000 Pa. On substrates with the highest and lowest rigidities, 322 and 111 000 Pa, the cells mainly formed pseudo-islets, while at rigidities of 1700-64800 Pa, including the rigidity of native pancreas tissue (3100 Pa), cells grew as a monolayer attached to the polyacrylamide gel surface. These observations provide evidence for an apparent mechanosensitivity of insulin-secreting INS-1E cells affecting morphology and cellular functions. The results can also provide practical advice regarding a selection of the materials appropriate for successful cell culture of insulin-secreting cells. Copyright © 2014 John Wiley & Sons, Ltd.

  13. In vivo IFN-γ secretion by NK cells in response to Salmonella typhimurium requires NLRC4 inflammasomes.

    Directory of Open Access Journals (Sweden)

    Andreas Kupz

    Full Text Available Natural killer (NK cells are a critical part of the innate immune defense against viral infections and for the control of tumors. Much less is known about how NK cells contribute to anti-bacterial immunity. NK cell-produced interferon gamma (IFN-γ contributes to the control of early exponential replication of bacterial pathogens, however the regulation of these events remains poorly resolved. Using a mouse model of invasive Salmonellosis, here we report that the activation of the intracellular danger sensor NLRC4 by Salmonella-derived flagellin within CD11c+ cells regulates early IFN-γ secretion by NK cells through the provision of interleukin 18 (IL-18, independently of Toll-like receptor (TLR-signaling. Although IL18-signalling deficient NK cells improved host protection during S. Typhimurium infection, this increased resistance was inferior to that provided by wild-type NK cells. These findings suggest that although NLRC4 inflammasome-driven secretion of IL18 serves as a potent activator of NK cell mediated IFN-γ secretion, IL18-independent NK cell-mediated mechanisms of IFN-γ secretion contribute to in vivo control of Salmonella replication.

  14. Neurotrophic effect of citrus 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone: promotion of neurite outgrowth via cAMP/PKA/CREB pathway in PC12 cells.

    Directory of Open Access Journals (Sweden)

    Hui-Chi Lai

    Full Text Available 5-Hydroxy-3,6,7,8,3',4'-hexamethoxyflavone (5-OH-HxMF, a hydroxylated polymethoxyflavone, is found exclusively in the Citrus genus, particularly in the peels of sweet orange. In this research, we report the first investigation of the neurotrophic effects and mechanism of 5-OH-HxMF in PC12 pheochromocytoma cells. We found that 5-OH-HxMF can effectively induce PC12 neurite outgrowth accompanied with the expression of neuronal differentiation marker protein growth-associated protein-43(GAP-43. 5-OH-HxMF caused the enhancement of cyclic AMP response element binding protein (CREB phosphorylation, c-fos gene expression and CRE-mediated transcription, which was inhibited by 2-naphthol AS-E phosphate (KG-501, a specific antagonist for the CREB-CBP complex formation. Moreover, 5-OH-HxMF-induced both CRE transcription activity and neurite outgrowth were inhibited by adenylate cyclase and protein kinase A (PKA inhibitor, but not MEK1/2, protein kinase C (PKC, phosphatidylinositol 3-kinase (PI3K or calcium/calmodulin-dependent protein kinase (CaMK inhibitor. Consistently, 5-OH-HxMF treatment increased the intracellular cAMP level and downstream component, PKA activity. We also found that addition of K252a, a TrKA antagonist, significantly inhibited NGF- but not 5-OH-HxMF-induced neurite outgrowth. These results reveal for the first time that 5-OH-HxMF is an effective neurotrophic agent and its effect is mainly through a cAMP/PKA-dependent, but TrKA-independent, signaling pathway coupling with CRE-mediated gene transcription. A PKC-dependent and CREB-independent pathway was also involved in its neurotrophic action.

