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Cell Survival (cell + survival)
Kinds of Cell Survival Terms modified by Cell Survival Selected AbstractsOptimization of Autologous Muscle Stem Cell Survival in the Denervated Hemilarynx,THE LARYNGOSCOPE, Issue 7 2008Stacey L. Halum MD Abstract Objective: Current treatments for vocal fold paralysis are suboptimal in that they fail to restore dynamic function. Autologous muscle stem cell (MSC) therapy is a promising potential therapy for vocal fold paralysis in that it can attenuate denervation-induced muscle atrophy and provide a vehicle for delivery of neurotrophic factors, thereby potentially selectively guiding reinnervation. The goal of this project was to characterize optimal conditions for injected autologous MSC survival in the thyroarytenoid (TA) muscle following recurrent laryngeal nerve (RLN) injury by local administration of adjuvant factors. Study Design: Animal experiment. Methods: Unilateral RLN transection and sternocleidomastoid muscle (,1 g) biopsies were performed in 20 male Wistar rats. One month later, 106 autologous MSCs labeled via retroviral-enhanced green fluorescent protein (EGFP) transduction were injected into the denervated hemilarynx of each animal with one of four adjuvant therapies: cardiotoxin [(CTX) 10,5 M], insulin-like growth factor-1 [(IGF- 1) 100 ,g/mL], ciliary neurotrophic factor [(CNTF) 50 ,g/mL], or saline. Animals were euthanized 1 month later and larynges harvested, sectioned, and analyzed for MSC survival. Results: All specimens demonstrate extensive MSC survival, with fusion of the MSCs with the denervated myofibers. Based on mean fluorescent intensity of the laryngeal specimens, IGF-1 and CNTF had the greatest positive influence on MSC survival. Myofiber diameters demonstrated myofiber atrophy to be inversely related to MSC survival, with the least atrophy in the groups having the greatest MSC survival. Conclusions: Autologous MSC therapy may be a future treatment for vocal fold paralysis. These findings support a model whereby MSCs genetically engineered to secrete CNTF and/or IGF-1 may not only promote neural regeneration, but also enhance MSC survival in an autocrine fashion. [source] Cell survival and apoptosis-related molecules in cancer cells in effusions: A comprehensive reviewDIAGNOSTIC CYTOPATHOLOGY, Issue 8 2009Lilach Kleinberg Ph.D. Abstract Spreading of cancer cells to effusions is a manifestation of advanced disease, for which the chances of achieving cure using conventional treatment are low. This emphasizes both the importance of improving early detection and the need for developing targeted therapy modes. Such approaches should be based on characterization of the antiapoptotic, survival and drug resistance mechanisms of the metastatic cells in addition to analysis of the primary tumor. This review presents current knowledge regarding the expression and clinical role of cell survival and apoptosis-related molecules in nonhematological cancers in effusions. Differences in the anatomic site-related expression and clinical role of these proteins are additionally discussed. The data presented highlight the complexity of the multiple molecular pathways that mediate tumor cell survival within the serosal cavities. Diagn. Cytopathol. 2009. © 2009 Wiley-Liss, Inc. [source] P2Y1 receptor signaling enhances neuroprotection by astrocytes against oxidative stress via IL-6 release in hippocampal culturesGLIA, Issue 3 2009Takumi Fujita Abstract Cell survival is a critical issue in the onset and progression of neurodegenerative diseases and following pathological events including ischemia and traumatic brain injury. Oxidative stress is the main cause of cell damage in such pathological conditions. Here, we report that adenosine 5,-triphosphate (ATP) protects hippocampal astrocytes from hydrogen peroxide (H2O2)-evoked oxidative injury in astrocyte monocultures. The effect of ATP was prevented by a selective antagonist of or siRNAs against P2Y1R. Interestingly, in astrocyte-neuron cocultures, ATP also produced neuroprotective effects against H2O2 -evoked neuronal cell death, whereas ATP did not produce any neuroprotective effects in monocultures. The ATP-induced neuroprotection in cocultures was completely inhibited by silencing of astrocytic P2Y1R expression, indicating that ATP acts on astrocytes and enhances their neuroprotective functions by activating P2Y1R. Furthermore, this neuroprotective effect was mimicked by applying conditioned medium from astrocytes that had been stimulated by ATP, implying an involvement of diffusible factors from astrocytes. We found that, in both purified astrocyte cultures and astrocyte-neuronal cocultures, ATP and the P2Y1R agonist 2-methylthioadenosine 5, diphosphate (2MeSADP) induced the release of interleukin-6 (IL-6), but this did not occur in neuron monocultures. Moreover, exogenous IL-6 produced a neuroprotective effect, and the neuroprotection induced by P2Y1R-stimulated astrocytes was prevented in the presence of an anti-IL-6 antibody. Taken together, these results suggest that P2Y1R-stimulated astrocytes protect against neuronal damage induced by oxidative stress, and that IL-6 is a crucial signaling molecule released from astrocytes. Thus, activation of P2Y1R in astrocytes may rescue neurons from secondary cell death under pathological conditions. © 2008 Wiley-Liss, Inc. [source] Alcohol inhibition of neurogenesis: A mechanism of hippocampal neurodegeneration in an adolescent alcohol abuse modelHIPPOCAMPUS, Issue 5 2010Stephanie A. Morris Abstract Adolescents diagnosed with an alcohol use disorder show neurodegeneration in the hippocampus, a region important for learning, memory, and mood regulation. This study examines a potential mechanism by which excessive alcohol intake, characteristic of an alcohol use disorder, produces neurodegeneration. As hippocampal neural stem cells underlie ongoing neurogenesis, a phenomenon that contributes to hippocampal structure and function, we investigated aspects of cell death and cell birth in an adolescent rat model of an alcohol use disorder. Immunohistochemistry of various markers along with Bromo-deoxy-Uridine (BrdU) injections were used to examine different aspects of neurogenesis. After 4 days of binge alcohol exposure, neurogenesis was decreased by 33 and 28% at 0 and 2 days after the last dose according to doublecortin expression. To determine whether this decrease in neurogenesis was due to effects on neural stem cell proliferation, quantification of BrdU-labeled cells revealed a 21% decrease in the dentate gyrus of alcohol-exposed brains. Cell survival and phenotype of BrdU-labeled cells were assessed 28 days after alcohol exposure and revealed a significant, 50% decrease in the number of surviving cells in the alcohol-exposed group. Reduced survival was supported by significant increases in the number of pyknotic-, FluoroJade B positive-, and TUNEL-positive cells. However, so few cells were TUNEL-positive that cell death is likely necrotic in this model. Although alcohol decreased the number of newborn cells, it did not affect the percentage of cells that matured into neurons (differentiation). Thus, our data support that in a model of an adolescent alcohol use disorder, neurogenesis is impaired by two mechanisms: alcohol-inhibition of neural stem cell proliferation and alcohol effects on new cell survival. Remarkably, alcohol inhibition of neurogenesis may outweigh the few dying cells per section, which implies that alcohol inhibition of neurogenesis contributes to hippocampal neurodegeneration in alcohol use disorders. © 2009 Wiley-Liss, Inc. [source] Role of the C5b-9 complement complex in cell cycle and apoptosisIMMUNOLOGICAL REVIEWS, Issue 1 2001Horea G. Rus Summary: Assembly of C5b-9 on cell membranes results in transmembrane channels and causes cell death. When the number of C5b-9 molecules is limited, nucleated cells are able to escape cell death by endocytosis and by shedding of membranes bearing C5b-9. Sublytic C5b-9 induces proto-oncogenes, activates the cell cycle, and enhances cell survival. In addition, C5b-9 reverses the differentiated phenotype of post-mitotic cells, such as oligodendrocytes and skeletal muscle cells. The signal transduction pathways responsible for cell cycle activation by C5b-9 include Gi-mediated activation of extracellular signal-regulated kinase 1 and phosphatidylinositol 3-kinase (PI3-K). Cell survival enhanced by C5b-9 is mediated by the PI3-K/Akt pathway, which inhibits apoptosis through regulation of BAD. These findings indicate that complement activation and membrane assembly of sublytic C5b-9 play an important role in inflammation by promoting cell proliferation and by rescuing apoptotic cells. This work was supported by NIH grants NS36231 and NS15662 and by multiple sclerosis pilot award PP-696. [source] Dexmedetomidine provides cortical neuroprotection: impact on anaesthetic-induced neuroapoptosis in the rat developing brainACTA ANAESTHESIOLOGICA SCANDINAVICA, Issue 6 2010R. D. SANDERS Background: Recent evidence has demonstrated the anti-apoptotic of dexmedetomidine in different brain injury models. Herein, we investigated whether dexmedetomidine could directly protect against cortical injury in vitro and in vivo. Methods: Apoptosis was induced by staurosporine or wortmannin treatment in cortical neuronal cultures in vitro or by 6 h of isoflurane (0.75%) administration to post-natal day 7 rat pups in vivo. Dexmedetomidine was then applied in escalating doses to assess the neuroprotective potential of this agent. Cell survival was quantified using an MTT assay in vitro and in vivo apoptosis was assessed using cleaved caspase-3 immunohistochemistry. Cortical Western blots were conducted for the cellular survival proteins Bcl-2 and phosphorylated extracellular signal-regulated protein kinase (pERK)1 and 2. Results: In vitro dexmedetomidine dose-dependently prevented both staurosporine- and wortmannin-induced injury in cortical neuronal cultures, indicating that dexmedetomidine can prevent apoptosis when applied directly. In vivo isoflurane induced cortical neuroapoptosis compared with air (327±80 vs. 34±9 caspase-3-positive neurons; P<0.05). Dexmedetomidine inhibited isoflurane-induced caspase-3 expression (P<0.05), although the protection achieved did not completely attenuate the isoflurane injury (P<0.05 vs. air). Isoflurane treatment decreased Bcl-2 and pERK protein expression relative to air, an effect reversed by dexmedetomidine treatment. Conclusions: Dexmedetomidine prevents cortical apoptosis in vitro and in vivo. However, using higher doses of dexmedetomidine does not further increase protection against isoflurane injury in the cortex than previously observed. [source] Cigarette smoke extract affects functional activity of MRP1 in bronchial epithelial cellsJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 5 2007Margaretha van der Deen Abstract Cigarette smoke is the principal risk factor for development of chronic obstructive pulmonary disease (COPD). Multidrug resistance-associated protein 1 (MRP1) is a member of the ATP-binding cassette (ABC) superfamily of transporters, which transport physiologic and toxic substrates across cell membranes. MRP1 is highly expressed in lung epithelium. This study aims to analyze the effect of cigarette smoke extract (CSE) on MRP1 activity. In the human bronchial epithelial cell line 16HBE14o,, MRP1 function was studied flow cytometrically by cellular retention of carboxyfluorescein (CF) after CSE incubation and MRP1 downregulation by RNA interference (siRNA). Cell survival was measured by the MTT assay. Immunocytochemically, it was shown that 16HBE14o, expressed MRP1 and breast cancer resistance protein. Coincubation of CSE IC50 (1.53% ± 0.22%) with MK571 further decreased cell survival 31% (p, = 0.018). CSE increased cellular CF retention dose dependently from 1.7-fold at 5% CSE to 10.3-fold at 40% CSE (both p < 0.05). siRNA reduced MRP1 RNA expression with 49% and increased CF accumulation 67% versus control transfected cells. CSE exposure further increased CF retention 24% (p = 0.031). A linear positive relation between MRP1 function and CSE-modulating effects (r = 0.99, p =0.089) was shown in untransfected, control transfected, and MRP1 downregulated 16HBE14o, cells analogous to blocking effects with MRP1 inhibitor MK571 (r = 0.99, p = 0.034). In conclusion, cigarette smoke extract affects MRP1 activity probably competitively in bronchial epithelial cells. Inhibition of MRP1 in turn results in higher CSE toxicity. We propose that MRP1 may be a protective protein for COPD development. © 2007 Wiley Periodicals, Inc. J Biochem Mol Toxicol 21:243,251, 2007; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20187 [source] Bcl-2 overexpression in hepatic stellate cell line CFSC-2G, induces a pro-fibrotic stateJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 7 2010Viridiana Y González-Puertos Abstract