  15. Characterization of stimulus-secretion coupling in the human pancreatic EndoC-βH1 beta cell line.

    Directory of Open Access Journals (Sweden)

    Lotta E Andersson

    Full Text Available Studies on beta cell metabolism are often conducted in rodent beta cell lines due to the lack of stable human beta cell lines. Recently, a human cell line, EndoC-βH1, was generated. Here we investigate stimulus-secretion coupling in this cell line, and compare it with that in the rat beta cell line, INS-1 832/13, and human islets.Cells were exposed to glucose and pyruvate. Insulin secretion and content (radioimmunoassay, gene expression (Gene Chip array, metabolite levels (GC/MS, respiration (Seahorse XF24 Extracellular Flux Analyzer, glucose utilization (radiometric, lactate release (enzymatic colorimetric, ATP levels (enzymatic bioluminescence and plasma membrane potential and cytoplasmic Ca2+ responses (microfluorometry were measured. Metabolite levels, respiration and insulin secretion were examined in human islets.Glucose increased insulin release, glucose utilization, raised ATP production and respiratory rates in both lines, and pyruvate increased insulin secretion and respiration. EndoC-βH1 cells exhibited higher insulin secretion, while plasma membrane depolarization was attenuated, and neither glucose nor pyruvate induced oscillations in intracellular calcium concentration or plasma membrane potential. Metabolite profiling revealed that glycolytic and TCA-cycle intermediate levels increased in response to glucose in both cell lines, but responses were weaker in EndoC-βH1 cells, similar to those observed in human islets. Respiration in EndoC-βH1 cells was more similar to that in human islets than in INS-1 832/13 cells.Functions associated with early stimulus-secretion coupling, with the exception of plasma membrane potential and Ca2+ oscillations, were similar in the two cell lines; insulin secretion, respiration and metabolite responses were similar in EndoC-βH1 cells and human islets. While both cell lines are suitable in vitro models, with the caveat of replicating key findings in isolated islets, EndoC-βH1 cells have the

  16. Influence of ketamine on amino acid neurotransmitters secretion by nerve cells in vitro

    Directory of Open Access Journals (Sweden)

    Mingxian Shi

    2016-04-01

    Full Text Available In order to study the influence of amino acid neurotransmitters secreted by the nerve cells after ketamine treatment, the nerve cells were cultured in vitro to exclude the interference of other factors in vivo and treated with three different doses of ketamine (1, 3 and 5 µg/mL. Then, the concentration of neuronal amino acid neurotransmitters was examined at 0, 15, 30, 45, 60, 90, 120 min after treatment. The trends of each amino acid concentration after ketamine treatment were nearly the same among the different treatment doses. After 15 min of adapting time, ketamine decreased the excitatory amino acid glutamic acid and aspartic acid concentration, and increased the concentration of the inhibitory amino acid glycine. Their concentrations showed a tendency to return approximately to the original level after 120 min.

  17. 34. Effect and the Possible Mediated Pathway of Cortisol Secretion in Adrenocortical Cells Induced by Lead and Cadmium in Vitro

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective: To understand the direct effect on the secretion of adreno-cortical cells induced by lead and cadmium and the possible mediated pathway. Methods: The adrenocortical cells of male guinea pigs were dispersed and primarily cultured, then the cells were incubated wich cadmiun chloride and lead acetate in dosage as 0,6.25, 12.5, 25, 50, 100 μmol/L respectively for different periods (30, 60, 120 and 240 minutes). The cortisol levels in culture medium and cellular cAMP concentration were measured with RIA. Results: Under the existence of ACTH, the levels of cortisol secreted from the cultured cells were showed significantly declined in dose-dependent manner when the cells were treated in 6.25-100μmol/L CdCl2 for 30 to 240 minutes. There would be an interaction for cortisol secretion between the dose of CdCl2 and the incubatal period. Nevertheless, it seemed to have no obvious linear relation in the alterations of cortisol secretion after 12.5~100μmol/L PbAc incubated for 30~240 minutes. It appeared to have a tendency of dual-phase response in a manner of inhibiting the cortisol secretion in low dose (lower than 25μmol/L) and stimulating the secretion function in high dose (50 and 100μmol/L). The cAMP level was presented a remarkably decrease after 6.25~100 μmol/L CdCl2 incubated with the cells. It was proved that the cAMP level had does-effect relations with the CdCl2 dose. PbAc appeared not only dual response with the tendency of cAMP inhibition in low dose and activating to raise in high dose but also dose-effect relationship. Conclusion: CdCl2 could directly inhibit the secretion of cortisol. PbAc is also of the toxic effect on the cortisol secretion with the characteristic of dual-response as inhibition in early phase and low dose while induction to raising in high dose. cAMP, as an important second messenger, play a role in synthesis and secretion of adrenocorticoids. The toxic effects on steroids secretion induced by cadmium and lead were