Background and Aim:, Development of hepatic fibrosis is a complex process that involves oxidative stress (OS) and an altered balance between pro- and anti-apoptotic molecules. Since Bcl-2 overexpression preserves viability against OS, our objective was to address the effect of Bcl-2 overexpression in the hepatic stellate cells (HSC) cell-line CFSC-2G under acetaldehyde and H2O2 challenge, and explore if it protects these cells against OS, induces replicative senescence and/or modify extracellular matrix (ECM) remodeling potential. Methods:, To induce Bcl-2 overexpression, HSC cell line CFSC-2G was transfected by lipofection technique. Green fluorescent protein-only CFSC-2G cells were used as a control. Cell survival after H2O2 treatment and total protein oxidation were assessed. To determine cell cycle arrest, proliferation-rate, DNA synthesis and senescence were assessed. Matrix metalloproteinases (MMP), tissue-inhibitor of MMP (TIMP), transglutaminases (TG) and smooth muscle a-actin (,-SMA) were evaluated by western blot in response to acetaldehyde treatment as markers of ECM remodeling capacity in addition to transforming growth factor-, (TGF-,) mRNA. Results:, Cells overexpressing Bcl-2 survived , 20% more than control cells when exposed to H2O2 and , 35% proteins were protected from oxidation, but Bcl-2 did not slow proliferation or induced senescence. Bcl-2 overexpression did not change ,-SMA levels, but it increased TIMP-1 (55%), tissue transglutaminases (tTG) (25%) and TGF-, mRNA (49%), when exposed to acetaldehyde, while MMP-13 content decreased (47%). Conclusions:, Bcl-2 overexpression protected HSC against oxidative stress but it did not induce replicative senescence. It increased TIMP-1, tTG and TGF-, mRNA levels and decreased MMP-13 content, suggesting that Bcl-2 overexpression may play a key role in the progression of fibrosis in chronic liver diseases. [source] Targeting of cell survival genes using small interfering RNAs (siRNAs) enhances radiosensitivity of Grade II chondrosarcoma cellsJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 6 2007Dae Won Kim Abstract The main treatment for chondrosarcoma is surgical resection with a wide margin. However, there are certain chondrosarcomas, such as those found in the pelvis and the spine, which cannot be resected adequately with surgery alone. Unfortunately, most chondrosarcomas are resistant to radiation and chemotherapy. Radiation and chemotherapy are thought to kill chondrosarcoma cells by inducing apoptosis, or programmed cell death. In this article, we hypothesize that antiapoptotic gene silencing enhances radiosensitivity in chondrosarcoma cells by facilitating apoptotic pathways. We knocked down antiapoptotic genes in chondrosarcoma cells using small interfering RNAs (siRNAs). Two well-established Grade II human chondrosarcoma cell lines were pretreated with siRNAs that specifically target mRNAs for Bcl-2, Bcl-xL, or XIAP. The cells were then treated with radiation. Cell death was assessed by flow cytometry. Cell survival and proliferation were measured by clonogenic survival assays. Chondrosarcoma cells exhibited radioresistance and increased the expression of Bcl-2, Bcl-xL, and XIAP in response to radiation. When one of the Bcl-2, Bcl-xL, or XIAP genes was silenced with the corresponding siRNA, radiosensitivity increased up to 9.2-fold (p,<,0.05). When two out of the three antiapoptotic mRNAs were knocked down simultaneously, there was an 11.3-fold increase in cell death after radiation (p,<,0.05). Our findings support a novel therapeutic concept that gene silencing may be used as a molecular adjuvant therapy for radioresistant sarcomas. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 25: 820,828, 2007 [source] Photobleaching of Melanosomes from Retinal Pigment Epithelium: II.PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2007Effects on the Response of Living Cells to Photic Stress Melanosomes of the retinal pigment epithelium (RPE) are long lived organelles that may undergo photobleaching with aging, which can diminish the antioxidant efficiency of melanin. Here, isolated porcine RPE melanosomes were experimentally photobleached with visible light to simulate aging and compared with untreated granules or control particles (black latex beads) for their effects on the survival of photically stressed ARPE-19 cultures. Particles were delivered to cultures for uptake by phagocytosis then cells were exposed to violet light and analyzed by a new live cell imaging method to identify the time of apoptotic blebbing as a dynamic measure of reduced cell survival. Results indicated that untreated melanosomes did not decrease photic injury to ARPE-19 cells when compared with cells lacking particles or with cells containing control particles, as might be expected if melanin performed an antioxidant function. Instead cells with untreated melanosomes showed reduced survival indicated by an earlier onset of blebbing and a lower fraction of surviving cells after photic stress. Cell survival was reduced even further in stressed cells containing melanosomes that were photobleached, and survival decreased with increasing photobleaching time. Photobleaching of RPE melanosomes therefore makes cells containing them more sensitive to light-induced cytotoxicity. This observation raises the possibility that aged melanosomes increase RPE cell photic stress in situ, perhaps contributing to reduced tissue function and to degeneration of the adjacent retina that the RPE supports. How melanosomes (photobleached or not) interact with their local subcellular environment to modify RPE cell survival is poorly understood and is likely determined by the physicochemical state of the granule and its constituent melanin. The live cell imaging method introduced here, which permitted detection of a graded effect of photobleaching, provides a sensitive bioassay for probing the effects of melanosome modifications. [source] Devil inside: does plant programmed cell death involve the endomembrane system?PLANT CELL & ENVIRONMENT, Issue 9 2010JEAN-LUC CACAS ABSTRACT Eukaryotic cells have to constantly cope with environmental cues and integrate developmental signals. Cell survival or death is the only possible outcome. In the field of animal biology, tremendous efforts have been put into the understanding of mechanisms underlying cell fate decision. Distinct organelles have been proven to sense a broad range of stimuli and, if necessary, engage cell death signalling pathway(s). Over the years, forward and reverse genetic screens have uncovered numerous regulators of programmed cell death (PCD) in plants. However, to date, molecular networks are far from being deciphered and, apart from the autophagic compartment, no organelles have been assigned a clear role in the regulation of cellular suicide. The endomembrane system (ES) seems, nevertheless, to harbour a significant number of cell death mediators. In this review, the involvement of this system in the control of plant PCD is discussed in-depth, as well as compared and contrasted with what is known in animal and yeast systems. [source] Prostaglandin E2 and its cognate EP receptors control human adult articular cartilage homeostasis and are linked to the pathophysiology of osteoarthritisARTHRITIS & RHEUMATISM, Issue 2 2009Xin Li Objective To elucidate the pathophysiologic links between prostaglandin E2 (PGE2) and osteoarthritis (OA) by characterizing the catabolic effects of PGE2 and its unique receptors in human adult articular chondrocytes. Methods Human adult articular chondrocytes were cultured in monolayer or alginate beads with and without PGE2 and/or agonists of EP receptors, antagonists of EP receptors, and cytokines. Cell survival, proliferation, and total proteoglycan synthesis and accumulation were measured in alginate beads. Chondrocyte-related gene expression and phosphatidylinositol 3-kinase/Akt signaling were assessed by real-time reverse transcription,polymerase chain reaction and Western blotting, respectively, using a monolayer cell culture model. Results Stimulation of human articular chondrocytes with PGE2 through the EP2 receptor suppressed proteoglycan accumulation and synthesis, suppressed aggrecan gene expression, did not appreciably affect expression of matrix-degrading enzymes, and decreased the type II collagen:type I collagen ratio. EP2 and EP4 receptors were expressed at higher levels in knee cartilage than in ankle cartilage and in a grade-dependent manner. PGE2 titration combined with interleukin-1 (IL-1) synergistically accelerated expression of pain-associated molecules such as inducible nitric oxide synthase and IL-6. Finally, stimulation with exogenous PGE2 or an EP2 receptor,specific agonist inhibited activation of Akt that was induced by insulin-like growth factor 1. Conclusion PGE2 exerts an antianabolic effect on human adult articular cartilage in vitro, and EP2 and EP4 receptor antagonists may represent effective therapeutic agents for the treatment of OA. [source] Valproate activates the Notch3/c-FLIP signaling cascade: a strategy to attenuate white matter hyperintensities in bipolar disorder in late life?BIPOLAR DISORDERS, Issue 3 2009Peixiong Yuan Objectives:, Increased prevalence of deep white matter hyperintensities (DWMHs) has been consistently observed in patients with geriatric depression and bipolar disorder. DMWHs are associated with chronicity, disability, and poor quality of life. They are thought to be ischemic in their etiology and may be related to the underlying pathophysiology of mood disorders in the elderly. Notably, these lesions strikingly resemble radiological findings related to the cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephelopathy (CADASIL) syndrome. CADASIL arises from mutations in Notch3, resulting in impaired signaling via cellular Fas-associated death domain-like interleukin-1-beta-converting enzyme-inhibitory protein (c-FLIP) through an extracellular signal-regulated kinase (ERK)-dependent pathway. These signaling abnormalities have been postulated to underlie the progressive degeneration of vascular smooth muscle cells (VSMC). This study investigates the possibility that the anticonvulsant valproate (VPA), which robustly activates the ERK mitogen-activated protein kinase (MAPK) cascade, may exert cytoprotective effects on VSMC through the Notch3/c-FLIP pathway. Methods:, Human VSMC were treated with therapeutic concentrations of VPA subchronically. c-FLIP was knocked down via small interfering ribonucleic acid transfection. Cell survival, apoptosis, and protein levels were measured. Results:, VPA increased c-FLIP levels dose- and time-dependently and promoted VSMC survival in response to Fas ligand-induced apoptosis in VSMC. The anti-apoptotic effect of VPA was abolished by c-FLIP knockdown. VPA also produced similar in vivo effects in rat brain. Conclusions:, These results raise the intriguing possibility that VPA may be a novel therapeutic agent for the treatment of CADASIL and related disorders. They also suggest that VPA might decrease the liability of patients with late-life mood disorders to develop DWMHs. [source] NADPH oxidase produces reactive oxygen species and maintains survival of rat astrocytesCELL BIOCHEMISTRY AND FUNCTION, Issue 2 2005Qing Liu Abstract Reactive oxygen species (ROS) produced by activated astrocytes have been considered to be involved in the pathogenesis of neurodegenerative diseases, while NADPH oxidase is an essential enzyme involved in ROS-mediated signal transduction. The goal of the present study was to determine whether NADPH oxidase plays a role in ROS generation and cell survival in rat astrocytes. We found that the release of ROS in rat astrocytes was significantly increased by stimulation with calcium ionophore or opsonized zymosan, which are known to trigger a respiration burst in phagocytes by the NADPH oxidase pathway. Further study indicated that diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, significantly suppressed the increase of ROS release caused by the calcium ionophore or opsonized zymosan. Cell survival assay and fluorescence double dyeing with acridine orange and ethidium bromide showed that DPI dose- and time-dependently decreased the viability of normal astrocytes, whereas exogenous supplementation of H2O2 can reverse the survival of DPI-treated astrocytes. For the first time, our results suggest that NADPH oxidase is an important enzyme for the generation of ROS in astrocytes, and the ROS generated by NADPH oxidase play an essential role in astrocyte survival. Copyright © 2004 John Wiley & Sons, Ltd. [source] Is neuroglobin (Ngb) a perspective for glaucoma?ACTA OPHTHALMOLOGICA, Issue 2009Y YOU Purpose To further explore the mechanisms of Ngb function in the retina, and investigate the possible neuroprotective potency of this O2-binding protein in glaucoma. Our project will potentially open exciting perspectives for the treatment and prognosis of the