  18. Antigen presentation by small intestinal epithelial cells uniquely enhances IFN-γ secretion from CD4{sup +} intestinal intraepithelial lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Hatano, Ryo; Yamada, Kiyoshi; Iwamoto, Taku; Maeda, Nana; Emoto, Tetsuro; Shimizu, Makoto; Totsuka, Mamoru, E-mail: atotuka@mail.ecc.u-tokyo.ac.jp

    2013-06-14

    Highlights: •Small intestinal epithelial cells (sIECs). •sIECs are able to induce antigen specific proliferation of CD4{sup +} IELs. •sIECs induce markedly enhanced IFN-γ secretion by CD4{sup +} IELs. •Induction of enhanced IFN-γ secretion by sIECs is uniquely observed in CD4{sup +} IELs. -- Abstract: Small intestinal epithelial cells (sIECs) express major histocompatibility complex class II molecules even in a normal condition, and are known to function as antigen presenting cells (APCs) at least in vitro. These findings raised the possibility that sIECs play an important role in inducing immune responses against luminal antigens, especially those of intestinal intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs). We herein showed that antigenic stimulation with sIECs induced markedly greater secretion of interferon-gamma (IFN-γ) by CD4{sup +} IELs, but not interleukin (IL)-4, IL-10 and IL-17 although the proliferative response was prominently lower than that with T cell-depleted splenic APCs. In contrast, no enhanced IFN-γ secretion by CD4{sup +} LPLs and primed splenic CD4{sup +} T cells was observed when stimulated with sIECs. Taken together, these results suggest that sIECs uniquely activate CD4{sup +} IELs and induce remarkable IFN-γ secretion upon antigenic stimulation in vivo.

  19. GDNF secreting human neural progenitor cells protect dying motor neurons, but not their projection to muscle, in a rat model of familial ALS.

    Directory of Open Access Journals (Sweden)

    Masatoshi Suzuki

    Full Text Available BACKGROUND: Amyotrophic lateral sclerosis (ALS is a fatal, progressive neurodegenerative disease characterized by rapid loss of muscle control and eventual paralysis due to the death of large motor neurons in the brain and spinal cord. Growth factors such as glial cell line derived neurotrophic factor (GDNF are known to protect motor neurons from damage in a range of models. However, penetrance through the blood brain barrier and delivery to the spinal cord remains a serious challenge. Although there may be a primary dysfunction in the motor neuron itself, there is also increasing evidence that excitotoxicity due to glial dysfunction plays a crucial role in disease progression. Clearly it would be of great interest if wild type glial cells could ameliorate motor neuron loss in these models, perhaps in combination with the release of growth factors such as GDNF. METHODOLOGY/PRINCIPAL FINDINGS: Human neural progenitor cells can be expanded in culture for long periods and survive transplantation into the adult rodent central nervous system, in some cases making large numbers of GFAP positive astrocytes. They can also be genetically modified to release GDNF (hNPC(GDNF and thus act as long-term 'mini pumps' in specific regions of the rodent and primate brain. In the current study we genetically modified human neural stem cells to release GDNF and transplanted them into the spinal cord of rats over-expressing mutant SOD1 (SOD1(G93A. Following unilateral transplantation into the spinal cord of SOD1(G93A rats there was robust cellular migration into degenerating areas, efficient delivery of GDNF and remarkable preservation of motor neurons at early and end stages of the disease within chimeric regions. The progenitors retained immature markers, and those not secreting GDNF had no effect on motor neuron survival. Interestingly, this robust motor neuron survival was not accompanied by continued innervation of muscle end plates and thus resulted in no