blinding condition of glaucoma. Methods 1) In vitro: We will set up cultures for primary RGCs. The cultured RGCs will be subjected to hypoxia by incubation in a controlled-atmosphere incubator. Cell survival and apoptosis will be assessed using MTT method and TUNEL staining. Ngb expression in normoxic and hypoxic cells will be compared. Additionally, the RGCs will be transfected with plasmids expressing Ngb mRNA to evaluate the neuroprotective effect of Ngb. 2) In vivo: The ischemia/reperfusion model and the staurosporine intravitreal injection model cause acute RGC loss. We will also use a laser model to induce chronic elevated IOP by obstructing the aqueous outflow pathway. We will first analyze upregulated Ngb expression in these models. The localization of both Ngb mRNA and protein will be assessed. Ngb-Tg mice will be subjected to the glaucoma models. We will measure cell density or retinal thickness and perform electroretinography to evaluate the physiological function of retina. We will also analyse the possible contribution of oxidative stress via immunostainings for ROS and determine possible effects on NO scavenging by evaluating the expression of NOS. Furthermore, we will transfect RGCs in vivo using intravitreal injections of a Ngb expressing adeno-associated virus (AAV) to test the therapeutic potential of Ngb. Conclusion Our project will not only provide us with an improved understanding of neuroglobin, but also open a new perspective for the treatment of retinal ganglion cell apoptosis and glaucomatous neuropathy. [source] Cytotoxicity of ophthalmic solutions with and without preservatives to human corneal endothelial cells, epithelial cells and conjunctival epithelial cellsCLINICAL & EXPERIMENTAL OPHTHALMOLOGY, Issue 6 2008Masahiko Ayaki MD Abstract Purpose:, The cytotoxicity of a range of commercial ophthalmic solutions in the presence and absence of preservatives was assessed in human corneal endothelial cells (HCECs), corneal epithelia and conjunctival epithelia using in vitro techniques. Methods:, Cell survival was measured using the WST-1 assay for endothelial cells and the MTT assay for epithelial cells. Commercially available timolol, carteolol, cromoglicate, diclofenac, bromfenac and hyaluronic acid ophthalmic solutions were assessed for cytotoxicity in the presence and absence of preservatives. The preservatives benzalkonium, chlorobutanol and polysorbate were also tested. The survival of cells exposed to test ophthalmic solutions was expressed as a percentage of cell survival in the control solution (distilled water added to media) after 48 h exposure. Results:, HCEC survival was 20,30% in ophthalmic solutions diluted 10-fold. The survival of HCEC was significantly greater in all solutions in the absence of preservative than in the presence of preservative. The survival of corneal and conjunctival epithelia was consistent with that of HCECs for all test ophthalmic solutions. The preservatives polysorbate and benzalkonium were highly cytotoxic with cell survival decreasing to 20% at the concentration estimated in commercial ophthalmic solutions. By comparison, the survival of cells exposed to chlorobutanol was 80% or greater. Conclusions:, The cytotoxicity of ophthalmic solutions to HCEC, corneal epithelia and conjunctival epithelia decreased in the absence of preservative. [source] PP2C family members play key roles in regulation of cell survival and apoptosisCANCER SCIENCE, Issue 7 2006Shinri Tamura Although unlimited proliferation of cancer cells is supported by multiple signaling pathways involved in the regulation of proliferation, survival, and apoptosis, the molecular mechanisms coordinating these different pathways to promote the proliferation and survival of cancer cells have remained unclear. SAPK and integrin-ILK signaling pathways play key roles in the promotion of apoptosis and cell proliferation/survival, respectively. Studies of TNF,- and H2O2 -induced apoptosis revealed that ASK1, a component of the SAPK system, mediates the TNF, and H2O2 signaling of apoptosis. ASK1 is activated by autophosphorylation of a specific threonine residue (T845) following TNF, stimulation. Our recent studies indicate that PP2C,, a member of the PP2C family, associates with and inactivates ASK1 by dephosphorylating T845. In contrast, PP2C,/ILKAP, a second PP2C family member, activates ASK1 by enhancing cellular phosphorylation of T845. PP2C,/ILKAP also forms a complex with ILK1 to inhibit the GSK3,-mediated integrin-ILK1 signaling in vivo, inhibiting cell cycle progression. These observations raise the possibility that PP2C,/ILKAP acts to control the cross-talk between integrin-induced and TNF,-induced signaling pathways, inhibiting the former and stimulating the latter, thereby inhibiting proliferation and survival and promoting the apoptosis of cancer cells. (Cancer Sci 2006; 97: 563,567) [source] TRAF6 knockdown promotes survival and inhibits inflammatory response to lipopolysaccharides in rat primary renal proximal tubule cellsACTA PHYSIOLOGICA, Issue 3 2010S. Liu Abstract Aim:, TRAF6 is a unique adaptor protein of the tumour necrosis factor receptor-associated factor family that mediates both tumour necrosis factor receptor (TNFR) and interleukin-1 receptor/Toll-like receptor (IL-1R/TLR) signalling. Activation of IL-1R/TLR and TNFR pathways in renal tubular cells contributes to renal injury. This study aimed to investigate if blockade of lipopolysaccharide (LPS)-triggered TLR4 signalling by small interfering RNA (siRNA) targeting TRAF6 protects survival and inhibits inflammatory response in isolated rat renal proximal tubular cells (PTCs). Methods:, PTCs isolated from F344 rat kidneys were transfected with chemically synthesized siRNA targeting TRAF6 mRNA. Real-time quantitative PCR was applied to measure mRNA level of TRAF6, TNF-,, IL-6 and monocyte chemoattractant protein-1 (MCP-1). Protein levels of extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase, caspase 3 and cleaved caspase 3 were evaluated by Western blotting. Cell viability was analysed with XTT reagents. Results:, We found that the TRAF6 gene was effectively silenced in PTCs using siRNA. TRAF6 knockdown resulted in reduced TNF-, and IL-6 mRNA expression upon LPS challenge. LPS-induced phosphorylation of JNK and p38 was attenuated in TRAF6 siRNA-transfected cells while the change in the phosphorylation of ERK was not remarkable. TRAF6 knockdown was associated with increased cell viability and reduced protein level of cleaved caspase-3, both, in the absence and presence of LPS. Conclusion:, Our studies suggest that TRAF6 knockdown may inhibit inflammatory response and promote cell survival upon LPS challenge in primary rat proximal renal tubular cells. [source] Nucleotides and epidermal growth factor induce parallel cytoskeletal rearrangements and migration in cultured adult murine neural stem cellsACTA PHYSIOLOGICA, Issue 2 2010I. Grimm Abstract Aim:, The adult subventricular zone (SVZ) contains neural stem cells that generate neuroblasts migrating to the olfactory bulb (OB) and differentiating into interneurones. The molecular cues controlling essential functions within the neurogenesis pathway such as proliferation, short and long distance migration, functional integration and cell survival are poorly understood. We have previously shown that cultured adult neural stem cells express a considerable variety of nucleotide receptors and that nucleotides and epidermal growth factor (EGF) induce converging intracellular signalling pathways that carry potential for synergism in the control of neural stem cell proliferation and cell survival. Here we investigate the role of EGF and the nucleotides ATP, ADP,S and UTP in neural stem cell migration. Methods:, Neural stem cells were prepared from adult mice and subjected to adherent culture. Labelling of F-actin was performed with tetramethylrhodamine isothiocyanate-phalloidin. Images were processed for quantitative evaluation of fluorescence labelling. Agonist-induced phosphorylation of AKT and focal adhesion kinase was analysed by quantitative Western blotting. Agonist-dependent cell migration was assayed using 48-well microchemotaxis chambers. Results:, Nucleotides and EGF induce the formation of stress fibres, an increase in the cortical actin cytoskeleton and in cell spreading. This is associated with increased phosphorylation of AKT and focal adhesion kinase. Using microchemotaxis chambers we demonstrate a parallel increase in cell migration. Conclusion:, Our results suggest that nucleotides and EGF acting as paracrine or autocrine signalling substances can be of relevance for structuring and maintaining the cytoarchitecture of the SVZ and the stream of neuroblasts migrating to the OB. [source] The death of cardiotonic steroid-treated cells: evidence of Na+i,K+i -independent H+i -sensitive signallingACTA PHYSIOLOGICA, Issue 1-2 2006S. N. Orlov Abstract Na/K-ATPase is the only known target of cardiotonic steroids (CTS) identified in plants, amphibians and later on in several mammalian species, including human. We focus our review on recent data implicating CTS in the tissue-specific regulation of cell survival and death. In vascular smooth muscle cells, CTS inhibited cell death triggered by apoptotic stimuli via a novel Na+i -mediated, Ca2+i -independent mechanism of expression of antiapoptotic genes, including mortalin. In contrast, exposure to CTS in vascular endothelial and renal epithelial cells led to cell death, showing combined markers of apoptosis and necrosis. This mode of cell death, termed oncosis, is caused by CTS interaction with Na/K-ATPase but is independent of the inhibition of Na/K-ATPase-mediated ion fluxes and inversion of the [Na+]i/[K+]i ratio. The intermediates of intracellular signalling involved in Na+i, K+i -independent oncosis of CTS-treated cells remain unknown. Recently, we found that this mode of cell death can be protected by modest intracellular acidification via the expression of H+i -sensitive genes. The molecular origin of intracellular Na+ and H+ sensor involvement in the development of apoptosis and oncosis is currently under investigation. [source] Regulation of focal adhesion targeting and inhibitory functions of the FAK related protein FRNK using a novel estrogen receptor "switch"CYTOSKELETON, Issue 2 2002Karen H. Martin Abstract Focal adhesion kinase (FAK) is a regulator of numerous adhesion-dependent processes including cell migration, cell proliferation, and cell survival. The C-terminal domain of FAK, FAK-related nonkinase (FRNK), is autonomously expressed and functions as an inhibitor of FAK signaling. Previous attempts to use FRNK as a tool to dissect FAK signaling have been limited because of an inability to temporally regulate the inhibitory functions of FRNK. In this report, we describe and characterize a conditionally targeted form of FRNK that was created by fusing the hormone-binding domain of the estrogen receptor (ER*) to the C-terminus of FRNK. In the absence of added hormone, FRNK-ER* was diffusely distributed throughout the cytoplasm of cells. Upon addition of hormone, the cytoplasmic pool of FRNK-ER* was rapidly redistributed to focal adhesions. We demonstrate that cells expressing FRNK-ER* show a hormone-dependent decrease in FAK tyrosine phosphorylation and cell migration. Furthermore, when cells expressing of FRNK-ER* were treated with hormone, the cells responded with a dramatic change in cell morphology, suggesting a role for FAK in the regulation of the adhesive properties of focal adhesions. Cell Motil. Cytoskeleton 51:76,88, 2002. © 2002 Wiley-Liss, Inc. [source] In Vivo Perfusion of Human Skin Substitutes With Microvessels Formed by Adult Circulating Endothelial Progenitor CellsDERMATOLOGIC SURGERY, Issue 2 2008ELAINE F. KUNG MD BACKGROUND At present, tissue-engineered human skin substitutes (HSSs) mainly function as temporary bioactive dressings due to inadequate perfusion. Failure to form functional vascular networks within the initial posttransplantation period compromises cell survival of the graft and its long-term viability in the wound bed. OBJECTIVES Our goal was to demonstrate that adult circulating endothelial progenitor cells (EPCs) seeded onto HSS can form functional microvessels capable of graft neovascularization and perfusion. MATERIALS AND METHODS Adult peripheral blood mononuclear cells (PBMCs) underwent CD34 selection and endothelial cell (EC) culture conditions. After in vitro expansion, flow cytometry verified EC phenotype before their incorporation into HSS. After 2 weeks in vivo, immunohistochemical analysis, immunofluorescent microscopy, and microfil polymer perfusion were performed. RESULTS CD34+ PBMCs differentiated into EPC demonstrating characteristic EC morphology and expression of CD31, Tie-2, and E-selectin after TNF,-induction. Numerous human CD31 and Ulex europaeus agglutinin-1 (UEA-1) microvessels within the engineered grafts (HSS/EPCs) inosculated