  20. Erwinia amylovora type three-secreted proteins trigger cell death and defense responses in Arabidopsis thaliana.

    Science.gov (United States)

    Degrave, A; Fagard, M; Perino, C; Brisset, M N; Gaubert, S; Laroche, S; Patrit, O; Barny, M-A

    2008-08-01

    Erwinia amylovora is the bacterium responsible for fire blight, a necrotic disease affecting plants of the rosaceous family. E. amylovora pathogenicity requires a functional type three secretion system (T3SS). We show here that E. amylovora triggers a T3SS-dependent cell death on Arabidopsis thaliana. The plants respond by inducing T3SS-dependent defense responses, including salicylic acid (SA)-independent callose deposition, activation of the SA defense pathway, reactive oxygen species (ROS) accumulation, and part of the jasmonic acid/ethylene defense pathway. Several of these reactions are similar to what is observed in host plants. We show that the cell death triggered by E. amylovora on A. thaliana could not be simply explained by the recognition of AvrRpt2 ea by the resistance gene product RPS2. We then analyzed the role of type three-secreted proteins (T3SPs) DspA/E, HrpN, and HrpW in the induction of cell death and defense reactions in A. thaliana following infection with the corresponding E. amylovora mutant strains. HrpN and DspA/E were found to play an important role in the induction of cell death, activation of defense pathways, and ROS accumulation. None of the T3SPs tested played a major role in the induction of SA-independent callose deposition. The relative importance of T3SPs in A. thaliana is correlated with their relative importance in the disease process on host plants, indicating that A. thaliana can be used as a model to study their role.

  1. Intracellular Ca2+ signals in human-derived pancreatic somatostatin-secreting cells (QGP-1N).

    Science.gov (United States)

    Squires, P E; Amiranoff, B; Dunne, M J

    1994-10-01

    Single-cell microfluorimetry techniques have been used to examine the effects of acetylcholine (0.1-100 microM) on the intracellular free calcium ion concentration ([Ca2+]i) in a human-derived pancreatic somatostatin-secreting cell line, QGP-1N. When applied to the bath solution, acetylcholine was found to evoke a marked and rapid increase in [Ca2+]i at all concentrations tested. These responses were either sustained, or associated with the generation of complex patterns of [Ca2+]i transients. Overall, the pattern of response was concentration related. In general, 0.1-10 microM acetylcholine initiated a series of repetitive oscillations in cytoplasmic Ca2+, whilst at higher concentrations the responses consisted of a rapid rise in [Ca2+]i followed by a smaller more sustained increase. Without external Ca2+, 100 microM acetylcholine caused only a transient rise in [Ca2+]i, whereas lower concentrations of the agonist were able to initiate, but not maintain, [Ca2+]i oscillations. Acetylcholine-evoked Ca2+ signals were abolished by atropine (1-10 microM), verapamil (100 microM) and caffeine (20 mM). Nifedipine failed to have any significant effect upon agonist-evoked increases in [Ca2+]i, whilst 50 mM KCl, used to depolarise the cell membrane, only elicited a transient increase in [Ca2+]i. Ryanodine (50-500 nM) and caffeine (1-20 mM) did not increase basal Ca2+ levels, but the Ca(2+)-ATPase inhibitors 2,5-di(tert-butyl)-hydroquinone (TBQ) and thapsigargin both elevated [Ca2+]i levels. These data demonstrate for the first time cytosolic Ca2+ signals in single isolated somatostatin-secreting cells of the pancreas. We have demonstrated that acetylcholine will evoke both Ca2+ influx and Ca2+ mobilisation, and we have partially addressed the subcellular mechanism responsible for these events.