with recipient murine circulation. Limitation of murine CD31 immunoreactivity to HSS margins showed angiogenesis was attributable to human EPC at 2 weeks posttransplantation. Delivery of intravenous rhodamine-conjugated UEA-1 and microfil polymer to HSS/EPCs demonstrated enhanced perfusion by functional microvessels compared to HSS control without EPCs. CONCLUSION We successfully engineered functional microvessels in HSS by incorporating adult circulating EPCs. This autologous EC source can form vascular conduits enabling perfusion and survival of human bioengineered tissues. [source] Novel functions of ribosomal protein S6 in growth and differentiation of Dictyostelium cellsDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 6 2009Kazutaka Ishii We have previously shown that in Dictyostelium cells a 32 kDa protein is rapidly and completely dephosphorylated in response to starvation that is essential for the initiation of differentiation (Akiyama & Maeda 1992). In the present work, this phosphoprotein was identified as a homologue (Dd-RPS6) of ribosomal protein S6 (RPS6) that is an essential member for protein synthesis. As expected, Dd-RPS6 seems to be absolutely required for cell survival, because we failed to obtain antisense-RNA mediated cells as well as Dd-rps6 -null cells by homologous recombination in spite of many trials. In many kinds of cell lines, RPS6 is known to be located in the nucleus and cytosol, but Dd-RPS6 is predominantly located in the cell cortex with cytoskeletons, and in the contractile ring of just-dividing cells. In this connection, the overexpression of Dd-RPS6 greatly impairs cytokinesis during axenic shake-cultures in growth medium, resulting in the formation of multinucleate cells. Much severe impairment of cytokinesis was observed when Dd-RPS6-overexpressing cells (Dd-RPS6OE cells) were incubated on a living Escherichia coli lawn. The initiation of differentiation triggered by starvation was also delayed in Dd-RPS6OE cells. In addition, Dd-RPS6OE cells exhibit defective differentiation into prespore cells and spores during late development. Thus, it is likely that the proper expression of Dd-RPS6 may be of importance for the normal progression of late differentiation as well as for the initiation of differentiation. [source] Whitesnake/sfpq is required for cell survival and neuronal development in the zebrafishDEVELOPMENTAL DYNAMICS, Issue 3 2008Laura Anne Lowery No abstract is available for this article. [source] Zebrafish dou yan mutation causes patterning defects and extensive cell death in the retinaDEVELOPMENTAL DYNAMICS, Issue 5 2007Anne E. Catalano Abstract The size of an organ is largely determined by the number of cells it contains, which in turn is regulated by two opposing processes, cell proliferation and cell death, however, it is generally not clear how cell proliferation and cell death are coordinated during development. Here, we characterize the zebrafish dou yanmi234 mutation that results in a dramatic reduction of retinal size and a disruption of retinal differentiation and lamination. The retinal size reduction is caused by increased retinal cell death in a non,cell-autonomous manner during early development. The phenotypic defect in dou yanmi234 arises coincident with the onset of retinal neurogenesis and differentiation. Interestingly, unlike many other small eye mutations, the mutation does not increase the level of cell death in the brain, suggesting that the brain and retina use different mechanisms to maintain cell survival. Identification and further study of the dou yan gene will enhance our understanding of the molecular mechanisms regulating retinal cellular homeostasis, i.e., the balance between cell proliferation and cell death. Developmental Dynamics 236:1295,1306, 2007. © 2007 Wiley-Liss, Inc. [source] Studies on epidermal growth factor receptor signaling in vertebrate limb patterningDEVELOPMENTAL DYNAMICS, Issue 2 2005Minoru Omi Abstract The epidermal growth factor receptor (EGFR) regulates multiple patterning events in Drosophila limb development, but its role in vertebrate limb morphogenesis has received little attention. The EGFR and several of its ligands are expressed in developing vertebrate limbs in manners consistent with potential patterning roles. To gain insight into functions of EGFR signaling in vertebrate limb development, we expressed a constitutively active EGFR in developing chick limbs in ovo. Expression of activated EGFR causes pre- and postaxial polydactyly, including mirror-image,type digit duplication, likely due to induction of ectopic expression and/or modulation of genes involved in anterior,posterior (AP) patterning such as Sonic hedgehog (Shh), dHand, Patched (Ptc), Gli3, Hoxd13, Hoxd11, bone morphogenetic protein 2 (Bmp2), Gremlin, and FGF4. Activation of EGFR signaling dorsalizes the limb and alters expression of the dorsal,ventral (DV) patterning genes Wnt7a, Lmx, and En1. Ectopic and/or extended FGF8 expressing apical ectodermal ridges (AERs) are also seen. Interdigital regression is inhibited and the digits fail to separate, leading to syndactyly, likely due to antiapoptotic and pro-proliferative effects of activated EGFR signaling on limb mesoderm, and/or attenuation of interdigital Bmp4 expression. These findings suggest potential roles for EGFR signaling in AP and DV patterning, AER formation, and cell survival during limb morphogenesis. Developmental Dynamics 233:288,300, 2005. © 2005 Wiley-Liss, Inc. [source] ROCK inhibitor (Y27632) increases apoptosis and disrupts the actin cortical mat in embryonic avian corneal epitheliumDEVELOPMENTAL DYNAMICS, Issue 3 2004Kathy K.H. Svoboda Abstract The embryonic chicken corneal epithelium is a unique tissue that has been used as an in vitro epithelial sheet organ culture model for over 30 years (Hay and Revel [1969] Fine structure of the developing Avian cornea. Basel, Switzerland: S. Karger A.G.). This tissue was used to establish that epithelial cells could produce extracellular matrix (ECM) proteins such as collagen and proteoglycans (Dodson and Hay [1971] Exp Cell Res 65:215,220; Meier and Hay [1973] Dev Biol 35:318,331; Linsenmayer et al. [1977] Proc Natl Acad Sci U S A 74:39,43; Hendrix et al. [1982] Invest Ophthalmol Vis Sci 22:359,375). This historic model was also used to establish that ECM proteins could stimulate actin reorganization and increase collagen synthesis (Sugrue and Hay [1981] J Cell Biol 91:45,54; Sugrue and Hay [1982] Dev Biol 