  2. Relationships among CFTR expression, HCO3- secretion, and host defense may inform gene- and cell-based cystic fibrosis therapies.

    Science.gov (United States)

    Shah, Viral S; Ernst, Sarah; Tang, Xiao Xiao; Karp, Philip H; Parker, Connor P; Ostedgaard, Lynda S; Welsh, Michael J

    2016-05-10

    Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. Airway disease is the major source of morbidity and mortality. Successful implementation of gene- and cell-based therapies for CF airway disease requires knowledge of relationships among percentages of targeted cells, levels of CFTR expression, correction of electrolyte transport, and rescue of host defense defects. Previous studies suggested that, when ∼10-50% of airway epithelial cells expressed CFTR, they generated nearly wild-type levels of Cl(-) secretion; overexpressing CFTR offered no advantage compared with endogenous expression levels. However, recent discoveries focused attention on CFTR-mediated HCO3 (-) secretion and airway surface liquid (ASL) pH as critical for host defense and CF pathogenesis. Therefore, we generated porcine airway epithelia with varying ratios of CF and wild-type cells. Epithelia with a 50:50 mix secreted HCO3 (-) at half the rate of wild-type epithelia. Likewise, heterozygous epithelia (CFTR(+/-) or CFTR(+/∆F508)) expressed CFTR and secreted HCO3 (-) at ∼50% of wild-type values. ASL pH, antimicrobial activity, and viscosity showed similar relationships to the amount of CFTR. Overexpressing CFTR increased HCO3 (-) secretion to rates greater than wild type, but ASL pH did not exceed wild-type values. Thus, in contrast to Cl(-) secretion, the amount of CFTR is rate-limiting for HCO3 (-) secretion and for correcting host defense abnormalities. In addition, overexpressing CFTR might produce a greater benefit than expressing CFTR at wild-type levels when targeting small fractions of cells. These findings may also explain the risk of airway disease in CF carriers.

  3. Brain-derived neurotrophic factor in neuroimmunology: lessons learned from multiple sclerosis patients and experimental autoimmune encephalomyelitis models.

    Science.gov (United States)

    Lühder, Fred; Gold, Ralf; Flügel, Alexander; Linker, Ralf A

    2013-04-01

    The concept of neuroprotective autoimmunity implies that immune cells, especially autoantigen-specific T cells, infiltrate the central nervous system (CNS) after injury and contribute to neuroregeneration and repair by secreting soluble factors. Amongst others, neurotrophic factors and neurotrophins such as brain-derived neurotropic factor (BDNF) are considered to play an important role in this process. New data raise the possibility that this concept could also be extended to neuroinflammatory diseases such as multiple sclerosis (MS) where autoantigen-specific T cells infiltrate the CNS, causing axonal/neuronal damage on the one hand, but also providing neuroprotective support on the other hand. In this review, we summarize the current knowledge on BDNF levels analyzed in MS patients in different compartments and its correlation with clinical parameters. Furthermore, new approaches in experimental animal models are discussed that attempt to decipher the functional relevance of BDNF in autoimmune demyelination.

  4. Measuring phospholipase D activity in insulin-secreting pancreatic beta-cells and insulin-responsive muscle cells and adipocytes.

    Science.gov (United States)

    Cazzolli, Rosanna; Huang, Ping; Teng, Shuzhi; Hughes, William E

    2009-01-01

    Phospholipase D (PLD) is an enzyme producing phosphatidic acid and choline through hydrolysis of phosphatidylcholine. The enzyme has been identified as a member of a