92:97,106; Sugrue and Hay [1986] J Cell Biol 102:1907,1916). Our laboratory has used the model to establish the signal transduction pathways involved in ECM-stimulated actin reorganization (Svoboda et al. [1999] Anat Rec 254:348,359; Chu et al. [2000] Invest Ophthalmol Vis Sci 41:3374,3382; Reenstra et al. [2002] Invest Ophthalmol Vis Sci 43:3181,3189). The goal of the current study was to investigate the role of ECM in epithelial cell survival and the role of Rho-associated kinase (p160 ROCK, ROCK-1, ROCK-2, referred to as ROCK), in ECM and lysophosphatidic acid (LPA) -mediated actin reorganization. Whole sheets of avian embryonic corneal epithelium were cultured in the presence of the ROCK inhibitor, Y27632 at 0, 0.03, 0.3, 3, or 10 ,M before stimulating the cells with either collagen (COL) or LPA. Apoptosis was assessed by Caspase-3 activity assays and visualized with annexin V binding. The ROCK inhibitor blocked actin cortical mat reformation and disrupted the basal cell lateral membranes in a dose-dependent manner and increased the apoptosis marker annexin V. In addition, an in vitro caspase-3 activity assay was used to determine that caspase-3 activity was higher in epithelia treated with 10 ,M Y-27632 than in those isolated without the basal lamina or epithelia stimulated with fibronectin, COL, or LPA. In conclusion, ECM molecules decreased apoptosis markers and inhibiting the ROCK pathway blocked ECM stimulated actin cortical mat reformation and increased apoptosis in embryonic corneal epithelial cells. Developmental Dynamics 229:579,590, 2004. © 2004 Wiley-Liss, Inc. [source] Induction of chondrogenesis in neural crest cells by mutant fibroblast growth factor receptorsDEVELOPMENTAL DYNAMICS, Issue 2 2002Anita Petiot Abstract Activating mutations in human fibroblast growth factor receptors (FGFR) result in a range of skeletal disorders, including craniosynostosis. Because the cranial bones are largely neural crest derived, the possibility arises that increased FGF signalling may predispose to premature/excessive skeletogenic differentiation in neural crest cells. To test this hypothesis, we expressed wild-type and mutant FGFRs in quail embryonic neural crest cells. Chondrogenesis was consistently induced when mutant FGFR1-K656E or FGFR2-C278F were electroporated in ovo into stage 8 quail premigratory neural crest, followed by in vitro culture without FGF2. Neural crest cells electroporated with wild-type FGFR1 or FGFR2 cDNAs exhibited no chondrogenic differentiation in culture. Cartilage differentiation was accompanied by expression of Sox9, Col2a1, and osteopontin. This closely resembled the response of nonelectroporated neural crest cells to FGF2 in vitro: 10 ng/ml induces chondrogenesis, Sox9, Col2a1, and osteopontin expression, whereas 1 ng/ml FGF2 enhances cell survival and Sox9 and Col2a1 expression, but never induces chondrogenesis or osteopontin expression. Transfection of neural crest cells with mutant FGFRs in vitro, after their emergence from the neural tube, in contrast, produced chondrogenesis at a very low frequency. Hence, mutant FGFRs can induce cartilage differentiation when electroporated into premigratory neural crest cells but this effect is drastically reduced if transfection is carried out after the onset of neural crest migration. © 2002 Wiley-Liss, Inc. [source] An agonistic mAb directed to the TrkC receptor juxtamembrane region defines a trophic hot spot and interactions with p75 coreceptorsDEVELOPMENTAL NEUROBIOLOGY, Issue 3 2010Veronique Guillemard Abstract The D5 domain of TrkC receptors is a docking site for Neurotrophin-3 (NT-3), but other domains may be relevant for function or harmonizing signals with p75NTR coreceptors. We report a monoclonal antibody (mAb) 2B7 targeting the juxtamembrane domain of TrkC. mAb 2B7 binds to murine and human TrkC receptors and is a functional agonist that affords activation of TrkC, AKT, and MAPK. These signals result in cell survival but not in cellular differentiation. Monomeric 2B7 Fabs also affords cell survival. Binding of 2B7 mAb and 2B7 Fabs to TrkC are blocked by NT-3 in a dose-dependent manner but not by pro-NT-3. Expression of p75NTR coreceptors on the cell surface block the binding and function of mAb 2B7, whereas NT-3 binding and function are enhanced. mAb 2B7 defines a previously unknown neurotrophin receptor functional hot spot; that exclusively generates survival signals; that can be activated by non-dimeric ligands; and potentially unmasks a site for p75-TrkC interactions. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2010. [source] Death and survival of heterozygous Lurcher Purkinje cells In vitroDEVELOPMENTAL NEUROBIOLOGY, Issue 8 2009Hadi S. Zanjani Abstract The differentiation and survival of heterozygous Lurcher (+/Lc) Purkinje cells in vitro was examined as a model system for studying how chronic ionic stress affects neuronal differentiation and survival. The Lurcher mutation in the ,2 glutamate receptor (GluR,2) converts an orphan receptor into a membrane channel that constitutively passes an inward cation current. In the GluR,2+/Lc mutant, Purkinje cell dendritic differentiation is disrupted and the cells degenerate following the first week of postnatal development. To determine if the GluR,2+/Lc Purkinje cell phenotype is recapitulated in vitro, +/+, and +/Lc Purkinje cells from postnatal Day 0 pups were grown in either isolated cell or cerebellar slice cultures. GluR,2+/+ and GluR,2+/Lc Purkinje cells appeared to develop normally through the first 7 days in vitro (DIV), but by 11 DIV GluR,2+/Lc Purkinje cells exhibited a significantly higher cation leak current. By 14 DIV, GluR,2+/Lc Purkinje cell dendrites were stunted and the number of surviving GluR,2+/Lc Purkinje cells was reduced by 75% compared to controls. However, treatment of +/Lc cerebellar cultures with 1-naphthyl acetyl spermine increased +/Lc Purkinje cell survival to wild type levels. These results support the conclusion that the Lurcher mutation in GluR,2 induces cell autonomous defects in differentiation and survival. The establishment of a tissue culture system for studying cell injury and death mechanisms in a relatively simple system like GluR,2+/Lc Purkinje cells will provide a valuable model for studying how the induction of a chronic inward cation current in a single cell type affects neuronal differentiation and survival. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009 [source] | |||||||||||||||||||||||||||||||||||||